Actividad Antioxidante de La Diosgenina de Dioscorea

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ANTIOXIDANT ACTIVITY OF DIOSGENIN FROM Dioscorea
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Oxidation Communications 39, No 1-I, 1–7 (2016)
Liquid-phase oxidation: hydrogen peroxide as oxidiser
EFFECT OF ETO AND HYDROGEN PEROXIDE PLASMA

STERILISATION METHODS ON CELL PROLIFERATION
ABILITY IN PLLA/GA SCAFFOLDS
HUANG HAOa, ZHOU XIAOLIa*, QIN NIANa, HE XIAOYANa,

XIAO ZHENGHUAb, ZENG QINGa, HE QIANa
a
Pulp Washing and Sterilisation Supply Centre in West China Hospital, Sichuan
University, 610 041 Chengdu City, Sichuan Province, China
E-mail: zhouxiaolicd@163.com
b
Department of Cardiac Surgery in West China Hospital, Sichuan University,
610 041 Chengdu City, Sichuan Province, China
ABSTRACT
The purpose of the research was to discuss if there is any difference between the
effects of two low-temperature sterilisation methods: ETO (ethylene oxide) and
hydrogen peroxide plasma to cell proliferation abilities in L-polylactic acid/gelatin
scaffold materials. By applying MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-
tetrazolium bromide) experiments and SEM (scanning electron microscope), we can
make a comparison between these two sterilisation methods in BMSCs (bone marrow
mesenchymal stem cells) proliferation on scaffolds at 4 timings: 2, 4, 6 and 8 days.
SEM suggests that both scaffolds are suitable for cell growth and cells are in
good shape; MTT suggests that OD (optical density) value of hydrogen peroxide
plasma group is higher than that of ETO group in 4, 6 and 8 days indicating that the
cell proliferation ability is strong. It was concluded that both those two sterilisation
methods can be applied to the sterilisation of cell proliferation scaffolds and hydrogen
peroxide plasma can make the cell grow better.
Keywords: ETO, hydrogen peroxide plasma, PLLA/GA, MTT.
AIMS AND BACKGROUND

In recent years, with the rapid development of tissue engineering technology, biologi-
cal scaffold materials have played an important role in tissue engineering. Similar to
ECM, it is the physical support and metabolic place of cell adhesion, proliferation and
differentiation, which can guide tissue regeneration and control tissue structures1,2.
*
For correspondence.
1
Sterilisation of scaffold materials has also become a key part in the building process
of extracorporeal tissue engineering, different sterilisation methods may affect cell
growth and proliferation in scaffold materials. The traditional pressure steam steri-
lisation method in the lab may result in scaffold degeneration due to high tempera-
tures3–5. Low-temperature sterilisation methods are applied to all varieties of precision
instruments which do not survive at high temperatures such as, medical devices and
disposable medical articles. The study on its principle and relative factors has been
reported already.
Currently, ETO (ethylene oxide) and hydrogen peroxide plasma are commonly
used in the hospital6. There is no report about relative studies on whether sterilis-
ing scaffold materials with low-temperature sterilisation methods will affect cell
proliferation. This study intends to compare cell proliferation abilities on L-PLA/
GA(levorotatory polylactic acid/gelatin) synthetic materials using two low-temperature
sterilisation methods, so as to provide a theory basis for choosing the optimum steri-
lisation method for lab scaffold materials.
EXPERIMENTAL
Production of PLLA/GA scaffold materials. Placing PLLA/GA electrospinning solu-
tion (the ratio of PLLA and GA is 1:1) into one 2 ml syringe through electrospinning
method. The electrospinning method requires that the voltage is 10–12 kV and the
receiving distance (distance between the needle and the receiver) is 15 cm. The electro-
spinning was applied with the humidity within 40–60% at room temperature. Then, the
fibre membrane samples were restored in a vacuum drying oven after electrospinning.
Sterilisation methods. PLLA/GA scaffold materials were divided into two groups. Two
groups were disinfected by ETO low temperature sterilisation methods (EO group)
and hydrogen peroxide plasma low temperature sterilisation methods (H2O2 group)
respectively (STERRAD type 200 hydrogen peroxide plasma sterilisation machines
and 3M 8XL ETO sterilisation machines offered by West China hospital sterilisation
and supply centre). Sterilisation instrument is shown in Fig. 1, and the sterilisation
parameter is generic parameter.
Inoculation of BMSCs. The PLLA/GA (Sigma Corporation of America) scaffold
materials disinfected by different sterilisation methods were imbed into 24-pore
plates, washed 3 times with PBS in sterile conditions and then soaked into the dul-
becco minimum essential medium culture for 24 h for standby. The cell suspension
was inoculated on the material of third generation BMSCs with the density of 4 ×
104 cm2. Then the cell suspension was placed into the incubator for static culture for
4 h at 37oC. The incubator should contain CO2 with 5% volume fraction. The culture
solution was added to 1 ml when the cells settled down and attached to the bottom,
the old culture should be replaced with new one every other day.
2
Fig. 1. ETO and hydrogen peroxide plasma sterilisation instruments
A – ETO sterilisation instruments; B – hydrogen peroxide plasma sterilisation instruments
SEM observation. Culture solution was drawn away from 24-pore plates. The 24-pore
plates were washed 3 times (the interval between each time was 5 min) with PBS
(phosphate buffer solution) whose pH is between 7.2–7.4. Then it was fixated with
3% glutaraldehyde PBS solution and laid aside for 1 h at 4oC. The 24-pore plates were
washed with PBS 3 times again after fixation in which the interval was 5 min. The
samples were placed right side up on the table and were treated with spray-gold after
ethanol gradient dehydration and being dried at room temperature. At last, the growth
situation of BMSCs on scaffold materials was observed by JSM-6700F FE-SEM.
MTT experiments. The principle of MTT is that succinate dehydrogenase and cy-
tochrome C in living cells can restore MTT into insoluble blue crystal, and precipitate
in the cells, while dead cells do not possess this function. DMSO can dissolve violet
crystal precipitating in the cells, and colour depth of the solution is correlated with
the amount of violet crystal. Testing of OD value at 490 nm through Alpha-1860P
UV-vis can reflect the number of living cells, so as to reflect cell proliferation. After
2, 4, 6 and 8 days, respectively, the cell proliferation situation on the scaffold with
MTT (AMRESCO Corporation of America) was detected. The statistical analysis
was conducted based on the average value of 5 samples at timing within each group.
Each group had 5 samples at each timing, then make statistical analysis with
average.
Statistic methods. Experimental results were expressed as means ± SD. For group
comparison were applied one-way Anova and post-hoct test. In case p < 0.05, the
difference is statistically significant. Repeated measurement data in each group were
statistically analysed by SPSS 13.0.
3
RESULTS
Observation of the growth situation of BMSCs on PLLA/GA scaffold materials with
SEM. By observing scaffold materials after 8-days sterilisation and culture with
these two methods, respectively, with SEM, we can discover that cells grow on both
materials, with great density and good spreading property, cells connect with each
other through pseudopodia, internal growth of some parts, and there is no difference
in numbers under the SEM (Fig. 2), which indicates that both methods are suitable
for cell growth.
Fig. 2. Growth form of BMSCs on scaffold materials with different sterilisation methods (×1200)
A – growth form of BMSCs in EO group; B – growth form of BMSCs in H2O2 group
Cell proliferation experiments with two different sterilisation methods (MTT). Com-
paring the growth and proliferation situation of BMSCs in 8 days of two groups,
results in Table 1 reveal that OD value shows no difference between two groups after
inoculation for 2 days (p > 0.05); with culture time being extended to 4 days, OD
value in H2O2 group is higher than that in EO group (p < 0.05), which indicates that
cell proliferation ability in H2O2 group is higher than that in EO group, so it is more
suitable for the growth of BMSCs.
Table 1. Comparison of BMSCs proliferation situation at different timings with two methods (x ± s)
Names OD value × 10–2 OD value × 10–2 OD value × 10–2 OD value × 10–2
2 d (n = 5) 4 d (n = 5) 6 d (n = 5) 8 d (n = 5)
H2O2 7.06±0.61 12.40±1.36* 27.10±0.59* 37.02±2.72D
EO 6.30±0.65 9.74±0.95* 20.76±2.43# 25.70±2.47D
Note: *, #, D means comparison between 2 timings among 4, 6, 8 days, p < 0.05.
DISCUSSION
Cells, scaffold material and microenvironment of specific tissues are three elements
of tissue engineering, among which, scaffold material plays a central role. To make
sure a long-term proliferation of cells in the scaffold material, sterilisation of scaffold
4
material is one of the key parts. At present, most materials used in tissue engineering
are biological material which cannot endure the traditional high temperature sterilisa-
tion methods. Otherwise, the alcohol and glutaraldehyde soaking methods may react
with some ingredients in the support materials, so as to change the nature of scaffold
material. Meanwhile, the remaining disinfectant will also bring danger to cell prolif-
eration. Low-temperature sterilisation methods have the advantage of low temperature
and simple operation, the common temperature is between 40–60oC, which is a new
approach for sterilisation of scaffold material in tissue engineering. Currently, the
common methods are hydrogen dioxide peroxide plasma low-temperature sterilisa-
tion methods and ETO.
ETO will have nonspecific alkylation reaction with carboxy, amidogen, sul-
phydryl, hydroxy, DNA and RNA in the microprotein, making it lose reactive group
hindering its normal chemical reaction and metabolism, so as to kill all kinds of
microorganisms and reach a good sterilisation effect, it has the advantage of strong
penetrability, broad-spectrum bactericidal effect, little damage to goods, reliable
sterilisation, long shelf-life, etc.8 Hydrogen dioxide peroxide plasma low tempera-
ture sterilisation method, by making use of a large amount of ultraviolet photon and
free radical with high activity, has reaction with protein and nucleic acid inside the
bacteria, leads to a quick death of microorganism, so as to reach a sterilisation effect9.
This study has applied these two sterilisation methods on PLLA/GA scaffold material
which is commonly used in tissue engineering currently, assesses cell proliferation
on scaffold material. The result of SEM shows that cells grow well on both scaffold
material, within 8 days, cells on scaffold material have not been polluted, which
indicates that both sterilisation methods reach ideal effects.
The principle of MTT is that succinate dehydrogenase and cytochrome C in living
cells can restore MTT into insoluble blue crystal, and precipitate in the cells, but dead
cells do not possess this function. DMSO can dissolve violet crystal precipitating in
the cells, colour depth of the solution is correlated with the amount of violet crystal.
Determination of OD value at 490 nm by ELIASA can reflect the number of living
cells, so as to reflect cell proliferation. The result of this study shows that cells in
H2O2 group are the same as those in EO in the second day, after 4 days, OD value in
the former group is obviously higher than that in the latter one, which shows that cell
proliferation in the scaffold material with hydrogen dioxide peroxide plasma is more
vigorous. There are two possible reasons: (1) the rate of hydrogen dioxide peroxiade
plasma sterilisation system is fast, requiring only 55 min, while ETO asks for longer
time, its average cycle period is 15 h, if sterilising too long, the shape of scaffold
material may change, so as to affect cell proliferation and growth on scaffold mate-
rial; (2) hydrogen peroxide plasma system does not require detoxification time in the
sterilisation process, it can take the dead microorganism and residue away through
gas-circulating system and decompose into water and oxygen, nothing will remain in
scaffold materials12,13. ETO sterilisation methods will have residue, ETO is biotoxical
itself, so it may cause hemolysis and bring damage to skin and mucosa, chlorhydrin,
5
the product of sterilisation, changes into glycol in the water, which will also damage
cell growth in scaffold materials14.
All in all, ETO sterilisation and hydrogen peroxide plasma sterilisation methods
are more commonly applied to medical goods than others. They both can have ideal
effects on scaffold materials commonly seen in tissue engineering. But they differ in
properties. hydrogen peroxide plasma sterilisation has the merits of short cycle and
no residue, cell proliferation ability on scaffold materials is stronger, and this result
offers theory basis to sterilisation methods in the building process of scaffold materi-
als of external tissue engineering.
CONCLUSIONS
Both sterilisation methods can be applied to the sterilisation of cell proliferation scaf-
folds, but hydrogen peroxide plasma can enhance this ability better.
ACKNOWLEDGEMENTS
This project was supported by Science and Technology Office in Sichuan Province
(No 14ZC1837).
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to Match the Mechanical Properties of Myocardial Tissue. Biomaterials, 29 (1), 47 (2008).
2. M. PATEL, J. P. FISHER: Biomaterial Scaffolds in Pediatric Tissue Engineering. Pediatr Res, 63
(5), 497 (2008).
3. E. A. CHRISTENSEN, H. KRISTENSEN: Biological Indicators for the Control of Ethylene Oxide
Sterilisation. Acta Path Micro Im B, 87 (3), 147 (1979).
4. R. R. ERNST, J. J. SHULL: Ethylene Oxide Gaseous Sterilisation. I. Concentration and Temperature
Effects. Appl Microbiol, 10, 37 (1962).
5. G. L. GILBERT, V. M. GAMBILL, D. R. SPINER, R. K. HOFFMAN, C. R. PHILLIPS: Effect of
Moisture on Ethylene Oxide Sterilisation. Appl Microbiol, 12, 496 (1964).
6. Q. X. WANG, W. R. LIU, S. H. LI: Application of Low Temperature Hydrogen Peroxide Plasma
Sterilizer in the Operation Room. Journal of Baotou Medical College, 28 (5), 93(2012).
7. B. S. KIM, D. J. MOONEY: Development of Biocompatible Synthetic Extracellular Matrices for
Tissue Engineering. Trends Biotechnol, 16 (5), 224(1998).
8. L. L. NIU: Comparison of Three Methods for the Sterilisation Effect of the Bladder. Chinese Journal
of Disinfection, 31 (2), 206 (2014).
9. X. B. LI, D. L. YUAN, S. M. TONG: Antibiotic Wastewater Treatment via Catalytic Oxidation.
Oxid Commun, 38 (4), 1896(2015).
10. S.ADLER, M.SCHERRER, D. DASCHNER: Costs of Low-temperature Plasma Sterilisation Com-
pared with Other Sterilisation Methods. J Hosp Infect, 40 (2), 125 (1998).
11. M. NAVYA, J. A. JUDITH, R. SUBRATA: Effect of Dielectric and Liquid on Plasma Sterilisation
Using Dielectric Barrier Discharge Plasma. PloS One, 8, 70840 (2013).
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12. Z. GUO’S. X. MAO: Discussion on the Risk Control of Ethylene Oxide Sterilisation. China Medical
Device Information, 11, 27 (2011).
13. M. FURUHASHI, T. MIYAMAE: Ethylene Oxide Sterilisation of Medical Devices – with Special
Reference to the Sporicidal Activity and Residual Concentration of Ethylene Oxide and Its Second-
ary Products. The Bulletin of Tokyo Medical and Dental University, 2, 23(1982).
14. L. Y. WEN, Q. S. HAI: Oxidation of Phenolic Wastewater with Glycol in Supercritical Water. Oxid
Commun, 38 (2), 796(2015).
Received 15 January 2016
Revised 5 February 2016
7
Solid-phase oxidation
INFLUENCE OF METHANOGENS AND METHANOTROPHS

ON METHANE GENERATION AND OXIDATION IN THE
PLANT-SOIL ECOLOGICAL SYSTEM
YANG KEa, JI HONGb,c*, XING ZHIXIANGa, LIU GECHENa, YU SIWEIa,

GU JINXINa
a
School of Environmental and Safety Engineering, Changzhou University,
213 164 Changzhou, China
b
School of Petroleum Engineering, Changzhou University, 213 164 Changzhou,
China
c
Jiangsu Key Laboratory of Oil-Gas Storage and Transportation Technology,
Jiangsu, 213 164 Changzhou, China
E-mail: jihong_academic@126.com
ABSTRACT
The mechanism of methane generation and oxidation process was investigated by us-
ing the fluorescence in situ hybridisation technique. The amounts of methanogens and
methanotrophs were analysed in the soil in a plant-soil·ecological system. The result
showed that methane generation and oxidation were closely related to the distribution
of methanogens and methanotrophs. The methanotrophs were mainly concentrated
in the plant rhizosphere, and the amount of methanogens were higher than that of
methanotrophs in the soil. Temperature fluctuation and oxidation-reduction potential
(ORP) greatly affected the methane generation and oxidation process in the plant–soil
ecological system. The methane emissions demonstrated the methane generation and
oxidation process directly. The annual average amount of methane emissions was
26.3 mg/(m2 h). The methane emissions peak occurred in the summer and was up to
194.2 mg/(m2 h).
Keywords: plant-soil systems, methane, fluorescence in situ hybridisation, methano-
gens, methanotrophs.
AIMS AND BACKGROUND

With the constant development of ecological engineering technology, the growing
number of plant soil ecological systems play a role in environmental safety disposal
*
For correspondence.
8
process. The plant soil ecological system will release methane to the atmosphere. If
we do not release methane regularly, it will be a great challenge to mitigating and
controlling the climate warming1–6. Microbial behaviour is obviously an important
factor that influences methane emissions, because methane is a by-product of micro-
bial metabolism. The relationship between soil, plant, miroorganism and the methane
emission in the soil-plant ecological system is an important aspect and it can reveal
the principle of methane emission. Fluorescence in situ hybridisation combines the
accuracy of molecular biology and the visibility of information under microscope. It
can monitor and evaluate different microorganisms in the natural or artificial environ-
ment of microorganisms, and evaluate the microbial community as well7.
In order to better explain the intrinsic relationship between microbial population
change and methane emission, this article has used fluorescence in situ hybridisation
technology to analyse the change and composition rules of the methanogens and
methanotrophs in the soil-plant ecological system, so as to provide reference material
for the design, promotion and evaluation of soil-plant ecological system.
EXPERIMENTAL
PLANT–SOIL ECOLOGICAL SYSTEM CONSTRUCTION
This experiment adapted the undercurrent type soil-plant ecological system. The
process unit was 88 cm deep and with 56 cm diameter, the soil horizon was a gravel
support with bottom depth of 18 cm. 10 reeds and 10 cattails with height of 80 cm
were transplanted to every process unit in early April. A process unit without plant was
set as comparative unite in the experiment. 5 ORP detection probes were buried in the
every process unite. The hydraulic loading of the process unite is 0.042 m³/(m2 day).
SAMPLE COLLECTION AND PROCESSING
The soil samples were collected once every two months. 5 sampling points were set
longitudinally and every sampling point was divided into 5 layers from top to bot-
tom. The depths of very sampling point were 0, –10, –20, –30 and –40 cm, respec-
tively. Put the samples on the ice immediately once the collecting processes were
completed. 5 g of soil samples were fixed in 4% paraformaldehyde fixed fluid for
18 h. The samples were disposed to remove the fixed fluid for 10 min. The samples
were fixed in the phosphate buttered solution and broken for 30 min in the ultrasonic
cell disruption system. Before fluorescence in situ hybridisation detection, the soil
samples were washed with phosphate buttered solution twice at normal temperature.
At last, the bacteria sample in the soil was suspended in the solution which contains
50% phosphate buttered solution and 50% ethyl alcohol solution.
9
FLUORESCENCE in situ HYBRIDISATION DETECTION
Selection of probes. In the experiment, the mass fraction of the probe and FA (for-
mamide) is shown in Table 1. The end of the probe 5 was labelled as Cy3. All of the
probes at –20°C are kept in TE buffer (10 mmol/l tris-HCl, 1 mmol/l, pH 7.2), and
its saving concentration is 4 pmol/μl. Transfect ion of nucleic acids of Green I SYBR
fluorescent dye (under the excitation of fluorescence microscope, green) was used to
label all the total microbial cells.
Table 1. Oligonucleotide probes and relevant characteristics

Probe Target microorganisms %
10-γ type I methanotrophs 0
9-α type II methanotrophs 0
MS1414 methanosarcina 35
MG 1200 methanomicrobiales 10
MB1174 methanomicrobia 35
MX825 methanosaetaceae 20
Hybridising. The soil sample was diluted to a suitable ratio with 200 mg/l tripolyphos-
phate solution and air dried on the glass slide at normal temperature. The samples
were dehydrated into 50, 80 and 98% ethanol solution for 3 min, respectively. Then,
the samples were hybridisated at 46oC with 16S rRNA probe hybridisation solution
for 2 h. The hybridisation temperature was increased to 48oC gradually, and each
sample was bath deposited for 20 min, so as to eliminate hybridisation or nonspecific
hybridisation of the probe. Green I 3 min stain SYBR after the excess stain, air dry. To
prevent the decolourisation, the 3 μl antigasfading agent was instilled on the circular
hole of the glass slide, and then the cover slip was placed, air bubbles were eliminated.
Quantitative observation of bacteria. TCS4D laser microscope (Leica Lasertechnik,
Germany) was used to observe the bacteria. Each soil sample was made into 3 parallel
hybrid samples. 10 visual fields were selected in every parallel hybrid sample. The
number of fluorescent spots was counted for every visual field. The quantity of the
sample can be obtained through the number of fluorescent spots from the sample.
Methane emission measurement. The emission flux of methane in the system was
detected by using closed chamber-gas chromatography technique every 20 days. In
order to eliminate emission flux difference caused by the daily change, the collection
time of the gas samples is fixed at 12:00. The gas samples were collected seven times
within 4 h. The transparent sampling box whose volume is 40 l was adopted in the
experiment. The body of the box is made from plastics, while the base and the top
are made from PVC material. In the measuring process, the sampling box was fixed
tightly on the top of the soil-plant ecosystem unit by using the screws. The sampling
box was sealed with the plastic colloidal cloth. There are two plugged pores which
are respectively switched in air tube and gas-collecting tube. When sampling, the two
10
tubes are switched in a small air pump simultaneously. The intake-tube penetrates into
the soil plant ecosystem surface layer by the air tube on the lid of the box.
The air entered into the sampling box by opening the pump and stirred 5 min
persistently to blend the gas in the box, so as to reduce the sampling error. Then the
air-entering switch was closed and the air-out switch was opened. The temperature
in the box was recorded for each sampling process. After sampling, the switches
should be closed immediately and gas-production tube should be clamped by clips
to prevent leakage. The gas sample is analysed by using gas chromatograph (Agilent,
GC7890A, USA). FID is chosen as the methane detector and the Poropak Q is chosen
as the chromatographic column.
RESULTS AND DISCUSSION

Longitudinal oxidation-reduction potential (ORP) distribution characteristics. ORP
is one of the important indices in the redox state of the reaction system. When pH
is certain, the ORP value varies with the content of the oxidised substances in the
system8,9. The vertical variation of ORP in the ecological bed can reflect the char-
acteristics of the oxygen species and the degradation of organic matter in different
water plants. Field test results of the plant bed ORP are shown in Fig. 1. It is seen that
ORP is similar from the surface to –15 cm in the growth and withering periods. In the
plant growth period, ORP is higher from the surface to –30 cm, providing favourable
conditions for methane oxidative bacteria. When the surface distance is 10–15 cm,
ORP gradually decreases in anaerobic condition. When there is no plant bed, due to
the water-dissolved oxygen, the surface is under aerobic condition, while other areas
are under anaerobic condition. The anaerobic environment is the primary condition
for the development of related bacteria while the aerobic environment is fit for the
growth and propagation of methanotrophs. The growth of plants has a great influence
on the content of ORP and substrate10.
100
50
0 withering period
–50 growing period
–100
ORP (mV)
–150
–200
–250
–300
–350
–400
–70 –60 –50 –40 –30 –20 –10 0 10
depth (cm)
Fig. 1. Distribution of ORP in soil-plant system
11
Distribution of methanogens in the methane generation and oxidation process. Us-
ing fluorescence in situ hybridisation technique and GREEN I SYBR background
staining, the fluorescent area was used as the index to detect the methanogens of the
corresponding probe. In the growing season of plants, the number and spatial distribu-
tion of methanogens detected by 16s rRNA probe, MX825, MB1174, MS1414 and
MG1200 are shown in Figs 2 and 3, respectively.
10
no plant bed
8
amount of methanogens × 108 (g–1)
reed profile
cattails profile
0
MS1414 MX825 MB1174 MG1200
16s rRNA
Fig. 2. Comparison of methanogens amounts by different probes
4 reed profile
cattails profile
no plant bed
amount of methanogens × 108 (g–1)
0
–45 –40 –35 –30 –25 –20 –15 –10 –5
depth (cm)
Fig. 3. Effect of plant species on methanogens distribution
In Fig. 2 there is a significant difference (MG1200) between the plant and the
soil in the soil of the plant and the soil moisture content under the same conditions as
the operating conditions and the soil moisture content (P = 0.013 < 0.05, T = 8.69).
Comparing the four kinds of target microorganisms detected by probes, the methanomi-
crobials (GM1200) distribution is the most extensive. In particular, the longitudinal
profile of the plant reached 1.0 × 109 g–1. There is a small number of methanogens in
the longitudinal section of cattail, and there was no significant difference between
12
the plant (P = 0.644 > 0.05, T = –0.54). This indicates that the growth environment of
methanogens in soil plant ecosystem is improved by the presence of the reed, which
is beneficial to the biosynthesis of methane. But the cattail may inhibit the growth
of methanogens. The number of methanogens and methanotrophs detected by probe
MS1414, MX825 and MB1174, respectively is much less, lower than 4.0 × 108 g–1.
Fluorescence in situ hybridisation test results (Fig. 3) reveal that the number
of methanogens in the longitudinal direction of the ecosystem increases with the
increasing of depth. In –40 cm place, the number of methanogens bacteria is larg-
est while in –20 cm place, the number of methanogens was higher than that in the
non-plant unit. The reason for the largest number of methanogens in –20 cm is that
oxygen is transported to the roots of the plants through the botanical photosynthesis.
The oxygen was released from the roots of the plants. ORP in the region is high and
it is not fit for the growth and reproduction of methanogens. At –40 cm place, the
number of methanogens in the plant unit is higher than that in the non-plant unit. It
indicated that ORP of the region is low and the main root of the plant is not distrib-
uted in the region. The distribution of ORP in soil-plant system was shown in Fig. 1.
Major factors affecting methanogen distribution in the soil are: (1) subsurface flow
flooding system creates the ideal living environment for methane bacteria, so as to
promote the proliferation of methanogens and methane formation; (2) plant growth
of methanogenic bacteria provides more growth of the substrate, but also promotes
the proliferation of methane oxidising bacteria11.
Distribution of methanotrophs in methane generation and oxidation process. In the
growing season of plants, the number and the spatial distribution of the type I and
type II methanotrophs tested by 165rRNA probe 10-γ and 9-α are shown in Figs 4 and
5, respectively. Figure 4 shows that the distribution of type I and type II methanotrophs
in the soil is different and type II were distributed more widely which revealed that
type II methanotrophs can survive better in plant root exudates and a nutrient poor
environment. Figure 4 also shows that the number of reed section type I and type II
methanotrophs were the highest, cattail profile of methane oxidising bacteria was
slightly higher than the total free plant bed.
Figure 5 shows that the amount of the methanotrophs is the highest at the –30 cm
in the plant. Planting can improve the soil, so as to improve the methanotroph breed
in different levels of growth and control the release of methane from the soil plant
ecosystem. It can be seen that from –40 to –30 cm, the number of methanotrophs in
the plant was significantly higher than that in the non-plant unit. Soil oxygen content
and plant growth in soil-plant ecosystem have a significant effect on the number of
species group. The metabolic activity during the plant growth period not only transport
and release the oxygen to the root, but also generates a large number of low molecular
organic compounds continuously. The process will provide a large number of nutrients
and energy in the rhizosphere, resulting in a significant rhizosphere effect12,13.
13
no plant bed
reed profile
7 cattails profile
amount of methanotroph × 108 (g–1)

5
0
10-γ 9-α
16s rRNA
Fig. 4. Amounts of type I and type II methanotrophs
no plant bed
reed profile
2.0 cattails profile
amount of methanotroph × 108 (g–1)
1.5
1.0
0.5
0.0
–45 –40 –35 –30 –25 –20 –15 –10 –5
depth (cm)
Fig. 5. Effect of plant species on Methanotrophs distribution
Relationship between bacteria distribution and methane emission. The methane emis-
sion flux of plant cultivation units and non-plant units is shown in Fig. 6. It is seen
that the annual average methane emission flux of plant bed and no plant bed are 26.3
and 6.1 mg/(m2 h), respectively. Seasonal fluctuations have a great influence on the
methane emissions of the plant beds, and the emission peaks occured in the summer
of July, as high as 194.2 mg/(m2 h). The plant withering period was from January to
November, mixed cultivation and no plant bed fluxes of methane plants were less
than 10.0 mg/(m2 h). Plants growing emission flux is 3 to 92 times at the withering
period, suggesting that the growth of reeds and cattails can stimulate the methane
emission. Ecological wastewater treatment system in the process released methane
with the range of 375–16757 mg/(m2 d). Anaerobic stabilisation pond process released
methane with the range of 16 000–32 000 mg/(m2 d), far higher than the emission
14
flux of ecological wastewater treatment process. This is mainly due to the anaerobic
environment conducive to the growth of methane bacteria. This shows that the system
ρ(DO) has a great influence on the methane emission flux.
180
160
with plant bed
140
CH4 emission flux (mg/(m2 h)) without plant bed
120
100
80
60
40
20
0
–20
1–15 3–18 5–17 7–19 9–20 11–21

time (month–day)
Fig. 6. Properties of methane emission from a soil plant system
CONCLUSIONS
After synthesising by the methanogens in the anaerobic environment, methane will
escape into the atmosphere through the soil and aquifer of the ecosystem. While
methane goes through the soil and aquifer, it could be oxidised by methanotrophs
in the above-mentioned system. The reason why methane is released is that more
methane is produced than that oxidised in the soil. Most plant-oil ecosystems are
under anaerobic condition. In addition, nutrient salt was input into the system, these
two factors create a very suitable condition for methane generating by methanogens
biological action. The growth of plants can enlarge the number of methanotrophs
surrounded by plant rhizosphere, so that those methanotrophs are able to raise the
oxidation rate. The amount of methanotrophs scattered on the plant rhizosphere is
much less than that of methanogens when they are measured at the same period. As
a result, the system produces fractional extra methane. The phenomenon shows that
it is necessary to cultivate plants and control the operating requirements in order to
reduce methane emissions of soil-botany ecosystem.
ACKNOWLEDGEMENTS
Financial support for this work is provided by Jiangsu Natural Science Foundation
(BK20140264, BK20150269), Changzhou University Funds (ZMF14020052, ZMF
14020055), National Natural Science Foundation of China (51276024, 51574046) and
Jiangsu key laboratory of oil and gas storage and transportation (SCZ1211200004/004).
15
REFERENCES
1. M. HRAD, M. PIRINGER, M. HUBER-HUMER: Determining Methane Emissions from Biogas
Plants–Operational and Meteorological Aspects. Bioresource Technol, 191, 234 (2015).
2. K. JOHN, F. JAUKER, J. MARXSEN, A. ZAITSEV, V. WOLTERS: Earthworm Bioturbation
Stabilizes Carbon in Non-flooded Paddy Soil at the Risk of Increasing Methane Emissions under
Wet Soil Conditions. Soil Biol Biochem, 91, 127 (2015).
3. J. A. VILLA, W. J. MITSCH: Methane Emissions from Five Wetland Plant Communities with
Different Hydroperiods in the Big Cypress Swamp Region of Florida Everglades. Ecohydrology &
Hydrobiology, 14 (4), 253 (2014).
4. E. J. WALETZKO, W. J. MITSCH: Methane Emissions from Wetlands: An in situ Side-by-side
Comparison of Two Static Accumulation Chamber Designs. Ecol Eng, 72, 95 (2014).
5. B. ZHANG, G. Q. CHEN: Methane Emissions in China 2007. Renew Sust Energ Rev, 30, 886
(2014).
6. M. ZHOU, B. ZHU, N. BRÜGGEMANN, X. WANG, X. ZHENG, K. BUTTERBACH-BAHL:
Nitrous Oxide and Methane Emissions from a Subtropical Rice–Rapeseed Rotation System in China:
A 3-Year Field Case Study. Agr Ecosyst Environ, 212, 297 (2015).
7. C. LENGAUER, M. R. SPEICHER, S. POPP, A. JAUCH, M. TANIWAKI, R. NAGARAJA, H.
C RIETHMAN, H. DONIS-KELLER, M. D’URSO, D. SCHLESSINGER: Chromosomal Bar
Codes Produced by Multicolor Fluorescence in situ Hybridization with Multiple Yac Clones and
Whole Chromosome Painting Probes. Hum Mol Genet, 2 (5), 505 (1993).
8. I. KIZILGOZ, E. SAKIN: Effects of Increasing Soil Application of Nitrogen and Phosphorus on
Dry Matter, Dry Weight, and Zinc and Boron Concentration in Wheat and Maize. Oxid Commun,
38 (3), 1504 (2015).
9. T. T. ONAY, M. ARSLAN, E. ATASOY: Determination of Metals, Carbon and Nitrogen Content
in the Sediments of The Black Sea. Oxid Commun, 37 (3), 853 (2014).
10. A. KAUR, H. C. BOGHANI, I. MICHIE, R. M. DINSDALE, A. J. GUWY, G. C. PREMIER:
Inhibition of Methane Production in Microbial Fuel Cells: Operating Strategies which Select Elec-
trogens over Methanogens. Bioresource Technol, 173, 75 (2014).
11. L. WU, K. MA, Y. LU: Rice Roots Select for Type I Methanotrophs in Rice Field Soil. Syst Appl
Microbiol, 32 (6), 421 (2009).
12. G. LIU, H. YU, J. MA, H. XU, Q. WU, J. YANG, Y. ZHUANG: Effects of Straw Incorporation
Along with Microbial Inoculant on Methane and Nitrous Oxide Emissions from Rice Fields. Sci
Total Environ, 518, 209 (2015).
13. C. NIU, Z. HE, Y. GE, J. CHANG, Z. LU: Effect of Plant Species Richness on Methane Fluxes
and Associated Microbial Processes in Wetland Microcosms. Ecol Eng, 84, 250 (2015).
16
Solid-phase oxidation
STUDY ON THE OXIDATION CHARACTERISTICS OF

METHANOTROPHS TO THE METHANE BEING ADSORBED
BY COAL
YANG KEa, JI HONGb,c*, XING ZHIXIANGa, LIU GECHENa, YU SIWEIa

a
213 164 Changzhou, China
b
School of Petroleum Engineering, Changzhou University, 213 164 Changzhou,
China
c
Jiangsu Key Laboratory of Oil-Gas Storage and Transportation Technology,
213 164 Changzhou, Jiangsu, China
E-mail: jihong_academic@126.com
ABSTRACT
In order to study the oxidation capacity of methanotrophs, methanotrophs bacteria
solution was used to adsorb and oxidise methane under certain conditions. The ex-
perimental results showed that the reduction of CH4 is related to the adsorption and
oxidation of methanotrophs bacteria in the coal sample. The formation of CO2 is the
result of oxidation metabolism of methanotrophs bacteria. When the methane pressure
was 6 MPa and the amount of methanotrophs bacteria solution 14.5%, the reduction
of methane in the mixed gas could reach 124.6%. It indicated that neither in aerobic
condition nor in anaerobic condition, methanotrophs bacteria solution could oxidise
methane. The adsorption and oxidation effect would increase with the amount of
methanotrophs bacteria and pressure. The existence of oxygen could promote the
adsorption and utilisation of methane.
Keywords: methanotrophs bacteria, methane, oxidation, coal.
AIMS AND BACKGROUND

Along with the development of the biotechnology, many researchers at home and
abroad carried out the use of microbial degradation of methane research, and obtained
obvious results1–6. Methane oxidising bacteria is a group of special bacteria to take
CH4 as the sole carbon and energy source of microorganisms, using a methane oxi-
dation capacity of methane oxidation bacteria for coal mine gas control research has
*
For correspondence.
17
achieved some results7,8. At present, the processing method of gas mainly is ventilation,
drainage and other physical methods. But due to the complexity of geological struc-
ture with coal that has seam permeability difference. Mine gas of coal with disaster
has not been primarily treated. Currently, the China gas control technology still has
some limitations. With the development of biotechnology, many domestic and foreign
researchers have carried out research on microbial degradation of surface methane,
and achieved remarkable results. But the present research in this aspect is less, and is
still in the initial stage. In gassy coal seam, CH4 is existing in the form of combined
with coal, and is released due to the dropped pressure in the process of mining. This
is the difficulty of coal mine gas control.
EXPERIMENTAL
Preparation. Before the experiment, the methane whose purity is 99.99% was stored
in the pressure cylinder with 13 ± 0.5 MPa. The oxygen cylinders whose purity is
99.999% was also stored in the pressure cylinder with 13 ± 0.5 MPa. FCTA2012
electronic balance which was produced in Shanghai Xinqiao Co., Ltd. was used in
the experiment. The gas sample is analysed by using gas chromatograph (Agilent,
GC7890A, U.S.A.). Raw coal sample was taken from the target No 3 coal seam from
Xinlong Coal Mine, which located in the southwest of China, and the selected coal
samples were classified and pretreated. The particles whose size is greater than 2 cm
should break to the size below 2 cm, and then the coal samples must be sieved for
the test by using 0.2 mm screen mesh.
Methods. High volumetric method was adopted for the determination of coal meth-
ane adsorption quantity. Dry coal samples handled were enclosed to adsorption tank,
with vacuum degassing and measuring the residual volume. For the pressure balance,
enough methane was inflated into the adsorption tank, one part of the methane was
adsorbed by the methanotrophic bacteria, and another part of the methane was in the
free condition. The volume of the inflated methane minus the volume of methane in
free condition leaves the adsorption volume of methane. The adsorption isotherm was
plotted after measuring the pressure and calculating the adsorption volume of methane.
Methanotrophs bacteria strains were separated and cultured from the soil sample.
the methanotrophic bacteria solution was cultured in the culture medium for 7 days
when the temperature is 30°C. The concentration of bacteria was measured by using
plate colony-counting methods and adjusted to 1×108 cfu ml–1 before the experiment.
Methane isothermal adsorption and desorption test. After weighing 50 g screened coal
sample, it was put into the adsorption tanks and seal-packed immediately. Then, the
tightness of the adsorption tanks was checked. The vacuum pump was started to extract
the adsorption tanks with mixed coal sample for 20 min. The methane was inflated
into the adsorption tanks, and the pressure was adjusted to reach the target pressure,
and then the valve was closed. Adsorption equilibrium experiment should be done at
18
30°C and the experiment time is usually 12 h. Then, the value should be recorded.
When the gas in the experimental box was released, the volume of gas released from
the tank and atmospheric pressure should be measured. The composition and content
of gas released from the tank were analysed by using the gas chromatography. The
gas adsorptive capacity should be calculated. The above-mentioned process should be
repeated to get equalised pressures and adsorptive capacity and draw the gas adsorp-
tion isotherm. 15 ml bacterial solution whose concentration is 1×108 cfu ml–1 should
be added into the coal sample in every experiment.

Adsorption property of coal to methane. The proximate analysis was carried out for
the raw coal of the target coal seam and the results are shown in Table1.
Table 1. Coal sample components industry analysis results

Density (g cm–3) Ash content (%) Volatile content (%)
1.96 30.54 10.35
Due to the coal seam gas pressure which is between 3 and 6 MPa in 600–1000 m
deep underground, the adsorption experiments in this paper were carried out in a
pressure range from 0 to 7 MPa. The adsorption fitting lines of dry coal sample and
moisture coal sample were got respectively (Figs 1 and 2). Meanwhile, the isothermal
adsorptive curves (Fig. 3) which conform with the Langmuir sorption model were
also plotted. Adsorption fitting lines from Figs 1 and 2 show that the fitting degree of
coal samples are all over 0.99, which is compliant with the request of the method of
measuring adsorbing capacity of coal sample to CH4 (High volumetric method) MT/
t757-1997, thus obtaining a qualified adsorption constant. The process of adsorption
and desorption that primary texture coal sample to CH4 is reversible. Higher pressure
resulted in increased adsorption quantity, when pressure was higher than 5.0 MPa
while the adsorption capacity did not increase. The adsorption of coal sample with
12.5 or 18.75% distilled water to CH4 conformed to the rule as well, but the adsorption
capacity significantly was lower than that of the dry coal samples. In the condition of
water injection with balance pressure higher than 5.0 MPa, the adsorption quantity on
CH4 gas increase is no longer apparent. According to the law of adsorption, inflation
pressure is determined to be 5.0 and 6.0 MPa in the subsequent adsorption experiments
with coal sample added methane oxidation bacteria. Setting pressure parameters are
6.0 and 5.0 MPa in the subsequent isothermal adsorption experiments.
Oxidation of methanotrophic bacteria to CH4 with oxygen. 9.5 and 14.5% bacteria
solution were added to the coal sample, CH4 was filled into the space, meanwhile, the
pressure becomes 5.0 and 6.0 MPa. After the system reaching equilibrium, the volume
of the emitted gas and the change of its composition were measured, the adsorption
quantity, the reduction quantity of CH4 and oxygen and the production quantity of
19
carbon dioxide were calculated. The variaiton of CH4 and CO2 during the adsorption
and desorption process with oxygen was shown in Table 2. In another experiment, we
replaced 9.5 and 14.5% bacteria solution with pure water. The isothermal adsorption
curve of the coal sample is shown in Fig. 3.
0.20
0.18 y = 0.0324x + 0.0524

0.16 R2 = 0.9994
0.14
y (Pg ml–1)
0.12
0.10
0.08
0.06
0.04
0.02
0 1 2 3 4 5 6
P (MPa)
Fig. 1. Dry coal sample adsorption curve
0.24
0.22 14.5% moisture

9.5% moisture
0.20
0.18
y (Pg ml–1)
0.16
0.14
0.12
0.10
0.08
0.06
0.5 1.0 1.5 2.0 2.5 3.0
P (MPa)
Fig. 2. Moisture coal sample adsorption curve
30
no water
9.5% moisture
14.5% moisture
adsorption (ml/g)
20
10
0 1 2 3 4 5 6 7
P (MPa)
Fig. 3. Isothermal adsorption curve of the coal sample
20
Table 2. Variation of CH4 and CO2 during the adsorption and desorption process under aerobic condition
Charge pres- Bacteria Adsorption CH4 adsorb- CO2 produc- O2 reduction CH4 reduc-
sure (MPa) solution equilibrium ing capacity tion (ml) (ml) tion (ml)
(%) pressure (ml g–1)
(MPa)
6 9.5 3.25 13.15 13.5 726 1408
5 14.5 2.85 10.23 10.4 685 1216
Table 3. Changes of CH4 and CO2 content in the adsorption process of non-additive coal samples
Charge pres- Moisture Coal com- Adsorption CH4 adsorp- CO2 produc- CH4 reduc-
sure (MPa) content bustible sub- equilibrium tion quantity tion (ml) tion (ml)
(%) stance (g) pressure (ml)
(MPa)
6 0 60 3.85 35.6 0 0
6 9.5 3.60 18.6 0 0
6 14.5 3.50 14.5 0 0
Experiments show that the concentration of CO2 and CH4 of whatever the dry
coal sample or control sample added 9.5 or 14.5% water releasing does not change
after the experiment of adsorbing in the absence of methanotrophic bacteria. On the
contrary, the concentration of CO2 and CH4 of the sample with methanotrophic bac-
teria releasing changes after the adsorption test, concentration of CO2 elevates while
that of CH4 decreases. The experimental results show that the decreasing of CH4
concentration is attributed to the absorption and oxidation of methanotrophic bacteria
in the coal sample. The formation of CO2 is the result of oxidation of methanotrophic
bacteria metabolism.
In the adsorption experiment without the sample of bacterium coal, pressure
decreases and reaches an equilibrium in a short period with an average period lasting
of 12 h. An obvious fluctuation can be observed during the equilibrium. The fluc-
tuation is due to the methanotrophic bacteria effect on the coal. The methane under
adsorption condition can be utilised by the bacteria on the surface of the coal, and it
causes the equilibrium unstable and makes the adsorption can undergo further. That
is the cause of the fluctuation of pressure. During the experiment, the proportion of
methane decreased significantly, meanwhile, the consumption of oxygen increased
and less carbon dioxide produced. The reduction of oxygen and the production of
carbon dioxide are less than the reduction of methane.
When the pressure is 5.0 MPa and the mass fraction of the methanotrophic bac-
teria solution is 12.5%, reduction of methane is 136.47% of the adsorption quantity
and the value accounts for 12.54% of total amount of methane. When the pressure
is 6.0 MPa and the mass fraction of the methanotrophic bacteria solution is 12.5%,
reduction of methane is 194.7% of the adsorption quantity and the value accounts
for 10.85% of total amount of methane. This result indicates that under high pressure
21
condition, the methane can be utilised by methanotrophic bacteria. The production
of carbon dioxide and reduction of oxygen can be explained as the effect of cellular
respiration mechanism, however, the higher amount of oxygen utilisation indicates
that methane is adsorbed and oxidised by the cell but most of them are not oxidised to
carbon dioxide. Two possible conclusions can be stated. Firstly, the energy is utilised
as the energy for cellular life history; secondly, the energy is stored as intermediate
product within the cell. Theoretically, one mol of methane requires one mol of oxygen
to be oxidised completely, in other words, their amount should be equal. If methane
is oxidised to intermediate product such as formaldehyde, the required amount of
oxygen is halved, which means that methane should be twice the amount of oxygen.
If we compare with the data provided is Fig. 3, we can make conclusion that under
5.0 MPa of pressure, the reduction of methane is 1.5 times of the oxygen; under 6.0
MPa, the reduction of methane is 1.9 times of the oxygen, which indicates that the
methane is adsorbed and exists as intermediate product within the cell most of the time.
Oxidation of methanotrophic bacteria to CH4 without oxygen. 9.5 and 14.5% bacteria
solutions were added to the coal sample, CH4 was filled into the space, meanwhile, the
pressure becomes 5.0 and 6.0 MPa. After the system reaching equilibrium, the volume
of the emitted gas and the change of its composition were measured, the adsorption
quantity, the reduction quantity of CH4 and oxygen and the production quantity of
carbon dioxide were calculated.
After the addition of 9.5 and 14.5% bacteria solution under 6.0 MPa, the amount
of methane reduction will be 73.28 and 104.87% of the maximum adsorbance under
these conditions. This result indicates that the reduction of methane is due to the
methanotrophs bacteria adsorbance and oxidised effect. The production of carbon
dioxide is due to the respiration of the bacteria. In contrast to the experiment under
adequate oxygen condition, oxygen deficient will decrease the ability of methanotrophs
bacteria of methane adsorbance and utilisation. For example, under 9.5% proportion
of bacteria solution and 6.0 MPa, the adsorbance of methane is 61% of that under
adequate oxygen condition. But the production of carbon dioxide is more than that
under adequate oxygen condition. At this time, the carbon dioxide is 0.204 times of
methane amount. The reason is that the respiration of cell is inhibited under oxygen-
deficient condition. The cell will obtain more energy using ferment effect.
The results showed that the production of carbon dioxide increased and meth-
ane was reduced significantly, while increasing the pressure of the experiment and
increasing methane adsorbance which promote the oxidation and adsorbance of
methane-oxidising bacteria. Meanwhile, although the increase of water content in
the coal sample will lead to the adsorption capacity of the coal sample decreased but
the increase of the bacteria solution will increase the adsorption capacity of the coal
sample. Theoretically, if 1 mol methane oxidised completely, it would produce 1 mol
carbon dioxide.
22
But in this experiment, the production of carbon dioxide was less than the produc-
tion of methane. Analysing the factors, two factors should be considered. Firstly, the
adsorbance system lack of oxygen; secondly, methane oxidation process in bacteria
involves multiple intermediate products, such as formaldehyde, methyl alcohol and
formic acid. The result indicates that increasing the proportion of bacteria solution and
environment pressure adequately could benefit the methane adsorbance and oxida-
tion. Besides, under oxygen deficient condition, the methane adsorbance by sample of
enzyme can also be utilised by methanotrophs bacteria as well as leading to methane
degradation. However, methanotrophs bacteria cannot oxidize methane completely,
the methane can be oxidised to intermediate produce instead.
CONCLUSIONS
Under the high pressure, with or without oxygen condition, methanotrophs bacteria
can adsorb and oxidise methane in the coal samples directly. Along with the increase
of the bacteria amount and pressure, the absorption and oxidation function of the
methanotrophs bacteria were enhanced. The consumption of oxygen increased and
less carbon dioxide produced. The existence of oxygen could promote the adsorbance
and utilisation of methane. It also indicates that methanotrophs bacteria can oxidise
the methane without oxygen. As a result, methanotrophs bacteria solution could still
preserve this ability in coal layer.
ACKNOWLEDGEMENTS
Financial support for this work was provided by Jiangsu Natural Science Foundation
(BK20140264, BK20150269), Changzhou University Funds (ZMF14020052, ZMF
14020055), National Natural Science Foundation of China (51276024, 51574046) and
Jiangsu key laboratory of oil and gas storage and transportation (SCZ1211200004/004).
REFERENCES
1. C. NIU, Z. HE , Y. GE, J. CHANG, Z. LU: Effect of Plant Species Richness on Methane Fluxes and
Associated Microbial Processes in Wetland Microcosms. Ecol Eng, 84, 250 (2015).
2. J. A. VILLA, W. J. MITSCH: Methane Emissions from Five Wetland Plant Communities with
Different Hydroperiods in the Big Cypress Swamp Region of Florida Everglades. Ecohydrology &
Hydrobiology, 14, (4), 253 (2014).
3. M. ZHOU, B. ZHU, N. BRÜGGEMANN, X. WANG, X. ZHENG, K. BUTTERBACH-BAHL:
Nitrous Oxide and Methane Emissions from a Subtropical Rice–Rapeseed Rotation System in China:
A 3-Year Field Case Study. Agr Ecosyst Environ, 212, 297 (2015).
4. K. JOHN K, F. JAUKER, J. MARXSEN, A. S. ZAITSEV, V. WOLTERS: Earthworm Bioturbation
Stabilizes Carbon in Non-flooded Paddy Soil at the Risk of Increasing Methane Emissions under
Wet Soil Conditions. Soil Biol Biochem, 91, 127 (2015).
23
5. A. KAUR, H. C. BOGHANI, I. MICHIE, R. M. DINSDALE, A. J. GUWY, G. C. PREMIER:
Inhibition of Methane Production in Microbial Fuel Cells: Operating Strategies which Select Elec-
trogens over Methanogens. Bioresource Technol, 173, 75 (2014).
6. C. GOU, Z. YANG, J. HUANG, H. WANG, H. XU, L. WANG: Effects of Temperature and Organic
Loading Rate on the Performance and Microbial Community of Anaerobic Co-digestion of Waste
Activated Sludge and Food Waste. Chemosphere, 105, 146 (2014).
7. V. A. SHARPATYI: On the Mechanism of Methane Emission by Terrestrial Plants. Oxid Commun,
30 (1), 48 (2007).
8. Y. TROTSENKO, V. KHMELENINA: Biology of Extremophilic and Extremotolerant Methano-
trophs. Arch Microbiol, 177 (2), 123 (2002).
24
Ionic oxidation
MINERALOGICAL AND IRON RECOVERY FROM

CCS FLOTATION TAILS BY PHYSICO-INTERFACIAL
SEPARATION: SEPARATION OF MAGNETITE AND IRON
SILICATES
TIANGUO LIa, JUNQING ANb, XIAOJUN XUa*, SHULI LIUa,

YANG ZHAOb
a
Faculty of Environmental Science and Technology, Kunming University of Science
and Technology, 650 500 Kunming, China
E-mail: xuxiaojun20091@126.com
b
Yunnan Solid Waste Management Centre, 210 036 Kunming, China
ABSTRACT
Copper converter slag (CCS) discharge causes significant environmental problems. In
this work, on the basis of a study of mineralogical characteristics, the recovery of iron
from CCS flotation tails using a combined process of magnetic separation and reverse
flotation was investigated. The study revealed that CCS flotation tails are primarily
composed of magnetite (21.49%) and iron silicates (72.52%). The iron content of
CCS flotation tails is as high as 49.60%, while only 30.31% of the iron distributed
in the magnetite and ferroalloy shows significant magnetic properties. Zeta-potential
studies of magnetite, quartz and fayalite samples have shown that amines are well
adsorbed, both on the surfaces of the magnetite and also on the surfaces of the iron
silicates, within a pH range of 5–10. The iron content of CCS flotation tails can be
effectively upgraded to 59.61% using magnetic separation. Amines with different
molecular structures performed different selectivity for iron silicates when reverse
flotation was used to remove iron silicates from the magnetic concentrate. The use
of mixtures of cationic and anionic collectors provided a formation of a stable hy-
drophobic adsorption layer on the surface of the iron silicates and magnetite–silicate
aggregates; this improved the flotation of these mineral particles, even in the pres-
ence of a starch, which acted as a depressant of Fe oxides. The scheme of combining
magnetic separation and reverse flotation is a promising method to use to (a) recover
iron and (b) produce iron concentrates with SiO2 content of 6.59% and Fe content of
up to 63.35% from CCS flotation tails with SiO2 content as high as 36.40%.
Keywords: CCS flotation tails, iron recovery, iron silicate removal, magnetic separa-
tion, reverse flotation.
*
For correspondence.
25
AIMS AND BACKGROUND
Copper slag is a by-product of the pyrometallurgical stages of metallic copper recovery
from copper concentrate. Production of 1t of copper results in the formation of 2.2 t
of copper slag, on average; the annual global output of copper-smelting slag is 25 Mt
(Ref. 1). CCS is a kind of copper slag generated from the converter-blowing process
of copper matte and discharged as industrial waste2. The mineralogical characteristics
of CCS depend on the natural properties of copper concentrate, the smelting condi-
tions in the conversion furnace, the cooling speed of the furnace slag, etc.2 Stockpiling
or disposing of CCS causes significant landfill and environmental problems1,3. The
effective utilisation and processing of such a large amount of copper slag has gradu-
ally become a bottleneck in the enterprise development of the circulation economy.
Recovery of metals is a primary method for the comprehensive utilisation of CCS,
because CCS contains copper, iron, nickel, cobalt and other valuable metals4,5. The
recovery and utilisation of these metals from the slag not only saves metal resources,
but also can protect the environment. The common ways of recycling metallic materi-
als are flotation1,6,7, leaching8,9 and roasting3,10.
Flotation is one of the most effective ways to recover non-ferrous metal from
CCS (Refs 11 and 12). The CCS flotation tails are a secondary waste produced after
the recycling of non-ferrous metal (such as copper, iron and nickel) from the CCS
using flotation separation. After the recovery of the non-ferrous metals, the slags may
be used in the production of construction mixtures and cements13, filling railways, etc.
However, an analysis of the chemical composition of CCS flotation tails shows that
they contain 30–50% iron. Today, mineral resources are gradually becoming scarce; the
recovery of valuable metals from industrial waste slag is a top priority. The methods
of recovering iron are roughly classified into the physical separation process and the
pyrometallurgical process14. Physical separation involves processes such as magnetic
separation, direct-reverse flotation, gravity separation and electrostatic separation;
these processes are comparatively simple, effective and environmentally friendly.
Magnetic separation in particular is a simple, inexpensive, non-destructive, rapid and
effective means of concentrating iron minerals15,16. The selection of the magnetic sepa-
ration technique depends on the assemblage characteristics of the minerals; namely,
their liberation size, corresponding magnetic susceptibility and other processing
factors17. To desilicate iron ore, reverse flotation has been shown to be an efficient
solution-both technologically and economically-to upgrade the iron concentrate, be-
cause the flotability of iron minerals (magnetite and hematite) is always lower than
that of quartz under the same physicochemical conditions18–21. The methods presently
used for the industrial reverse flotation of iron ore are anionic and cationic reverse
flotation18,21. Comparing anionic and reverse cationic flotation is difficult, because the
processes are governed by the chemical, physical and metallurgical properties of the
iron ore22–24. In the process of reverse flotation, the iron-silicates are floated while the
target iron mineral (magnetite and hematite) is depressed18. Key to the success of the
26
process is the selective depression of the iron mineral and the effective collection of
the associated silicate gangue minerals18. Generally speaking, the recovery potential of
iron from the slag using the physical separation process is determined by the texture,
mineralogy and chemical characteristics of the iron minerals19,25.
Yunnan is known in China for having extraordinary non-ferrous metal resources.
As copper production has increased, massive amounts of depleted slag have been
stockpiled in slag fields in Yunnan. There is only one copper smelting slag field in
Yunnan, China, which currently holds approximately 7 million t of CCS. The CCS
was processed using flotation separation to recover metallic copper, and it was dis-
covered that the CCS contains approximately 49.60% iron. However, the grade and
mineralogical characteristics of the iron contained in the flotation tails obtained from
Yunnan waste CCS using the flotation process, and the possible recovery of iron, have
not been widely studied or described. Therefore, the aim of this work is to investigate
the mineralogical characteristics of recovery iron, and to evaluate the economic po-
tential and technological feasibility of the recovery of iron via magnetic separation or
reverse flotation technologies. This study aims to provide a reference point regarding
the recovery of iron and the complete utilisation of CCS flotation tails, as well as to
provide direction for further research.
EXPERIMENTAL
Materials. The CCS flotation tails used in this study were derived from the copper
flotation process applied to CCS from a copper smelting plant in Yunnan, China; they
were slowly water-air-cooled. The chemical composition of the CCS flotation tails
sample is: 0.34% Cu, 49.60% Fe, 0.05% Zn, 0.095% Pb, 0.03% As, 1.87% S, 36.40%
SiO2, 5.44% CaO, 1.26% MgO and 4.85% Al2O3. Zeta-potential studies used pure
mineral. Dodecylamine (DDA), cetyltrimethyl ammonium bromide (CTAB), quater-
nary ammonium salts (DTAL), sodium butyl xanthate (SBX) and ammonium dibutyl
dithiophosphate (ADD) were used as collectors, and CaO was used as an activator
for the iron-silicate minerals. Starch was used as a depressant for the iron minerals.
No two oils (NTO) were used as a frother. All reagents (NaOH, DDA, CTAB, SBX,
ADD, CaO and starch) used in the experiment were analytical grade.
Batch experiments. The CCS sample was crushed and ground to a particle size of
(P90% ≤ 48 µm). After recovery of the copper by flotation separation, the CCS flotation
tails for testing were obtained. Through the process of dehydration and drying, the
CCS flotation tails were divided into two groupings for mineralogical characteristics
analysis and iron recovery using magnetic separation and reverse flotation, respectively.
The closed-circuit flowsheet of the magnetic separation and reverse flotation tests is
shown in Fig. 1. The wet magnetic separation tests were performed using a cydariform
wet weak magnetic separator (XCRS-74) with a magnetic field strength of 0–0.12 T.
In the magnetic separation experiments, the tails sample was continuously fed into a
magnetic field. Then, the non-magnetic product was thrown off the roll into the tailings
27
compartment while the magnetic product was gripped and deposited into the magnetic
compartment. The optimum magnetic field strength (MST) and regrinding fineness of
the crude concentrate used as the cleaner have been discussed. After magnetic separa-
tion, both magnetic and non-magnetic products were analysed. Similarly, the reverse
flotation experiments were conducted in a flotation separator (XFD) with a volume
of 1.5–3.0 l, and the possibility of upgrading the iron in the magnetic concentrates
using the reverse flotation process was investigated. The optimum collector scheme
(DDA, CTAB, DTAL, SBX, ADD) was studied. The iron grade and recovery ratio
were used to comprehensively analyse the ore dressing efficiency.
Analytical methods. The chemical composition of the copper slag was determined
by wavelength dispersive X-ray fluorescence spectrometry (XRF). The phase com-
position of the CCS flotation tails was confirmed by X-ray diffraction (XRD). The
liberation and assemblage characteristics of minerals were analysed using a mineral
liberation analyser (MLA) set to the XBSE measurement mode. The zeta-potential
of the mineral surface was measured using a Zeta potential analyser (Delsa-440SX,
Beckman Coulter).
MINERALOGICAL CHARACTERISTICS
Mineral phase and composition. Chemical analysis of the CCS flotation tails showed
that the major constituents were iron, silica, copper, zinc and the oxides of calcium,
magnesium and aluminum. The mineral phase of the tails sample is shown in Fig. 2.
The primarily mineral phases are fayalite (60.32%), garnet (12.20%) and magnetite
(21.49%); the sample also contains some copper sulphide, quartz, glass and other
materials. The X-ray diffraction pattern shown in Fig. 3 confirms that fayalite and
magnetite are crystal phases. The garnet mineral is the only significant amorphous
silicate in the chemical composition. The photomicrograph of the sample tails shown
in Fig. 4 reveals that iron and copper are finely disseminated in the iron silicate matrix
(fayalite), which makes the enrichment of iron and copper difficult, as they must be
liberated from the fayalite gangue mineral. The weight content of recycled ingredients
(copper sulphide and magnetic iron) was 21.49%. The average iron content of the CCS
flotation tails was 49.60%. The mineral forms of elemental iron present in the samples
are given in Table 1. The percentage of iron occurring in the magnetite and ferroalloy
that showed excellent magnetic properties is only 30.31%. There were several other
forms of iron distributed in the iron-silicates. This suggests that recovery of iron by
magnetic separation has a certain potential, and that the main impure minerals are
iron-silicates (fayalite, garnet).
28
Table 1. Forms of the mineral occurrence of iron in CCS flotation tails
Phases Mag- Fer- Hema- Copper Fayalite Garnet Glass Others Total
netite roalloy tite sulphide
Distribution of 27.49 2.82 1.34 1.78 58.34 7.72 0.45 0.04 100.00
Fe (%)
Fe grade (%) 71.41 100.00 42.40 14.24 42.10 26.28 26.20 – 49.60
b Magnetic concentrates
a
CCS flotation tailings
Grinding :P 90% ≤ 48um DDA, CTAB , SBX, ADD

Grinding :P 90% ≤ 48um NaOH : 750, CaO : 30
MFS starch : 150, NTO : 15
Rough er
DDA, CTAB , SBX, ADD
Grinding :P ?% ≤ 38um Rougher
Starch : 50, NTO : 5
MFS
Starch : 100 Cleaner

MFS Cleaner 1
Scavenger
Cleaner 2
Concentrate Tailings Tailings Concentrate
Fig. 1. Closed-circuit flowsheet of magnetic separation and reverse flotation experiments

a – magnetic separation test, unit of MFS: T; b – reverse flotation test, unit of reagents: g/t
Quartz and glass (1.77%)

Others (0.13%) Copper sulphide（2.71% ）
Garnet
(12.20%) H em atite （1.36% ）
Target mineral
Fayalite (25.56 %)
(60.32%)
Magnetite and ferroalloy
(21.49%)
Fig. 2. Primary mineral constituents of CCS flotation tails
29
Fayalite (2FeO .SiO 2)
Magnetite （Fe 3O 4）
intensity (CPS )
2θ(º)
Fig. 3. X-ray diffraction pattern of CCS flotation tails
fayalite
magnetite
hematite
copper sulphide
garnet
100 mm ferroalloy
Fig. 4. Photomicrographs of CCS flotation tails
Size and liberation analysis. The size distribution of both the sample ore and the
magnetite was analysed. Compared with the sample ore, the size distribution of the
magnetite was finer (Table 2). The liberation degree of magnetite and ferroalloy are
given in Table 3. There was 57.52% of magnetite and ferroalloy with a liberation
degree above 80%. The size distribution and liberation studies of the sieve fractions
indicated that the magnetite grains may have an optimum liberation of particles in the
size range of 15.0–25.0 μm. The lower degree of liberation demonstrates a relatively
smaller average diameter of mineral particle size. This suggests that the parts of the
iron minerals with an elaborate embedded characteristic are difficult to liberate. Even
though the size distribution of minerals containing magnetite and ferroalloy without
liberation is already fine, increasing the grinding fineness seems to be the only effective
method to improve the liberation degree. The specific gravity increased slightly with
the increasing of the liberation degree, indicating that gravity separation is unlikely
to achieve the enrichment of the magnetite.
30
Table 2. Size distribution of tails sample and magnetic iron minerals (magnetite and ferroalloy)
Index Eigenvalue of size P (µm) Size distribution (%)
P10 P20 P50 P80 P90 +19 µm +9.6 to –9.6 µm
–19 µm
Sample ore 10.83 16.00 34.06 60.04 74.28 74.43 20.81 4.76
Magnetic iron 8.70 12.47 25.67 45.69 57.92 63.76 22.78 13.46
ore
Table 3. Mineral liberation degree of magnetite and ferroalloy

Liberation degree Average diameter Specific gravity Distribution Cumulative
(%) (μm) (t/m3) (%) distribution (%)
0–20 8.27 4.31 4.30 100.00
20–40 14.35 4.47 8.12 95.70
40–60 19.85 4.60 12.44 87.58
60–80 24.27 4.78 17.62 75.14
80–100 25.13 5.02 20.40 57.52
100 16.01 5.11 37.12 37.12
Embedded characteristics. The mineral interlocking and inclusion relationship of

magnetite and ferroalloy of the CCS flotation tails is given in Table 4. Under the
grinding fineness of a particle size of (P90% ≤ 48 µm), 42.48% of the magnetite and
ferroalloy were not liberated and instead remained interlocked with the fayalite and
garnet gangue minerals (Fig. 5e). The inclusion relationships of magnetite and fer-
roalloy were binary (Fig. 5a, sign 6) and multiple (Fig. 5a, sign 5), and the inclusions
of the binary were the dominant form. This can be clearly seen in Fig. 5. Microscopic
studies reveal that the tails samples mostly contained iron mineral phases like mag-
netite, hematite, ferroalloy and other gangue minerals (Fig. 5). Most of the minerals
occurring in coarse particles (particles with a diameter larger than 35 µm) were liber-
ated, except for the fayalite (Fig. 5a, sign 4). In this size range, magnetite remained
encapsulated in the gangue minerals of the fayalite (Fig. 5a, sign 1) and garnet (Fig.
5a, sign 2). Most of the magnetite and ferroalloy was interlocked with fayalite, cop-
per sulphide and garnet (Fig. 5a). In the range of medium size particles, some of the
magnetite was liberated to the mineral monomer (Fig. 5b, sign 3), and most of the
minerals existed in the form of mineral interlocking. There were almost no inclusions
in the fine mineral particles (Fig. 5c).
31
a 2 5 b 2 c 1 2
1
3 1
6
200 um 200 um 100 um
d e f
100 um 100 um
Fig. 5. Photomicrographs of the embedded characteristics of CCS flotation tails under the condition of
a grinding fineness of P90% ≤ 48 µm (equivalent diameter): a – coarse particles: de ≥ 35 µm, b – medium
size particles: 35 µm > de ≥ 10 µm, c – fine particles: de < 10 µm, d – mineral monomer of magnetite and
ferroalloy, e – mineral interlocking of magnetite and ferroalloy, f – legend
Table 4. Mineral interlocking and inclusion relationship of magnetite and ferroalloy of CCS flotation
failings
Mineral phase Magnetite and ferroalloy
interlocking binary multiple
Copper sulphide 8.75 4.42 1.79
Antimonide 0.03 0.00 0.00
Hematite 3.09 1.32 0.81
Fayalite 61.82 53.02 11.07
Garnet 20.59 14.76 7.51
Quartz and glass 5.59 3.05 2.18
Oxides 0.03 0.05 0.00
Others 0.09 0.02 0.00
Total 100.00 76.64 23.36
Magnetic separation studies. In order to achieve the desired grade of concentrate

with appreciable weight recovery, beneficiation studies were undertaken using wet
magnetic separation techniques. The effects of the magnetic field strength on the
iron grade and recovery of roughing using magnetic separation are shown in Fig. 6.
Figure 6 shows that the recovery of iron increased as the current of the magnetic
field strength increased. For the roughing, the recommended magnetic field strength
is 0.10 T, and the grade and recovery ratio of iron in the rough concentrates obtained
from the magnetic roughing were 53.13 and 55.10%, respectively. As the magnetic
field strength increased, the recovery of iron increased at the expense of reducing the
iron grade. The low percentage of the iron recovery and grade might have been due
32
to the low distribution of magnetic iron in the CCS flotation tails, and the fact that
liberation was less effective at the grinding fineness of (P90% ≤ 48 µm). Similarly, the
study suggested that the optimum magnetic field strength for the cleaner 1 and cleaner
2 stages of magnetic separation (Fig. 1a) were 0.06 and 0.03 T, respectively.
60
57
54
percentage (%)
51
grade
recovery
48
45
42
0.03 0.06 0.09 0.12

magnetic field strength (T)
Fig. 6. Effect of magnetic field strength on iron grade and recovery of roughing (open-circuit test)
Table 5 shows the effects of regrinding fineness of the roughing concentrates on

the iron grade, recovery ratio and yield of the iron concentrates obtained. We found
that regrinding the rough concentrates was beneficial to improving the iron grade and
ensuring the weight recovery. The optimum magnetic separation route was single-
stage roughing and double-stage cleaner. The grade and recovery of iron concentrate
enriched in this work were 59.58 and 38.31%, respectively, at a roughing concentrate
regrinding fineness of (P95% ≤ 38 μm)). These results indicate that the recovery of iron
was relatively poor. From the characteristics of the mineralogy of the CCS flotation
tails, it can be concluded that this may have been due to the limited distribution of
magnetic iron (magnetic and ferroalloy) in the CCS flotation tails. Also, the recovery
and grade of the iron may be significantly improved by further optimising the param-
eters of the magnetic separation. It seems that to improve the potential distribution of
magnetic iron is one effective way to recover iron from CCS flotation tails and further
studies focus on this. For example, regulating the cooling crystallisation process of
copper converter slag should be examined, and other effective methods of converting
iron silicates (fayalite, garnet) to magnetic iron should be sought.
33
Table 5. Effect of the regrinding fineness of roughing concentrates on iron recovery (open-circuit test)
Regrinding fineness Detail Yield Fe grade Fe recovery
(%) (%) (%)
feed (roughing concentrate) 100.00 53.13 100.00
P90%≤48 µm magnetic fraction 44.10 56.67 47.04
middling 4.80 50.47 4.56
nonmagnetic fraction 51.10 50.32 48.40
middling 9.60 54.46 9.84
middling 11.00 57.04 11.81
P95% ≤ 38 µm magnetic fraction 34.16 59.58 38.31
middling 13.60 51.65 13.22
REVERSE FLOTATION OF IRON SILICATES REMOVAL
Zeta-potential studies. Figure 7 shows the zeta-potential values of magnetite, quartz

and fayalite as a function of pH with or without additions of amine acetate. As can be
seen, the isoelectric point of the magnetite sample occurs at pH 5.5, and that of the
fayalite sample occurs at pH 4. The point of zero charge occurs within pH values that
are less than 2.0, depending on the mineral crystalline structure. The zeta-potential and
pH of the isoelectric point increased after the introduction of amine (dodecylamine) at
a 10–4 mol/l concentration in the mineral/water system; this may be due to the fact that
the cationic species adsorbed into the double electric layer on the negatively-charged
surface. The character of the zeta-potential curves for all minerals after the addition
of dodecylamine clearly indicates that the adsorption of the cationic species occurred
only on negatively-charged surfaces. This is accompanied by a zeta-potential increase
along with an increase of surface electronegativity. Electrokinetic research has shown
that amines are adsorbed equally well on the surface of both silicates (quartz and
fayalite) and magnetite in a pH range of 5.0–10.0. This indicates that both silicates
(quartz and fayalite) and magnetite have a strong surface hydrophobic effect due to
collector (amines) adsorption. Therefore, the effective removal of the silicate complex
from the magnetic concentrates requires the application of a depressant that prevents
the magnetite from adsorbing the collector.
34
60
quartz
40 quartz
fayalite
fayalite
magnetite
20
zeta-potential (mV)
magnetite
–20
–40
–60
2 4 6 8 10 12
pH
Fig. 7. Zeta-potential of magnetite, quartz and fayalite as a function of pH in the absence (dotted line)
and presence (solid line) of 10–4 mol/l dodecylamine acetate
Effects of starch on iron silicates removal. Research on magnetite reverse flotation

in the presence of a starch indicates an almost complete depression of iron-oxides
and demonstrates the impossibility of their flotation by amines21,23. Filippov et al.23,24
reported that starch can depress iron silicates and lead to flocculation of fine particles
and a consequential decrease in the flotation of quartz and iron silicates by amines.
Therefore, starch dosage is one of the factors that determine the flotation results.
Table 6 shows the effect of a starch dosage on the reverse flotation of the magnetic
concentrate with dodecylamine. The use of starch can decrease the Fe losses in the
tails, but desilicates also are subdued along with an increase in the dosage of starch.
The scheme without the addition of starch (test 1) provides a more effective elimina-
tion of the silicates as compared to tests 2–5, although Fe losses in the tails increased
by 3–12%. This may be due to the fact that SiO2 in the magnetic concentrates was
distributed in the iron silicates instead of in the quartz; it also may be due to the exist-
ence of undissociated aggregates of Fe oxides in the silicates. The higher starch dosage
causes not only the depression of Fe oxides, but also the depression of iron silicates
and mineral aggregates. The appropriate dosage of starch can provide a subtle adjust-
ment of the hydrophilisation on the surface of the Fe oxides, and allows the creation
of a less dense adsorption layer of the depressant on the surface of the iron silicates
and aggregates (Fe oxides and iron silicates). To cause more efficient de-silication, a
starch dosage of 100 g/t (test 2) was chosen for our studies.
35
Table 6. Results of flotation of the magnetic concentrate with dodecylamine as a function of starch dosage
No Production Yield Grade (%) Recovery (%) Conditions
(%) Fe SiO2 Fe SiO2
1 froth 20.58 53.06 41.53 18.32 55.25 starch: 0 g/t,
concentrate 79.42 61.31 8.72 81.68 44.75 DDA: 300 g/t,
total 100.00 59.61 15.47 100.00 100.00 pH=10, Ca2+: 100 g/t,
NTO: 20 g/t
2 froth 17.74 51.01 45.82 15.17 52.64 starch: 100 g/t,
concentrate 82.26 61.51 8.89 84.83 47.36 DDA: 300 g/t
total 100.00 59.65 15.44 100.00 100.00 pH=10, Ca2+: 100 g/t,
NTO: 20 g/t
3 froth 14.61 50.57 52.14 12.4 49.43 starch: 300 g/t, DDA:300 g/t
concentrate 85.39 61.12 9.13 87.6 50.57 pH=10, Ca2+: 100 g/t,
Total 100.00 59.58 15.41 100.00 100.00 NTO: 20 g/t
4 froth 10.42 49.98 68.10 8.73 46.14 starch: 500 g/t, DDA:300 g/t
total 100.00 59.66 15.38 100.00 100.00 NTO: 20 g/t
5 froth 7.83 44.54 85.86 5.85 43.57 starch: 1000 g/t,
concentrate 92.17 60.90 9.45 94.15 56.43 DDA: 300 g/t
total 100.00 59.62 15.43 100.00 100.00 pH=10, Ca2+: 100 g/t,
NTO: 20 g/t
Effects of collector scheme on removal iron silicates. Silica content requires strict
regulation in the process of making steel from metallised pellets, as well as in the
process of electric-arc smelting23,24. The silica content in the magnetic concentrates
studied in this work was as high as 15.40%. Reverse cationic flotation successfully
removes silicates such as quartz, amphiboles, mica and feldspars from iron oxides
(magnetite, hematite)26. Various amines (DDA, CTAB, DTAL) are used as cationic
collectors during the reverse flotation of iron ores and show a high selectivity for
silicates. The mineralogical properties of the CCS flotation tails show mineral impu-
rities (fayalite and garnet) and a small amount quartz. However, little research has
been conducted on the removal of fayalite and garnet using reverse flotation. Hence,
in this study, we focused discussing different collector schemes that can be used with
the reverse flotation process to effectively separate fayalite and garnet from the mag-
netic concentrates. The effects of the different collector schemes on silica recovery
are shown in Fig. 8. As Fig. 8 shows, the recovery of silica from the froth products
of reverse flotation increased as the dosage of collectors of different molecular struc-
tures increased. At a dosage of approximately 300 g/t, silica recovery had a result of
approximately 51.60, 54.05 and 61.12%, respectively, with the use of DDA, CTAB
and DTAL, respectively. DTAL showed a higher silica recovery than CTAB and
DDA for the reverse flotation tests. Silica removal from the magnetic concentrates
during reverse flotation with cationic collectors is determined by the effectiveness
of the collectors to: (a) partially destroy the hydrated starch layer on the iron silicate
36
surface and (b) shift the hydrophilic–hydrophobic balance toward hydrophobisation
of the surface. This suggests that DTAL has better selectivity and could be a suitable
cationic collector for the flotation of iron silicate minerals such as fayalite and garnet.
60
50
recovery of SiO2 (%)
40
30
DDA
20
CTAB
DTAL
10
0
50 100 150 200 250 300 350 400 450
dosage of collectors (g/t)

Fig. 8. Silica recovery of froth products affected by the type of amines collector and their dosage
The use of mixtures containing cationic and anionic collectors was suggested to
improve the metallurgical results for the cationic flotation of iron-bearing silicates27.
Hence, the removal of iron silicates by reverse flotation with a mixture of collectors
of different molecular structures has been investigated, and the results are presented in
Table 7. The mixed use of anionic collectors (SBX and ADD) with cationic collectors
(DTAL) can improve the silica recovery from the froth products. The formulations
of mixed SBX and ADD with DTAL (tests 2–3) allowed for more effective removal
of poorly floated silicates and mineral aggregates, as compared to the use of DTAL
alone (test 1), and the SiO2 obtained decreased by 3.11–6.62%. Comparatively speak-
ing, in terms of strengthening the removal of silica, ADD performed more effectively
than SBX when mixed with DTAL. Iron concentrates with 6.59% SiO2 and 63.35%
Fe were produced, with Fe recovery of 76.44% from the magnetic concentrates (test
4), when the collector mixture contained both SBX and ADD with DTAL. These
phenomena: (a) contributed to the adsorption effect of the molecular structures of the
mixture of cationic and anionic collectors (SBX + DTAL, ADD + DTAL or SBX +
ADD + DTAL), (b) provided a more stable adsorption layer of collectors on the het-
erogeneous surface of the fayalite, garnet and magnetite–silicate aggregates, and (c)
enhanced the performance of adsorbed starch by means of varying surface energy. The
co-absorption of cationic and anionic molecules results in the increasing floatability of
these mineral particles, and leads to the formation of a denser adsorption layer due to
decreased electrostatic repulsion. However, the Fe loss in the tails was approximately
5% with a 5% increase in the silica removal rate, and the Fe grade in the improved
concentrate that was obtained did not change significantly. This is potentially due to
37
the fact that the iron silicates contain a certain amount of Fe, and that the magnetite
and iron silicates were incompletely liberated; the presence of other gangue minerals
was confirmed by the liberation and embedded analysis data of the magnetite. On the
other hand, the use of collectors and starch provides a stable adsorption layer on the
surface of the iron silicates that helped to aggregate the magnetite and iron silicates.
The flotation of the silica also caused a certain amount of loss of magnetite in ag-
gregate. However, it can be concluded that a scheme of mixed cationic and anionic
collectors in a reverse flotation process is a promising technology for the removal of
iron silicates from the magnetic concentrates of CCS.
Table 7. Results of reverse flotation of the magnetic concentrates with a collector mixture
No Production Yield Grade (%) Recovery (%) Conditions
(%) Fe SiO2 Fe SiO2
1 froth 22.89 47.99 41.12 18.43 61.12 DTAL: 300 g/t, starch: 100 g/t,
total 100.00 59.60 15.40 100.00 100.00 NTO: 20 g/t
2 froth 24.76 46.95 40.68 19.51 65.23 DTAL: 250 g/t, SBX: 50 g/t
concentrate 75.24 63.75 7.14 80.49 34.77 starch: 100 g/t , pH=10,
total 100.00 59.59 15.44 100.00 100.00 Ca2+: 100 g/t, NTO: 20 g/t
3 froth 27.64 49.86 37.82 23.12 67.74 DTAL: 250 g/t, ADD: 50 g/t
concentrate 72.36 63.33 6.88 76.88 32.26 starch: 100 g/t , pH=10,
total 100.00 59.61 15.43 100.00 100.00 Ca2+: 100 g/t, NTO: 20 g/t
4 froth 28.02 50.16 38.07 23.56 69.23 DTAL: 200 g/t, ADD: 50 g/t
concentrate 71.98 63.35 6.59 76.44 30.77 SBX: 50 g/t; starch: 100 g/t,
total 100.00 59.65 15.41 100.00 100.00 pH = 10, Ca2+: 100 g/t,
NTO: 20 g/t
CONCLUSIONS
The iron mineral phases in CCS flotation tails are magnetite, ferroalloy and hema-
tite, and the main impure minerals are fayalite, garnet, copper sulphide and quartz.
The average iron content of CCS flotation tails can be as high as 49.60%, but only
30.31% of the iron distributed in magnetite and ferroalloy show significant magnetic
properties. Mineral liberation and embedded characteristics indicate that magnetite
is finely disseminated in the fayalite, and that magnetite grains can have an optimum
liberation of at a particle size in the range of 15.0–25.0 μm. The degree of liberation
has been defined as one of the factors that determines the difficulties encountered in
the re-treatment of CCS flotation tails using magnetic and flotation separation pro-
cesses. The liberation degree of magnetite is relatively low, and magnetite mainly
interlocks with, or is included in, fayalite at a grinding fineness of (P90% ≤ 48 µm).
Zeta-potential research of magnetite, quartz and fayalite samples has demonstrated
that there is little difference between iron silicates and magnetite with respect to the
38
adsorption degree of amines on the surface in the alkaline pH range. DTAL has bet-
ter selectivity than DDA or CTAB for the reverse flotation of iron-silicate (fayalite,
garnet). Using formulations with amines (DDA, CTAB and DTAL) and a mixture of
cationic and anionic collectors likely forms a more stable adsorption layer of collec-
tors on the heterogeneous surfaces of iron silicates, and helps to aggregate magnetite
and iron silicates with adsorbed starch (due to co-adsorption). Thus, it improves the
flotation of these mineral particles. Magnetic separation can effectively upgrade the
iron content in the CCS flotation tails, and a magnetic concentrate containing 59.61%
iron with a total iron recovery rate of 37.31% was obtained. The poor recovery of iron
by magnetic separation may be due to the relatively limited distribution and liberation
of magnetic iron in the CCS flotation tails. Iron concentrates with 6.59% SiO2 and Fe
content of 63.35% were produced, with Fe recovery of 76.44% from the magnetic
concentrates. Thus, the combination of magnetic separation and reverse flotation is a
promising method for the recovery of iron and the reduction of smelting slag discharge;
it must be further confirmed using industrial scale studies.
ACKNOWLEDGEMENTS
This study was supported by the Scientific Research Project of the Yunnan Solid
Waste Management Centre (20140153), and also by the Analysis and Measurement
Foundation of Kunming University of Science and Technology (20140550, 20150460).
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Ferric Sulphate. Hydrometallurgy, 44, 261 (1997).
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ylammonium Chloride as a Collector. Int J Min Sci Technol, 22 (2), 285 (2012).
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Iron Ore Sample. Int J Min Sci Technol, 23, 1 (2013).
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Received 8 June 2015
Revised 7 August 2105
40
Ionic oxidation
PERIODATE OXIDATION OF
AQUA-N-(2-ACETAMIDO)IMINODIACETATO-COBALT(II)
COMPLEX INVOLVING MALEIC ACID CO-LIGANDS
H. A. EWAISa, M. A. NAGDYb, S. A. HAMEEDa*

a
Chemistry Department, Faculty of Science, King Abdulaziz University,
P.O. Box 80 203, 21 589 Jeddah, Saudi Arabia
E-mail: drshameed@hotmail.com
b
Chemistry Department, Faculty of Science, Qassim University, Saudi Arabia
ABSTRACT
The kinetics of oxidation of the ternary complexes [CoII(ADA)(L)(H2O)]2– (ADA =
N-(2-acetamido)iminodiacetate, L = maleic acid) by periodate has been investigated
spectrophotometrically in aqueous medium over 30–50ºC range, pH 3.72–4.99 and
I = 0.2 mol dm–3. The reaction was carried out under pseudo-first order conditions by
using a tenfold excess of [IO4–] over [CoII(ADA)(L)(H2O)2–]. The kinetics of oxidation
of [CoII(ADA)(L)(H2O)]2– obeyed the rate law:
d[CoIII]/dt = [CoII(ADA)(L)(H2O)2–] [H5IO6] {k4K5 + (k5K6K2/[H+])}.
The system was studied at different concentrations of acetate ion over a tempera-
tures range (25–40)oC and the kinetics of oxidation of [CoII(ADA)(L)(H2O)]2– obeyed
the rate law:
d[CoIII]/dt ={k8K8 + k9K7K9[CH3CO2–]} [CoII(ADA)(L)(H2O)2–] [IO4–].
The initial Co(III) products were slowly converted to the final products, fitting
an inner-sphere mechanism. Thermodynamic activation parameters were calculated
using the transition state theory equation.
Keywords: periodate oxidation, cobalt(II) complex, kinetics, inner-sphere mechanism.
AIMS AND BACKGROUND

Periodate oxidations have been reported to play an important role in biological de-
terminants, they are used to degrade carbohydrate determinants in proteins without
altering protein or lipid epitopes1. Oxidation by periodate exerts a number of biological
effects including the enhancement of lymphocyte activation and increased frequency
*
For correspondence.
41
of effecter to target cell binding2. Periodate is a stronger oxidant in acid medium than
alkaline medium. The reduction potential of the IO4–/IO3– couple is +1.6 and +0.70 V
in acid and alkaline media, respectively3. The action value of periodate as an oxidis-
ing agent under suitable conditions of pH and temperature is largely restricted to the
oxidation of certain types of compounds4.
Oxidations of inorganic substrates5 and transition metal complexes6–9 by pe-
riodate are reported to proceed through an inner-sphere mechanism either labile or
inert complexes possessing at least one bridging ligand. Oxidations by periodate
are catalysed with trace amounts of metal ions6,10. Iron(II) catalyses oxidation of
chromium(III)–DL–aspartic acid complex by periodate in acetate buffer, due to the
formation of iron(III) which acts as the oxidising agent10. Catalysis by Cu(II) ions
in the oxidation of Fe(CN)64– by periodate in acid medium is well established and
is seen to result from the oxidation of Cu(II) to Cu(III) which acts as an oxidising
agent6. Periodate oxidation of ternary chromium(III) complexes involving guanosine
and aspartic acid was studied11. In all cases the electron transfer proceeds through an
inner-sphere mechanism via coordination of IO4– to chromium(III).
The kinetics of oxidation of cobalt(II) complexes of trans-1,2-diaminocyclohex-
anetetraacetate (CDTA) (Ref. 12), propylenediaminetetraacetate (PDTA) (Ref. 13),
1,3-diamino-2-hydroxypropanetetraacetate (HPDTA) (Ref. 13), trimethylenediamine-
tetraacetate (TMDTA) (Ref. 14) and ethyleneglycol,bis(2-aminoethyl)ether,N,N,N',N'-
tetraacetate (EGTA) (Ref. 14) by periodate in aqueous medium have been investigated.
In all cases, the electron transfer takes place through an inner-sphere mechanism and
gave only the final product13,14.
Oxidation of a ternary complexes, [CoII(nta)(M)(H2O)2] (nta = nitrilotriacetate
and M = maleate) and [CoII(nta)(T)(H2O)] (T = tartarate) (Refs 3–16) by periodate
were studied. The initial cobalt(III) products were formed and changed slowly to final
cobalt(III) products. It is proposed that the reaction obeys an inner-sphere mechanism
which suggested that the relatively faster rates of ring closure were compared to the
oxidation step15,17.
In this work, the kinetics of oxidation of [CoII(ADA)(L)(H2O)]2– (ADA = N-(2-
acetamido)iminodiacetate and L = maleic acid) by periodate have been investigated,
in order to study the effect of maleic acids as secondary ligands on the stability of
[CoII(ADA)(H2O)3] (Ref. 7) towards oxidation and also to deduce the reaction path-
ways.
EXIPERIMENTAL
Materials and solutions. All chemicals were of reagent grade (Analar, BDH, Sigma).
Buffer solutions were prepared from acetic acid and sodium acetate of known con-
centrations. Sodium nitrate was used to adjust ionic strength in the different buffer
solutions. A stock solution of sodium periodate (Aldrich) was prepared by accurate
weighing and wrapped in aluminum foil to avoid photochemical decomposition4.
42
Na2[CoII(ADA)(L)(H2O)] was synthesised using the same methods as for
the preparation of [CoII-nta] (Ref. 18). The elemental analysis data of the com-
plex Na2[CoII(ADA)(L)(H2O)] are: Found: C, (27.6); H, (2.3); N, (6.2), [Na2CoII
C10H11O10N2]. Calcd: C, (28.3); H, (2.6); N, (6.6) %). To confirm the formula of the
complex, IR spectra and TGA data were recorded. In the IR spectrum, bands in the
3158–3404 cm–1 region, were attributed to ν(OH–) of the coordinated water molecule.
The OH– band disappeared and a new COO– band appeared in the 1647–1422 cm–1
region. The thermal analysis of [Na2CoIIC10H11O10N2] shows a weight loss (31.5%)
beginning at 286oC corresponding to the loss of one water ligand plus two molecules
of CO2, and one molecule of CO (calcd. 32%).
Kinetic procedures. The reaction rates were measured by monitoring the initial Co(III)
complex absorbance at 565 nm on a Milton-Roy SP 601 spectrophotometer. Dur-
ing the oxidation, the pink aqueous solution of the complexes gradually changed to
blue. The pH of the reaction mixture was measured using a G-C825 pH meter. The
temperature of the cell compartment was maintained at the required temperature by
a circulatory water arrangement from a thermostat (Gallen Kamp Griffen, BGL 240
V). Pseudo-first order conditions were maintained in all runs by the presence of a
large excess of IO4– (> 10-fold). The ionic strength was kept constant by addition of
NaNO3 solution. The pH of the reaction mixture was found to be always constant
during the reaction run. Pseudo-first order rate constants, kobs, were obtained from
the slopes of plots of ln (A∞ – At) versus time, where At and A∞ are the absorbencies
at time t and infinity, respectively.
Oxidation products. The UV-vis. absorption spectra of complexes and the oxidation
products of complex (Fig. 1) were recorded on a JASCO UV-530 spectrophotometer
as a function of time over the 325–725 nm range as shown in Fig. 1. The maxima and
molar absorption coefficients of final cobalt(III) product at pH = 4.99 were emax573 =
127 mol–1 cm–1 dm3.
43
0.16
8
7
9
6
0.12
5
3
absorbance
0.08
1
0.04
0
350 450 550 650 750
wavelength (nm)
Fig. 1. Variation of absorbance as a function of time

Curves 1–8 were recorded at 5, 15, 25, 40, 60, 100, 150 and 180 min, respectively, from the time of
initiation of the reaction. Curve 9 represents the final products

Effect of pH on the oxidation of [CoII(ADA)(L)(H2O)]2– by periodate. The oxidation
rate of [CoII(ADA)(L)(H2O)2–] by periodate was studied over pH = 3.72–4.99, ionic
strength range 0.20–0.60 mol dm–3 at 30–50oC using various periodate and complex
concentrations. Plots of ln (A∞ – At) versus time were linear up to > 85% of the reac-
tion, where At and A∞ are the absorbances at time t, and infinity, respectively. Pseudo-
first order rate constants, kobs, obtained from the slopes of these plots are collected in
Table 1. The constant value of kobs over the complex concentration range (1.0–3.0) ×
10–3 mol dm–3 at constant periodate concentration indicates first order dependence on
the complex concentration, as shown in Table 1 and equation (1).
d[CoIII]/dt = kobs [CoII(ADA)(L)(H2O)2–] (1)
Plots of kobs against [IO4–] were found to be linear without intercept (Fig. 2). The
dependence of kobs on [IO4–] is represented by equation (2):
kobs = k1[IO4–]. (2)
44
Table 1. Dependence of the reaction rate constant, kobs, on [CoII(ADA)(L)(H2O)]2– a, [IO4–], Ib and tem-
peratures at pH = 4.99
[IO4–] × 102 kobs× 104 (s–1)
(mol dm–3) 30oC 35oC 40oC 50 oC
0.5 0.65±0.01 0.83±0.01 1.16±0.02 1.60±0.01
1.0 1.13±0.01 1.67±0.02 2.83±0.02 3.72±0.03
2.0 2.33±0.02 3.50±0.03 4.50±0.04 5.67±0.05
3.0 3.38±0.06 5.33±0.04 7.83±0.06 9.12±0.10
4.0 4.60±0.06 7.00±0.05 10.00±0.08 12.37±0.12
5.0 5.93±0.05 8.50±0.07 12.33±0.10 16.08±0.15
a
[CoII(ADA)(L)(H2O)2–] = 1.0 × 10–3 mol dm–3; Ib = 0.20 mol dm–3; kobs ×104= 4.63 ± 0.05, 4.43 ± 0.04,
4.66 ± 0.06 and 4.57 ± 0.04 s–1 at 10–3 [CoII(ADA)(L)(H2O)2–] = 0.50, 1.50, 2.0 and 2.50 mol dm–3, re-
spectively at 40oC, and [IO4–] = 0.02 mol dm–3; at I = 0.3, 0.4, 0.5 and 0.6 mol dm–3, 40ºC, [CoII(ADA)
(L)(H2O)2–] and [IO4–] = 0.02 mol dm–3, kobs × 104 = 4.73 ± 0.03, 5.17 ± 0.05, 5.67 ± 0.06 and 6.16 ± 0.10
s–1, respectively.
16 50oC
40oC
12
kobs × 104 (s–1)
35oC
8
30oC
0
0 1 2 3 4 5 6
[IO4– ] × 102 (mol dm–3)
Fig. 2. Plots of kobs versus [IO4–] at different temperatures
The kinetics of the reaction was studied over a pH range of 3.72–4.99 at differ-
ent temperatures. The variation of k1 with [H+] at different temperatures is listed in
Table 2 and shows that the rate increased gradually with increasing pH. Plots of the
slope k1 versus 1/[H+] were linear, with intercepts as shown in Fig. 3. This behaviour
can be described by equation (3):
k1 = k2 + k3/[H+]. (3)
45
Table 2. Variation of k1 with pH
[CoII(ADA)(L)(H2O)]2– = 1.0 ×10–3 mol dm3, I = 0.20 mol dm–3, different [IO4–] and different temperatures
pH k1 ×102 (mol–1 dm3 s–1)
30ºC 35ºC 40ºC 50ºC
3.72 0.35±0.02 0.44±0.01 0.65±0.03 0.87±0.04
4.05 0.44±0.05 0.62±0.03 0.85±0.06 1.09±0.03
4.63 0.74±0.04 0.90±0.05 1.28±0.04 1.76±0.07
4.99 1.13±0.06 1.73±0.07 2.48±0.08 2.95±0.10
3 50 oC
40 oC
2
k1 × 102 (mol–1 dm3 s–1)
35 oC
30 oC
0
0 2 4 6 8 10 12
[H+]–1 × 10–4 (mol–1 dm3)
Fig. 3. Variation of k1 with 1/[H+] at different temperatures
Table 3 shows the variation of k2 and k3 with temperature. The enthalpy and
entropy of activation associated with k2 and k3 were calculated using the transition
state theory equation. The enthalpies of activation, ΔH2* and ΔH3* were calculated
as 35.6 ± 1.5 and 36.5 ± 1.2 kJ mol–1, respectively. The corresponding entropies of
activation, ΔS2* and ΔS3* were –174.4 ± 3.6 and –136 ± 4.2 J K–1 mol–1, respectively.
Table 3. Variation of k2 and k3 with temperature

T (οC) 103/T (K–1) k2 × 103 (mol–1 dm3 s–1) k3 × 107 (s–1)
30 3.30 3.40 ±0.03 0.81 ±0.02
35 3.25 4.00 ±0.05 1.31 ±0.04
40 3.19 5.64 ±0.08 1.88 ±0.08
50 3.10 8.06 ±0.12 2.15 ±0.07
The reaction rate increased with increasing in ionic strength over a 0.20–0.60
mol dm–3 range (Table 1). From equations (1), (2) and (3) the experimental rate law
was derived as follows:
d[CoIII]/dt = (k2 + k3/[H+]) [CoII(ADA)(L)(H2O)2–]0[IO4–] (4)
46
In the ternary complex, [CoII(ADA)(L)(H2O)]2–, ADA is tridentate through two
carboxylate groups and one imino group, while maleic acid is bidentate through two
carboxylate groups. Oxidation of ternary cobalt(II) complexes, [CoII(ADA)(L)(H2O)]2–,
by periodate proceeds via an inner-sphere mechanism, to give initial cobalt(III) prod-
ucts, which slowly convert to the final products15–17. Periodate is capable of acting as
a ligand, as evidenced from its coordination to copper(III) (Ref. 19) and nickel (Ref.
20). The liability of the cobalt(II) reactants and the inertness of the cobalt(III) products
can be utilised as a diagnostic tool for ascertaining inner-sphere electron transfer15–17.
In aqueous medium the [CoII(ADA)(L)(H2O)]2– complex is in equilibrium:
[CoII(ADA)(L)(H2O)]2– [CoII(ADA)(L) (OH)]3– + H+ (5)
The value of K1 associated with equation (8) was determined potentiometrically
as 5.20 × 10–6 at 35oC; this indicates that at the pH range employed in this study, the
protonated CoII-complex species will be predominant.
Also, in aqueous solution, periodate undergoes the following equilibria:
H5IO6 H4IO6– + H+ K2 (6)
H4IO6– 2H2O + IO4– K3 (7)
H4IO6 H3IO6 2– + H+ K4 (8)
The equilibrium constants K2, K3 and K4 associated with equations (6), (7)
and (8) are 5.1 × 10–4, 40 and 2.0 × 10–7, respectively21. Thus, it may be concluded
that over the pH range used, periodate will be present as H5IO6 and H4IO6–.
The hydrogen ion dependence of the reaction rate is in agreement with the involve-
ment of both deprotonated and protonated forms of periodate in the rate-determining
steps, proceeded by a rapid equilibrium. The observation that the rate increased with
ionic strength, is consistent with a reaction between charged species of the same sign
in the rate-determining step.
In view of the above considerations, the oxidation of [CoII(ADA)(L)(H2O)]2–
by periodate proceeds by one first-order pathway in each reactant, as described by
equations (9)–(13).
H5IO6 H4IO6– + H+ (9)
[Co (ADA)(L)(H2O)] + H5IO6
II 2–
[Co (ADA)(L)(H5IO6)] + H2O K5 (10)
II 2–
[CoII(ADA)(L)(H2O)]2– + H4IO6– [CoII(ADA)(L)(H4IO6–)]3- + H2O K6 (11)

[CoII(ADA)(L)(H5IO6)]2– → [CoIII(ADA)(L)(H5IO6)]– k4 (12)
[CoII(ADA)(L) H4IO6–)]3– → [CoIII(ADA)(L)(H4IO6–)]2– k5 (13)
From the above mechanism, the rate of the reaction can be described by equa-
tion (14):
rate = k4K5[CoII(ADA)(L)(H2O)2–] [H5IO6] + k5K6[CoII(ADA)(L)(H2O)2–] [H4IO6–] (14)
Substitution [H4IO6–] from equation (10) gives,
47
d[CoIII]/dt = [CoII(ADA)(L)(H2O)2–][H5IO6](k4K5 + k5K6K2/[H+]) (15)
which is identical to the experimental rate law, equation (4), and therefore,
kobs = (k4K5 + k5K6K2/[H+]) [H5IO6] (16)
Comparison of equations (17) and (2) gives:
k1 = k4K5 + k5K6K2/[H+]) (17)
The values of k2 and k3 were obtained by comparison of equations (17) and
(3), as follows:
k2 = k4K5 and k3 = k5K6K2
Effect of acetate ion on the reaction rate. Oxidation reaction of [CoII(ADA)(L)(H2O)]2–

by periodate was carried out at constant pH and ionic strength by using different acetate
ion concentrations for a temperature range 25–40oC. Table 4 and Fig. 4 show that k1
depends on acetate ion concentration. It can be seen from Fig. 4 that the variation of
k1 with [CH3CO2–] is described by equation (18) at the temperature employed.
k1 = k6 + k7[CH3CO2–] (18)
Table 4. Variation of k1 with acetate ion concentration at different temperatures

[CoII(ADA)(L)(H2O)2–] = 1.0 × 10–3 mol dm–3, I = 0.50 mol dm–3, pH = 4.99
[CH3CO2–] k1 × 102 (dm3 mol–1 s–1)
(mol dm–3) 25.0oC 30.0oC 35.0oC 40.0oC
0.09 1.00±0.02 1.52±0.01 2.08±0.02 2.74±0.03
0.18 1.25±0.01 2.10±0.02 2.96±0.04 3.62±0.05
0.27 1.54±0.02 2.54±0.02 3.43±0.06 4.60±0.07
0.36 2.06±0.03 3.05±0.03 4.02±0.05 5.72±0.09
0.45 2.24±0.05 3.50±0.07 4.80±0.05 6.38±0.12
0.54 2.48±0.04 4.26±0.05 5.50±0.07 7.09±0.10
0.63 3.00±0.05 4.57±0.06 6.24±0.10 8.26±0.14
k1 = kobs/[IO4–].
From equations (1), (2) and (18) the rate law for the oxidation of [CoII(ADA)(L)
(H2O)]2– by IO4– in acetate medium is given by equation (19):
d[CoIII]/dt = (k6 + k7[CH3CO2–])[CoII(ADA)(L)(H2O)2–]0[IO4–] (19)
The values of k6 and k7 collected in Table 5 at the temperature used were obtained
from the intercepts and the slopes respectively of Fig. 4. The enthalpy of activation
ΔH4* and ΔH5* associated with k4 and k5, were calculated from the Eyring equation
as 53.6 ±3.2 and 48.7 ± 2.40 kJ mol–1, respectively. The corresponding entropies of
activation ΔS2* and ΔS3* were calculated as –106.3 ± 4.0 and –109 ± 4.6 J K–1 mol–1,
respectively.
48
10
40°C
8
35°C
6
k1 (mol–1 dm3 s–1)
30°C
25°C
0
0 0.2 0.4 0.6
[CH3CO2–] (mol dm–3)
Fig. 4. Variation of k1 with [CH3CO2–] at different temperatures
Table 5. Variation of acetate-independent rate constant k6 and acetate-dependent rate constant k7 with
temperature
Temperature (oC) k6 ×102 (dm3 mol–1 s–1) k7 ×102 (dm6 mol–2 s–1)
25.0 0.63±0.04 3.64±0.08
30.0 1.02±0.06 5.73±0.14
35.0 1.44±0.08 7.51±0.20
40.0 1.88±0.10 10.03±0.25
Periodate oxidation of [CoII(ADA)(L)(H2O)]2– in the presence of acetate ions

proceeds via formation of cobalt(III) products. The rate of reaction increases with
increasing acetate concentration (Table 4), indicating [CoII(ADA)(L)(H2O)]2– is the
reactive species. Coordination of acetate to [CoII(ADA)(L)(H2O)]2– prior to oxida-
tion by periodate. The rate law obtained also requires acetate in a step preceeding the
rate-determining one.
The mechanism of the [CoII(ADA)(L)(H2O)]2– oxidation by periodate in acetate
medium may be described by the following equations:
[CoII(ADA)(L)(H2O)]2– + [CH3CO2]– [CoII(ADA)(L)(CH3CO2)]3– + H2O K7 (20)
[CoII(ADA)(L)(H2O)]2– + IO4– [CoII(ADA)(L)(OIO3)]3– + H2O K8 (21)
[CoII(ADA)(L)(CH3CO2)]3– + IO4– [CoII(ADA)(L)(CH3CO2)(OIO3)]4– + H2O K9 (22)
[CoII(ADA)(L)(OIO3)]3– → [CoIII(ADA)(L)(OIO3)]2– k8 (23)
[CoII(ADA)(L)(CH3CO2)(OIO3)]4– → [CoIII(ADA)(L)(CH3CO2)(OIO3)]3– k9 (24)
49
supposing that K7, K8 and K9 are small, the rate law given by equation (25), which is
similar to the experimental results, is derived from the above mechanism.
d[CoIII]/dt = {k8K8 + k9K7K9[CH3CO2–]} [CoII(ADA)(L)(H2O)2–] [IO4–] (25)
A comparison of equations (19) and (25) shows that k6 = k8K8 and k7 = k9K7K9.
The initial cobalt(III) products may be converted to the final products according
to equations (26)–(28):
[CoIII(ADA)(L)(IVII)]2- + H2O → [CoIII(ADA)(L)(H2O)]2- + IVI (26)
[CoIII(ADA)(L)(CH3CO2)(IVII)]3– + H2O → [CoIII(ADA)(CH3CO2)(H2O)]2– + IVI (27)
2IVI → IO4– + IO3– (28)
From the above reactions, I(VI) in the initial product is probably substituted by
a water molecule with a very slow rate due to the inertness of Co(III) and Co(II)–
OIO3 bond is being stronger than Co–H2O bond22. The mechanism of oxidation of
[CoII(ADA)(L)(H2O)]2– by periodate proceeds via an inner-sphere electron transfer
in which the initial cobalt(III) product is slowly converted to a final cobalt(III) prod-
uct15–17. Outer-sphere electron transfer would lead directly to the formation of a final
cobalt(III) product12–14.
Comparison of the oxidations of [CoII(ADA)(H2O)3] (Ref. 13), [CoII(ADA)(M)
(H2O)]2– (Ref. 23) and [CoII(ADA)(L)(H2O)]2–, under the same conditions, shows that
the protonated complexes are significantly more reactive. The rate of oxidation of the
binary complex [CoII(ADA)(H2O)3] is greater than those of the ternary complexes
[CoII(ADA)(M)(H2O)]2– and [CoII(ADA)(L)(H2O)]2–, hence the secondary ligand in
the ternary complexes increases the stability of these complexes towards oxidation.
The high negative entropies of activation for these reactions were suggested to be
largely the result of charge concentration upon encounter complex formation, which
causes substantial mutual ordering of the solvated water molecules24. The intramo-
lecular electron transfer steps are endothermic, as indicated by the value of ∆H*. The
contributions of ∆H* and ∆S* to the rate constant seem to compensate each other,
suggesting that the factors controlling ∆H* must be closely related to those control-
ling ∆S*. Therefore, the solvation state of the encounter complex would be important
in determining ∆H* (Ref. 24). Thus, the relatively small enthalpy of activation, ∆H*,
can be explained in terms of the formation of a more solvated complex.
CONCLUSIONS
The oxidation of [CoII(ADA)(L)(H2O)]2– by periodate is assigned an inner-sphere
mechanism via an electron transfer in which the initial cobalt(III) product is slowly
converted to a final cobalt(III) product. The rate of oxidation of [CoII(ADA)(L)(H2O)]2–
is lower than that of [CoII(ADA)(H2O)3]. The rate of reaction increases with increasing
acetate concentration. The intramolecular electron transfer steps are endothermic, as
indicated by the value of ∆H*.
50
REFERENCES
1. M. P. WOODWARD, W. W. YOUNGY, R. A. BLOODGOOD: Detection of Monoclonal Antibodies
Specific for Carbohydrate Epitopes Using Periodate Oxidation. J Immunal Meth, 78, 143 (1985).
2. I. NODA, S. FUJIEDA, H. SAITO, T. SAITO, T. OTSUBOO, M. YAGITA: Enhancement of Cyto-
lytic Activity of Human Peripheral Blood Lymphocytes by Sodium Periodate Possible Involvement
of Protein Kinase C. Int J Immunopharm, 20, 15 (1998).
3. F. A. COTTON, G. WILKINSON: Advanced Inorganic Chemistry. 3rd ed. Wiley, 1972, p. 849.
4. M. C. R. SYMONS: Photodecomposition of Periodate. J Chem Soc, 2794 (1955).
5. F. R. El-ZIRI, Y. SULFAB: Oxidation of Hexaaquoiron(II) by Periodate in Aqueous Acidic Solution.
Inorg Chim Acta, 25, 15 (1977).
6. A. Y. KASSIM, Y. SULFAB: Kinetics and Mechanism of Oxidation of Hexacyanoferrate(II) by
Periodate in Acidic Solutions. Evidence for Copper Catalysis. Inorg. Chim Acta, 22, 169 (1977).
7. A. A. ABDEL-KHALEK, I. M. ISMAIL, M. A. NAGDY, H. A. EWAIS: Kinetics and Mechanism of
Oxidation of N-(2-acetamido)iminodiacetatocobaltate(II) by Periodate in Presence of Manganese(II)
Catalyst in Acetate and Aqueous Micellar Media. Oxid Commun, 35, 327 (2012).
8. H. A. EWAIS, M. A. HABIB, S. A. K. ALROBY: Kinetics and Mechanism of Periodate Oxidation of
Two Ternary Nitrilotriacetatochromium(III) Complex Involving Histidine and Asparate Co-ligands.
Trans Met Chem, 35, 73 (2010).
9. A. A. ABDEL-KHALEK, Y. SULFAB: Kinetics and Mechanism of the Oxidation of Cobalt(II)
Aminopolycarboxylate Complexes by Periodate. J Inorg Nucl Chem, 43, 3257 (1981).
10. A. A. ABDEL-KHALEK, A.A. MOHAMED, H. A. EWAIS: Kinetics and Mechanism of Oxidation
of Chromium (III)-DL-aspartic Acid Complex by Periodate. Evidence Iron(II) as a Catalyst. Trans
Met Chem, 24, 233 (1999).
11. H. A. EWAIS, I. M. ISMAIL , S. A. AHMED, A. A. ABDEL-KHALEK: Mechanism of Electron
Transfer for the Oxidation of Ternary Complex of Chromium(III) Involving Guanosine and DL-as
partic Acid with Periodate. Oxid Commun, 37, 209 (2014).
12. A. A. ABDEL-KHALEK, Y. SULFAB: Kinetics and Mechanism of Oxidation of Trans-1,2-
diaminocyclohexaneteteraacetatocobalt(II) by Periodate. Trans Met Chem, 7, 297 (1982).
13. R. M. NAIK, J. SARKER, D. D. CHATURVEDI, A. VERMA, S. K. SINGH: Kinetics of Oxidation of
Cobalt(II) Complexes of Propylenediaminetetraacetate and 1,3-diamino-2-hydroxyprpane-tetraacetate
by Periodate. Ind J Chem Sect A, 42, 1639 (2003).
14. R. M. NAIK, A. SRIVASTAVA, A. K. TIWARI, S. B. S. YADAV, A. K. VERMA: Kinetic and Mecha-
nistic Studies of Oxidation of Amine-N-polycarboxylates Complexes of Cobalt(II) by Periodate Ions
in Aqueous Medium. Iran Chem Soc, 4, 63 (2007).
15. S. M. HASSUN, H. A. EWAIS: Kinetics of Periodate Oxidation of a Ternary Nitrilotriacetatocobalt(II)
Complex Involving Maleate Co-ligand. Oxid Commun, 35, 340 (2012).
16. A. A. ABDEL-KHALEK, E. S. HASSAN, R. A. MOHAMED: Mechanism of Electron Transfer
Reactions of Ternary Nitrilotriacetatocobaltate(II) Complexes Involving Tartarate by Periodate.
J Coord Chem, 61, 1 (2007).
17. M. A. HUSSEIN, A. A. ABDEL-KHALEK, Y SULFAB: Inner-sphere Oxidation of Diaqua(nitri
lotriacetato)cobalt(II) by Periodate in Acetate Medium. J Chem Soc, Dalton Trans, 839, 317 (1983).
18. H. MIZUOCHI, S. SHIRAKATA, E. KYUBO, R. TSCHIYA: Studies on the Synthesis of Cobalt(II)
Complex of Nitrilotriacetic Acid. Bull Chem Soc Jap, 43, 397 (1970).
19. I. HADINCE, L. JENOVSKY, A. LINEK, V. SYNECEK: The Structure of Complex of Percuprates.
Naturwiss, 47, 377 (1960).
20. P. RAY: Sodium and Potassium Nickel (IV) Paraperiodates. Inorg Synth, 5, 201 (1957).
51
21. C. E. CROUTHAMEL, A. M. HAYES, D. S. MARTIN: Ionization and Hydration Equilibria of
Periodic Acid. J Am Chem Soc, 73, 82 (1951).
22. V. S. SHARMA, D. L. LEUSSING: Rapid Formation of Ternary Complexes of Copper(II) with
Serinate, Histamine and Ethylenediamine. Inorg Chem, 11, 138 (1972).
23. H. A. EWAIS, M. R. SHEHATA, M. A. NAGDY, A. A. ABDEL-KHALEK: Kinetics and Mechanism
of Oxidation of the Ternary Complex of Cobalt(II) Involving N-(2-acetamido)iminodiacetic Acid
and Malonate by Periodate in Acetate Medium. Inorg Chem An Indian J, 5, 84 (2010).
24. M. J. WEAVER, E. L. YEE: Activation Parameters for Homogeneous Outer-sphere Electron-transfer
Reactions. Comparisons between Self-exchange and Cross Reactions Using Marcus’ Theory. Inorg
Chem, 19, 1936 (1980).
Revised 13 September 2015
52
Ionic oxidation
OXIDATIVE DECARBOXYLATION AND DEAMINATION OF

GLYCINE, ALANINE AND VALINE BY N-IODOSUCCINIMIDE
IN AQUEOUS ACETIC ACID MEDIUM. A KINETIC STUDY
K. VIVEKANANDAN, A. NAGARAJAN*
PG and Research Department of Chemistry, National College (Autonomous),
620 001 Tiruchirappalli, Tamil Nadu, India
E-mail: nagarajanraja@gmail.com
ABSTRACT
The kinetics of oxidation of glycine, alanine and valine using N-iodosuccinimide
in acetic acid-water medium in the presence of hydrochloric acid at 303 K has been
studied. The reaction shows inverse first order with respect to oxidant and [H+]. In-
crease in [amino acid] has a slight positive effect on the rate, indicating first order
dependence. Addition of salts like K2SO4, Na2SO4, KCl to the reaction medium has
no effect on the rate, increase in temperature increases the rate of the reaction. The
amino acids were oxidised to corresponding aldehydes, carbon dioxide, and ammonia.
The products obtained were isolated and identified. The activation parameters have
been computed. A mechanism confirming to the kinetic observations is suggested.
Keywords: oxidation, kinetics, amino acid, N-iodosuccinimide, mechanism.
AIMS AND BACKGROUND

The oxidation of amino acids is of interest due to their biological importance. Amino
acids play a significant role in a number of metabolic reactions. Specific metabolic roles
of amino acids include the biosynthesis of polypeptides and proteins and the synthesis
of nucleotides. Thus the mechanism of analogous non-enzymatic chemical processes
in the oxidation of amino acids is a potential area for intensive investigations1.
An extensive literature survey reveals that kinetics and mechanism of oxidation
of amino acids have been studied using various N-halo compounds2–19 like N-bromon-
icotinamide11–15, N-chlorobenzamide16, N-bromobenzamide17, N-bromoacetamide18,
N-bromosuccinimide19, N-chlorosuccinimide20. They have been used in a variety of
reactions like oxidation, halogenation, etc. N-iodosuccinimide is such a compound.
It is a mild, efficient, stable and less expensive oxidant21. Its chlorine analog has been
*
For correspondence.
53
investigated by Ramachandran et al.20 There is no significant data on the NIS oxidation
of glycine, alanine and valine. Hence, the present investigation proposed to examine
its utility as an oxidant by investigating the kinetics of oxidation of amino acids such
as glycine, alanine, and valine by NIS in aqueous acetic acid medium in presence
of hydrochloric acid. Also possible stoichiometry, product analysis and mechanism
have been proposed.
EXPERIMENTAL
Materials. All the essential amino acids like glycine, alanine and valine (A.R LOBA)
were used as purchased. Oxidant N-iodosuccinimide (A.R Sigma) with purity 98%
were purchased and used. Hydrochloric acid (Merck) used as a source of hydrogen
ions. Conductivity water was used throughout the study. Other chemicals used were
of analytical grade.
Kinetic measurements. The reaction was carried out under pseudo-first order conditions
([amino acid] >> [NIS]). The reaction was followed potentiometrically by setting up a
cell made up of the reaction mixture into which the platinum electrode and reference
electrode (SCE) were dipped. The electromotive force (e.m.f.) of the cell was measured
periodically using an Equip-Tronics (EQ-DGD) potentiometer. The pseudo-first order
rate constants computed from the linear (r2 > 0.9990) plots of lg (Et - E∞) against time.
Duplicate kinetic runs showed that the rate constants were reproducible within ±3%.
The course of the reaction was studied for more than two half-lives.
Stoichiometry. A mixture of amino acid (0.01 mol dm-3), NIS (0.001 mol dm-3) dis-
solved in methanol and HCl (1.0 mol dm-3) was made up to 100 ml with water and
acetic acid mixture (1:1). After the reaction was complete, the excess of NIS was
determined iodometrically and indicated 1:1 stoichiometry. The overall stoichiometry
of the oxidation reaction may be represented as follows:
NIS + amino acid → aldehyde + I– + CO2 + NH3 + succinimide (1)
Product analysis. In a typical experiment, a mixture of amino acid (Gly, Ala,Val

1 mol dm-3 ) and NIS (1.56 g, 0.2 mol dm-3) was made up to 50 ml with acetic acid-
water mixture (1:1) in the presence of HCl (1.0 mol dm-3). The mixture was allowed to
stand for 12 h in the dark to ensure completion of the reaction. It was then treated with
excess (125 ml) of a saturated solution of 2,4-dinitrophenylhydrazine in 2 mol dm-3
HCl and set aside for 10 h. The precipitated 2-dinitrophenylhydrazone (DNP) was
filtered off, dried, recrystallised from ethanol and weighed. The melting points of the
derivatives of the products obtained were identical with an authentic sample of DNP
of formaldehyde. The yield was 80%. In similar experiment with other amino acids the
corresponding carbonyl compounds were identified as their DNP derivatives20. In all
the cases carbon dioxide and ammonia were detected by baryta water and the Nessler
54
reagent, respectively. The presence of corresponding aldehydes and ammonium ions
were also confirmed by chromotropic acid.

The kinetic results for the oxidation of amino acid by NIS can be summarised as fol-
lows. The kinetic studies were carried out under pseudo-first order conditions with
[amino acid] >> [NIS].
Effect of variation of [oxidant]. The oxidation was carried out with different initial
concentrations of NIS. The pseudo-first order rate constants decrease with increase
in the initial concentration of the oxidant. But in each kinetic run, the reaction shows
no deviation whatsoever from the first order plot (Table 1).
Table 1. Effect of variation of [NIS] on reaction rate

[aminoacid] – 0.01 mol dm–3; [HCl] – 1.0 mol dm–3; H2O:CH3COOH (1:1); temperature 302 K
Variation kobs × 104 (s–1)
[NIS] × 103 (mol dm–3) [Sub] = 0.01 mol dm–3 [HCl] = 1.0 mol dm–3
Gly Ala Val
1.0 4.82 4.34 4.22
2.0 4.69 3.75 4.17
3.0 4.54 2.39 4.02
4.0 4.42 1.70 3.93
[Sub] × 102 (mol dm–3) [NIS] = 0.001 (mol dm–3) [HCl] = 1.0 mol dm–3
1.0 4.82 4.34 4.22
2.0 5.32 5.13 4.94
3.0 5.77 5.51 5.66
4.0 6.39 6.97 6.39
[HCl] (mol dm–3) [Sub] = 0.01 mol dm–3 [NIS] = 0.001 (mol dm–3)
1.0 4.82 4.34 4.22
1.1 3.72 3.75 3.72
1.2 2.67 2.39 2.67
1.3 1.98 1.70 1.98
Effect of variation of [amino acid]. At constant [H+], with the [aminoacid] in excess,
the plot of lg (Et - E∞) (where Et is the e.m.f. of the cell at time t and E∞ – the cor-
responding value at the completion of the reaction) versus time is linear, indicating
a first order dependence of rate on [NIS]. Increase in [amino acid] has a slight posi-
tive effect on the rate, indicating fractional order dependence of rate on [amino acid]
(Table 1) (Fig. 1).
55
Fig. 1. Plot of lg k1 versus 3 + lg [S]
Effect of variation of [HCl]. The rates decreased with increase in [HCl], at fixed
[NIS] and [amino acid] (Fig. 2), showing inverse order dependence in [H+] (Table 1).
Fig. 2. Plot of 4 + lg k1 versus 3+lg [H+]
Effect of variation of dielectric constant of the medium. An increase in the rate constant
is noticed on decreasing the dielectric constant of the medium (Table 2). An increase in
the amount of acetic acid in the solvent results in an increase in the rate of oxidation.
Table 2. Effect of variation of [CH3COOH:H2O] on reaction rate

[Sub] – 0.01 mol dm–3; [HCl] – 1.0 mol dm–3; H2O:CH3COOH (1:1); temperature 302 K
CH3COOH H 2O D kobs ×104 (s–1)
(%) (%) Gly Ala Val
50 50 37.50 4.82 4.34 4.22
55 45 34.75 5.74 4.88 4.88
60 40 31.50 6.52 5.74 5.45
65 35 28.50 7.68 6.21 6.21
56
Effect of addition of succinimide. The rate of reaction decreases on adding succinimide.
Thus added succinimide has a retarding effect on the rate of oxidation22.
Effect of added salts on reaction rate. The effect of added salts like Na2SO4 , KCl,
BaCl2 and K2SO4 on the reaction rate was studied by adding various concentrations
of these salts, keeping the concentrations of amino acid, HCl and NIS constant. It was
observed that the rate of oxidation was not altered by the addition of these neutral salts.
Test for free radicals. The possibility of free radical intervention in the reaction was
tested as follows: The reaction mixture containing acrylonitrile scavenger was kept
for 24 h in an inert atmosphere and then diluted. On dilution formation of precipitate
was not observed indicating the absence of free radical intervention in the reaction.
Effect of temperature. Increase in temperature increases the rate of oxidation and
plot of lg kobs versus reciprocal of temperature is linear. The oxidation of amino acid
was studied at temperatures from 302 to 317 K (Table 3) (Fig. 3) and the activation
parameters were evaluated (Table 4).
Table 3. Effect of temperature on reaction rate

[aminoacid] – 0.01 mol dm–3; [HCl] – 1.0 mol dm–3; H2O:CH3COOH (1:1); temperature 302 K
Temperature (K) kobs ×104 (s–1)
Gly Ala Val
302 4.82 4.34 4.22
307 5.51 4.62 5.22
312 6.14 5.52 6.14
317 7.09 6.86 7.09
Fig. 3. Plot of lg (K2/T) versus 1/T
57
Table 4. Activation parameters
Substrate Ea (kJ mol–1) DH* (kJ mol–1) DS* (J K–1 mol–1) DG* (kJ mol–1)
Gly –25.93 –28.44 –193.63 30.04
Ala –27.12 –29.63 –194.82 29.20
Val –21.44 –23.96 –194.95 34.92
Mechanism and rate law. Addition of succinimide decreases the rate of oxidation21.
This retarding effect suggests that the pre-equilibrium step involves a process in which
succinimide is one of the products.
NIS + H2O succinimide + HOI (2)
In acid medium, amino acid exists in its protonated form (SH+) which is resistant
to attack by NIS. It is observed that the rate has inverse dependence on [H+]. Thus the
only species possibly controlling the rate of oxidation seems to be −OOC–CH(NH2)
CH2–S–S–CH2CH(NH3)+COO−.
The electrophilic attack of HOI on the dianion of amino acid results in the
formation of an intermediate which cleaves in fast steps to give the final product,
corresponding acid.
It may be pointed out that in the present study, oxidation by iodine was completely
suppressed as the oxidative studies were carried out in presence of mercuric acetate
which combines with iodide ions formed in the reaction. Thus kinetics of only NIS
oxidation was followed.
The first order dependence on [amino acid] and [NIS] reveals that overall rate
may involve the interaction of HOI and amino acid in the rate-determining step. First
order in the [amino acid] and a definite intercept in the 1/kobs versus 1/[sub] plot sug-
gest that the decomposition of the complex formed from the substrate and HOI is the
rate-determining step21 as shown below:
k2
SH+ S + H+ (3)
k–2
S + HOI [RCH(NH2COOI] + H2O (4)

k–3 complex

[RCH(NH2)COOI] → R-C+H(NH2) + CO2 + I– (5)

R-C+H(NH2) → RCH=NH + H+ (6)
RCH=NH + H2O → RCHO + NH3 (7)
where R = H for Gly, CH3 for Ala, CH3CH (CH3) for Val.
The rate law for the above mechanism may be derived as follows:
rate = – d[NIS]/dt
= k4 [complex] (8)
58
kdk1k2k3[NIS][SH+]
= (9)
K–1K–2K–3[SA][H+]
[NIS]T = [NIS] + [HOI] + [complex] (10)
k1[NIS] kdk1k2k3[NIS][SH+]
= [NIS] + + (11)
k–1[SA] k–1k–2k–3[SA][H+]
[NIS]T
NIS = (12)
k–1k–2k–3[SA][H ] + k1k–2k–3[H+] + kdk1k2k3[NIS][SH+]
+
k–1k–2k–3[SA][H+]
Substituting [NIS] in equation (9):

d[NIS]T kdk1k2k3[NIS][SH+]
– = (13)
dt k–1k–2k–3[SA][H+] + k–1k–2k–3[H+] + k1k2k3[SH+]
On re-arranging equation (13) we obtain:

k–1k–2k–3[SA][H+] k–2k–3[H+] 1 1 1
+ + = (14)
kdk1k2k3 kdk2k3 [SH ] +
kd kobs
The formation of the complex involves the charge separation which leads to a
negative solvent effect. This has been confirmed by the increase in rate with decreas-
ing dielectric constant of the medium. The involvement of substrate molecule in the
rate-determining step leads to different values of kobs for different initial concentrations
of amino acid under study, namely, glycine, alanine and valine.
The proposed mechanism is well supported by the moderate values of energy of
activation and thermodynamic parameters. The negative entropy of activation indi-
cates the complex formation as suggested in the above reaction mechanism, and also
indicates that the complex is more ordered than reactants23. High positive values of
the free energy of activation and the enthalpy of activation show that the transition
state is highly solvated (Table 4).
CONCLUSIONS
The rate of the oxidation of amino acids by NIS depends on the first power of con-
centration of H+ and NIS. Increase in acetic acid proportion increases the rate. Added
succinimide retards the reaction. Addition of salts to the reaction medium has no
effect on the rate. Increase in temperature increases the rate of reaction. The activa-
tion parameters are evaluated from the study of oxidation at different temperatures.
The products obtained (in this case the corresponding α-ketoacid) were isolated and
characterised.
59
ACKNOWLEDGEMENTS
The authors thank the Management, National College (Autonomous), Trichy and
Godrej Consumer Products Ltd., Pondicherry for the facilities and supports provided.
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Lysine and Tyrosine by N-chloronicotinamide in Aqueous Acetic Acid Medium. A Kinetic Study.
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(2004).
8. N. A. MOHAMED FAROOK, G. A. SEYED DAMEEM, A. MURUGESAN, M. KANAGARAJ:
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Medium. E J Chem, 1 (2), 132 ( 2004).
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N-bromosuccinimide. J Al-Nahrain University, 10 (2), 66 ( 2007).
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60
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Received 27 July 2015
Revised 10 September 2015
61
Ionic oxidation
FORMATION AND CHARACTERISATION OF A STABLE

AQUEOUS DISPERSION OF SILVER NANOPARTICLES IN
AQUEOUS AND MICELLAR MEDIA. A KINETIC STUDY
I. M. ISMAILa,b, H. A. EWAISa*
a
Department of Chemistry, Faculty of Science, King Abdulaziz University,
E-mail: hshalby2002@yahoo.co
b
Centre of Excellence in Environmental Studies, King Abdulaziz University,
ABSTRACT
The kinetics and mechanism of the formation of silver nanoparticles by reduction
with arabinose are studied spectrophotometrically in aqueous and micellar media at
different temperatures. The reaction was carried out under pseudo-first-order condi-
tion by taking the [arabinose] >10-fold the [Ag+]. The rate of reaction increases with
increasing in the [OH–]. A mechanism of the reaction between silver ion and arabinose
was proposed and the rate equation derived from the mechanism was consistent with
experimental rate. Cetyltrimethylammoniuom bromide (CTAB) as capping agent
stabilised the formed silver sol. The formation of silver nanoparticles by this method
gave an easy and viable strategy for the obtained silver sols with well controlled shape
and size. Enthalpy and entropy of activation were calculated. The particle size of silver
sols is characterised by transmission electron microscopic (TEM), physicochemical
and spectroscopic methods.
Keywords: nanoparticles, silver sol, kinetics and mechanism, surfactants, TEM.
AIMS AND BACKGROUND

Silver nanoparticles were formed and stabilised by chemical and physical methods;
the chemical method, such as electrochemical techniques, chemical reduction, and
photochemical reduction is most widely used1,2. The chemical reduction is the most
used method for the formation of silver nanoparticles as stable, colloidal dispersions
in aqueous or non-aqueous media3,4. Hydrazine, aniline, DL-tryptophan, galactose and
fructose are used as reducing agents in the formation of nanoparticles5–9. The stabilising
*
For correspondence.
62
agents, such as cetyltrimethylammoniuom bromide, sodiumdodecyl sulphate, Tritron
X-100 and poly(vinyl alcohol) are used as capping agent to control the shape and
size of silver nanocrystals5,8,10. The reduction of silver ion (Ag+) in aqueous medium,
gave a colloidal silver sol with a particle diameter of several nanometers4. The reduc-
tion of silver ion with various reducing agents leads to the formation of silver atoms
(Ag0), which is followed by agglomeration in to oligomeric clusters11.These clusters
eventually lead to the formation of yellow colloidal silver nanoparticles solution11.
Silver nanoparticles have many applications in biotechnology12, medicine13,14,
biosensor15, catalysis and biochemical studies16. The evolution and technological
progress of silver nanoparticles in the manufacture are due to their antiviral and
antibacterial properties. There are many industrial applications of silver nanoparti-
cles such as microelectronics, cosmetics, adhesives and catalysis to enhanced solar
cells16–19. The formation of nanoparticles using biological entities has great important
in biotechnology due to their individual shape dependent on optical, electrical and
chemical properties12.
In this paper, the characterisation and kinetic studies of the silver nanoparticles
formation by reduction method are carried out in order to obtain a stable form of na-
noparticles in aqueous and micellar media. Also, the formation of silver nanoparticles
by this method gave an easy and viable strategy for the obtained silver sols with well
controlled shape and size.
EXPERIMENTAL
Materials and instruments. Silver nitrate, arabinose, cetyltrimethylammonium bromide
(CTAB), sodium hydroxide and sodium dodecyl sulphate (SDS) (Fluka and BDH )
were used without further purification. The solutions of silver nitrate and arabinose
were prepared daily (to arrest the aerial oxidation). Doubly distilled water was used
for the preparation of all solutions.
UV-vis. LABOMED, INC UVD-2960 spectrophotometer and Perkin Elmer
EZ-150 spectrophotometer recording spectrophotometer were used to monitor the
absorbance of the formation of silver sols. TEM images for the determination of the
size of silver particle were recorded using transmission electron microscope (JEOL,
JEM-1011, Japan). The preparation of samples was carried out by adding a drop of
working solution on a carbon-coated standard copper grid (300 mesh) operating at
80 kV. The Fourier transform infrared (FT-IR) spectra were recorded on a Bruker
Equinox 55 spectrophotometer. A few drops of the silver sol were added on potassium
bromide pellets, leave to dry and IR spectra recorded. The particles were imaged by
LEO 440i Scanning Electron Microscopy (SEM) at an accelerating voltage of 20 kV.
Kinetic measurements. The ultraviolet-visible absorption spectra of the products
(silver sol) were followed spectrophotometrically for a definite period of time using
the LABOMED, INC UVD-2960 spectrophotometer. All reactants were thermally
equilibrated for ca 20 min in an automatic circulation thermostat, thoroughly mixed
63
and quickly transferred to an absorption cell. The reaction rates were measured by
monitoring the absorbance of product at 415 nm, using a Perkin Elmer EZ-150 spec-
trophotometer, where the absorption of the products is maximum. The temperature
of the reacting solution was adjusted, using automatic circulation thermostat. The
thermostat was provided with a special pumping system for circulating water at
regulated temperature in the cell holder.
Pseudo-first order conditions were maintained in all runs by the presence of a large
excess of arabinose (>10-fold). Pseudo-first order rate constants kobs were obtained
from the slopes of plots of ln a/(1 – a) versus time with a fixed-time method where
a = At/A∞ and At and A∞˛ are the absorbancies at times and infinity, respectively20.
Thermodynamic activation parameters DH* and DS* were calculated using the Eyring
equation by plotting ln (k/T) versus 1/T:
ln (k/T) = ln (K/h) + DS*/R – DH*/RT
where K is the Boltzmann constant, h – the Plank constant, R – the universal gas
constant, T – the absolute temperature, and k – the rate constant.
The preparation of silver nanoparticles by the reduction of silver nitrate solution
with arabinose in the presence of micelles in aqueous medium was investigated. A
series of runs were carried out, using a different concentration of reductant, oxidant
and CTAB to obtain a perfectly clear silver sol. In a similar procedure, 2.0 ml of a
0.001 mol dm−3 solution of silver nitrate and 4.0 ml 0.1 mol dm−3 sodium hydroxide
solution were mixed with 4.0 ml of 0.01 mol dm−3 CTAB solution. The pale yellow
colour of the silver sol is formed, when 2.0 ml of 0.02 mol dm−3 solution of arabinose
were added to the reaction mixture at the beginning of reaction. The total volume of
the reaction mixture was always 40 ml. The presence of pale yellow colour indicated
the formation of Ag-nanoparticles17,21.
The solid sample of silver nanoparticles for FT-IR, EDS and SEM analysis was
synthesised using the CTAB as stabiliser for silver nanoparticles that were formed from
the aqueous colloidal solution via centrifugation by adding acetone as anti-solvent
for several times, then dried.

Formation and characterisation of silver nanoparticles. The formation of the stable
silver nanoparticles using a suitable reducing agent and stabiliser is very important
because the shape and size depend on the nature of stabiliser and reducing agent23.
Metal nanoparticles were synthesised by the moderate reductants, was comparatively
more stable than produced by strong reductants22. Arabinose is one of the relatively
moderate reducing agent used for the reduction of silver ions in presence of CTAB
as stabiliser.
The UV-vis. absorption spectra of the silver nanoparticles products (Fig. 1) indi-
cate the formation of a yellow colour of silver sol at 415 nm. The absorption band at
64
415 nm is characteristic of spherical or roughly spherical shape of Ag-nanoparticles
synthesis. FT-IR is used to characterise the state of silver nanoparticles present in the
colloidal solution. Figure 2 shows IR spectra of silver nitrate and silver nanoparticles.
The bands at 3291 and 1629 cm−1 are broad and sharp, and can be assigned to the
hydroxyl groups from adsorbed moisture. These bands disappear in the silver nitrate
spectrum. SEM image of silver nanoparticles prepared by the reduction of silver
ion with arabinose is represented in Fig. 3. From SEM image, it is observed that the
particles had a relatively narrow size and spherical shape. The size of silver nanopar-
ticle is ranging from 16.67 to 48.72 nm and has various shapes: sphere and irregular
with broader size distribution (Fig. 3). TEM image of the prepared Ag-nanoparticles
is represented in Fig. 4. From Fig. 4, it clear that the size of the nearly spherical na-
noparticles ranges between 10 and 46.87 nm and their size distribution is relatively
wide. The TEM image confirms that CTAB stabilises the spherical non-hexagonal
forms of silver nanocrystals during the reduction of silver ions with hydrazine and
glucose, respectively3,23.
0.6
7
0.5
0.4
5
absorbance
0.3
4
0.2
3
0.1 2
0
300 400 500 600
wavelength (nm)
Fig. 1. Absorbance spectra of reaction mixtures versus time

Curves 1–7 were recorded at 1, 5, 10, 20, 30, 40 and 60 min at [Ag+] = 5.0 × 10–5 mol dm–3, [arabinose] =
1.0 × 10–3 mol dm–3, [CTAB] = 1.0 × 10–3 mol dm–3, [OH–] = 8.0 × 10–3 mol dm–3 and T 50oC
65
120
100
a
80
T (%)
60
40
20
0
4000 3500 3000 2500 2000 1500 1000 500 0
wavenumber (cm–1)
Fig. 2. FT-IR spectra for silver nitrate (a) and silver nanoparticles (b)
Fig. 3. SEM images of silver nanoparticles that were obtained as the final products under the optimum
conditions mentioned under Fig. 1
Fig. 4. TEM images of silver nanoparticles that were obtained as the final products under the optimum
conditions mentioned under Fig. 1
66
Kinetics of formation of silver nanoparticles. The kinetics of the formation of Ag na-
noparticles by reduction with arabinose was studied at different concentrations of Ag+,
arabinose, NaOH and CTAB for a temperature range 45–60oC. Plotting absorbance
versus time (Fig. 5) shows that autocatalysis is involved in the silver sol formation.
The observation of autocatalysis in Fig. 5 is due to the formation of metal nucleation
centre which acts as a catalyst for the reduction of other silver ions present in solution.
In the present study it is necessary to point out that the plots of ln a/(1–a) against time
are linear up to 87% (Fig. 6), where a = At/A∞ and At and A∞ are the absorbancies at
times t and infinity ∞, respectively21. The values of the rate constants were obtained
from the slopes of ln a/(1–a) versus time plots (Table 1). The constant values of kobs
over the [Ag+] range (2.50–7.50) × 10–5 mol dm–3 at fixed arabinose concentration
indicate first-order dependence on the [Ag+] (Table 1).
rate = kobs [Ag+] (1)
Table 1. Dependence of the [Ag+]/[arabinose] reaction rate on [Ag+], [arabinose] and [CTAB] = 1.0 ×
10–3 mol dm–3 at T = 50.0oC
[OH–] × 103 [Ag+] × 105 [Arabinose] × 103 kobs × 103
(mol dm–3) (mol dm–3) (mol dm–3) (s–1)
8.0 5.00 2.00 4.37
1.75 3.85
1.50 3.32
1.25 2.92
1.00 2.18
0.75 1.48
0.50 1.15
2.0 1.00 1.17
4.0 1.00 1.49
6.0 1.00 1.86
10.0 1.00 2.79
12.0 1.00 3.41
8.0 2.50 1.00 2.13
3.75 1.00 2.32
5.00 1.00 2.18
6.25 1.00 2.43
7.50 1.00 2.37
67
0.7 + -5 -3
[arabinose] x 103 mol dm-3 [Ag ] = 5.0 x 10 mol dm
-3
[NaOH] = 8.0 x 10-3 mol dm
0.5 -3 -3
[CTAB ] = 1.0 x 10 mol dm
0.75
T = 50 oC
1
0.6 1.25
1.5
1.75
2
0.5
0.4
absorbance
0.3
0.2
0.1
0
0 10 20 30 40 50
time (min)
Fig. 5. Absorbance – time curves of silver sol formation at different [arabinose]
The dependence of the observed rate constant, kobs, on arabinose (R-CHO) was
studied over the concentration range (0.5–2.0) × 10–3 mol dm–3 at fixed [Ag+] (= 5.0 ×
10–5 mol dm–3), [NaOH] (= 8.0 × 10–3 mol dm–3) and [CTAB] (= 1.0 × 10–3 mol dm–3)
at T = 50oC. A plot of kobs versus [R-CHO] was linear without intercept, obeying the
linear equation: y = mx with a correlation coefficient of R = 0.9987. This can be de-
scribed by the following equation:
kobs = k1[R-CHO] (2)
The kinetics of the formation of silver nanoparticles was studied over a [NaOH]
range of (2.0–12.0) × 10–3 mol dm–3 at different temperatures. The values of k1 with
[NaOH] at different temperatures are represented in Table 2 and show that the rate
increased gradually with increasing in [NaOH]. Plot of k1 against [OH–] was linear, with
intercepts as shown in Fig. 7. This state can be described by the following equation:
k1 = k2 + k3[OH–] (3)
68
11
[Ag+] = 5.0 x 10-5 mol dm-3
[arabinose] x 103 mol dm-3
[NaOH] = 8.0 x 10-3 mol dm-3
0.5
[CTAB] = 1.0 x 10-3 mol dm-3
0.75 T = 50 oC
1
9 1.25
1.5
1.75
2
5
ln a/(1 – a)
-1
-3
0 10 20 30 40 50
time (min)
Fig. 6. Plots of ln a/(1–a) versus time at different [arabinose]
Table 2. Variation of k1 with hydroxide ion concentration

[Ag+] = 5.0 × 10–5 mol dm–3, [arabinose] = 1.0 × 10–3 mol dm–3, [CTAB] = 1.0 × 10–3 mol dm–3, at dif-
ferent temperatures
[OH–] × 103 k1 (dm3 mol–1 s–1)
(mol dm–3) 45.0oC 50.0oC 55.0oC 60.0oC
2.0 0.91 1.22 1.54 1.75
4.0 1.15 1.53 1.97 2.38
6.0 1.51 2.01 2.62 2.87
8.0 1.84 2.22 3.25 3.69
10.0 2.16 2.62 3.77 4.25
12.0 2.42 3.09 4.17 4.74
The hydroxide-independent rate constant k2 and hydroxide dependent rate constant

k3 were obtained from the intercepts and the slopes of Fig. 7, respectively. The k2 and
k3 values are collected in Table 3 at the temperature used, and both are dependent on
temperature.
69
6
5
o
T = 60 C
o
T = 55 C
4
k1 (dm3 mol–1 s–1)
o
T = 50 C
3
o
T = 45 C
0
0 2 4 6 8 10 12 14
[OH–] × 103 (mol dm–3)
Fig. 7. Plot of k1 versus [OH–] at different temperatures
From equations (1), (2) and (3), the rate law for the reduction of [Ag+] by arab-
inose is given by equation (4):
rate = (k2 + k3[OH–]) [Ag+] [R-CHO] (4)
and
kobs = [R-CHO] (k2 + k3[OH–]) (5)
The enthalpy of activation ΔH2* and ΔH3* related to k2 and k3, were calculated
from a least-squares fit to the transition state theory equation as 36.5 and 40.3 kJ mol–1,
respectively. The corresponding entropies of activation ΔS2* and ΔS3* were calculated
as –134.4 and –76.5 J K–1 mol–1, respectively.
70
Table 3. Variation of hydroxide-independent rate constant k2 and hydroxide dependent rate constant k3
with temperature
Temperature (oC) k2×10 (dm3 mol–1 s–1) k3×10–2 (dm6 mol–2 s–1)
45.0 5.74 1.56
50.0 8.29 1.83
55.0 9.69 2.74
60.0 11.40 3.05
Mechanism. The formation of silver sol in aqueous medium included a different spe-
cies of silver metal particles. Several species of silver ions like Ag2+, Ag42+, Ag32+,
Ag64+ and Ag9+ exist in aqueous medium24,25, as suggested by Henglein et al.24 and
other researchers25. Ag42+ is the more stable species for a long time in presence of
active surfactants. Equation (6) involved a complex formation between R-CHO and
Ag+ in alkaline medium. In the rate-determining step, the complex, R-COOAg, de-
composes unimolecularly through one electron oxidation–reduction mechanism to
the formation of Ag0 (equation (8)). In the following step, Ag0 reacts with an Ag+ to
yield a silver sol (Ag42+) (equation (10)) (Ref. 25). A possible mechanism is described
by the following equations:
R-CHO + Ag+ + OH– → R-COOAg + H2 K1 (6)
R-CHO + Ag+ + H2O → R-COOH + Ag0 + H2 k4 (7)
R-COOAg → R-COO. + Ag0 k5 (8)
R-COO + Ag → R + CO2 + Ag (Ref. 7)
. + 0
(fast) (9)
2Ag0 + 2Ag+ → Ag42+ (fast) (10)
(silver sol. yellow colour)
From the above mechanism the rate law is given by:
rate = k4[R-CHO][Ag+] + k5[R-COOAg] (11)
From equilibrium (6), we obtained:
R-COOAg = K1[R-CHO] [Ag+][OH–] (12)
rate = k4[R-CHO][Ag+] + k5K1[R-CHO] [Ag+][OH–] (13)
rate = [R-CHO][Ag+] (k4 + k5K1[OH–]) (14)
and
kobs = [R-CHO] (k4 + k5K1[OH–]) (15)
From the comparison of equations (15) and (5), one obtained k2 = k4 and k3 =
K1k5. Equation (15) contains two terms, the first term represents path independent of
[OH–] and the second one – path dependent on [OH–].
71
Effect of micelles on the rate of formation of silver nanoparticles. The effect of
micelles such as cetyltrimethylammonium bromide (CTAB), sodium hydroxide and
sodium dodecyl sulphate (SDS) on the formation of silver nanoparticle by reduction
with arabinose was studied at [Ag+] = 5.0 × 10–5 mol dm–3, [arabinose] = 1.0 × 10–3
mol dm–3, [OH–] = 8.0 × 10–3 mol dm–3, [micelles] = (0.5–4.0) × 10–3 mol dm–3 and
T = 50.0oC. Table 4 shows that the reaction rate increases with increasing [SDS] up
to ≥ 3.0 × 10−3 mol dm−3 and remains constant at higher [SDS]. This may be due to
the dilution effect. The role of SDS micelles in catalysis can be explained by incor-
poration/solubilisation of Ag+/arabinose in the Stern layer of SDS micelles through
electrostatic and hydrophobic interactions. These results are in good agreement with
our previous observations26. Also, the reaction rate decreases with increasing [CTAB]
up to 2.5 × 10–3 mol dm–3 (Table 4). At higher [CTAB] = 2.5 × 10–3 mol dm–3, the
reaction mixtures becomes turbid and transparent silver sol was not formed due to
dilution effect.
Table 4. Effect of micelles in the formation of silver nanoparticles

[Ag+] = 5.0 × 10–5 mol dm–3, [arabinose] = 1.0 × 10–3 mol dm–3, [NaOH] = 8.0 × 10–3 mol dm–3 and T = 50oC
[Micelles] × 103 (mol dm–3) kobs (CTAB) × 103 (s–1) kobs (SDS) × 103 (s–1)
0.50 2.84 1.85
1.00 2.18 2.38
1.50 1.83 2.69
2.00 1.61 2.95
2.50 1.47 3.09
3.00 turbid 3.13
4.00 turbid 3.08
The role of micelles in catalysis and inhibition of some reactions is due to the
solubilisation and/or incorporation of reactants into the small volume of micelles
through electrostatic, hydrophobic, hydrogen bonding and Van der Walls forces27,28.
The different behaviour of SDS and CTAB micelles in the formation of silver sol
due to the presence of positive charge on the Ag+ and its metal particles in the reac-
tion medium. The formation of ion pair, (–OSO3– Ag+), from positive charge on Ag+
with the negative head group of SDS micelles which concentrate the Ag+ through
the electrostatic interactions into the reaction site (i.e. the Stern layer as most of the
ionic micelle mediated reactions are believed to occur in this region). Furthermore,
electrostatic repulsion between the positive head group (–N+ (CH3)3) of CTAB mi-
celles with silver ion, Ag+ is present in the bulk aqueous pseudo-phases. According
to the following observations, the reaction rate increases in the presence of SDS but
decreases in the presence of CTAB.
72
CONCLUSIONS
This study describes the formation of stable and transparent silver sol in aqueous
and micellar media. The rate of formation increases with increasing [NaOH] and the
rate of reaction is derived which is consistent with experimental rate law. The rate
of formation of silver nanoparticles decreases with increasing of [CTAB], while it
increases with increasing of [SDS]. The particle size of silver sols is characterised
by FT-IR, SEM and TEM.
ACKNOWLEDGEMENTS
This work was supported by the Deanship of Scientific Research (DSR), King Ab-
dulaziz University, Jeddah, under grant No (130-032-D1433). The authors, therefore,
gratefully acknowledge the DSR technical and financial support.
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Received 16 March 2015
Revised 16 April 2015
74
Analytical section
A NEW SURFACTANT-BASED APPROACH FOR THE

DETERMINATION OF MALACHITE GREEN
WANG LANJIE
College of Chemistry and Environmental Engineering, Yangtze University,
JingZhou, China
E-mail: wanglanjie1999@126.com
ABSTRACT
The effects of different types of surfactants on the determination of Malachite Green
(MG) by using electrochemical method were studied, and a type of anionic surfactant
of sodium dodecyl benzene sulphonate (SDBS) was screened out, proving to have
played a significant sensitising effect in the determination of MG in pH 6.5 phosphate
buffer solution. Cyclic voltammetry was applied in the research on the electrochemical
behaviour of MG in carbon paste electrode, which optimised the supporting electrolyte,
types and consistency of surfactant, time of concentration and experimental param-
eters. The peak current of MG and its concentration presented good linear relationship
within the range of 1.0×10–8–2.0×10–6 mol/l, and the detection limit was 8.0 × 10–9
mol/l after 4 min of enrichment. A fast and sensitive electrochemical method for the
determination of MG was established.
Keywords: surfactant, Malachite Green (MG), carbon paste electrode.
AIMS AND BACKGROUND

MG (Malachite Green), also known as basic green or peacock green, is a kind of
N-methylated diaminotriphenymethane soluble in water and ethanol, with the pres-
entation of dark green crystalline solid. Its structural formula is shown in Fig. 1. In
aquaculture, MG is mainly used to resist fungal infection and kill parasites. For the
fish farming, MG is generally used for the prevention and cure of saprolegniasis,
rotten gill disease, parasites, etc. The MG entering the bodies of aquatic animals for
excessively long residence time possesses high toxic, high residual, carcinogenic,
teratogenic and mutagenic effects. Moreover, it can also cause pollution to the envi-
ronment, and pose a certain degree of threat to human health. In view of this, many
countries have listed MG as banned drugs in aquiculture, and thus it is of practical or
immediate significance in the exploration of new detection method of MG.
75
Fig. 1. Molecular structure of MG
Currently, the main detection methods of MG comprise high performance liquid

chromatography-mass spectrometry (HPLC/MS) (Refs 1–6), and ultraviolet visible
light or fluorescent combined detection. These methods have very high sensitivity and
good selectivity, but the sample pre-processing requirements are high, the operations
are tedious and time-consuming, and the instruments are highly expensive. Accord-
ing to reports, the glassy carbon electrode modified by multi-wall carbon nanotube
possesses high sensitivity and selectivity of MG. The carbon paste electrode has
such advantages as simple preparation method, handy treating process and low price.
However, there are few reports on its usage in the determination of MG.
Due to its peculiar amphipathic structure and long C–H chain, the surfactant can
significantly reduce the surface tension of solution and change the system surface
state. Thus, it finds an increasingly wider application in the electrochemical analy-
sis7–10. The common cationic surfactant of CTMAB has been successfully applied in
the determination of estrogen, ethinyl estradiol and other drugs, and played signifi-
cant sensitising effect. The anionic surfactants of SDBS and SDS have improved the
sensitivity in the determination of palladium, bismuth, terbium and other metal ions.
The non-ionic surfactant of Triton X-100 has obviously increased the dissolution
peak height of lead11–14.
In this experiment, it is aimed to probe into the effects of different types of sur-
factants on MG determination system and screen out a kind of surfactant which can
play a significant sensitising effect. In the aqueous solution, the hydrophobic long
C–H chain of anionic surfactant of sodium dodecyl benzene sulphonate (SDBS) can
be absorbed on the surface of carbon paste electrode and play the role of lead wire
in the electron transfer. On the other hand, the negatively-charged hydrophilic group
extends to the bulk solution and absorbs the positively-charged MG molecule through
the electrostatic induction. As a result, the accumulation content of MG on electrode
surface is increased, and its size of oxidation current density is thus enhanced. When
SDBS has reached its critical micelle concentration, the adsorption quantity of MG
will reach saturation and the peak current increases no longer. Therefore, by basing
upon the low concentration SDBS sensitising effect, it is possible to establish a kind
of sensitive and quick electrochemical method for the determination of MG.
76
EXPERIMENTAL
Instruments. The voltammetric determination was conducted at a 660C CHI electro-
chemical workstation (Shanghai Chen Hua Instrument Company). Three electrode
systems were used: carbon paste electrode (φ 3 mm) serving as working electrode,
platinum electrode as auxiliary electrode, Ag/AgCl electrode as reference electrode.
PB-10 sartorius acidimeter, 78-1 magnetic stirrer (Jincheng Guosheng Laboratory
Apparatus Factory at Jintan City, Jiangsu Province), ultrasonic cleaner, electronic
balance, microsample injector, ultrapure water processor were used in the study.
Reagents. MG standard solution: (Tianjin Fuchen Chemical Reagent Plant M = 364.92)
1.0 × 10–2 mol/l stock solution was prepared by using dehydrated alcohol as solvent and
kept in dark place. The standard solutions with other concentrations were prepared at
the time of application. Surfactant: SDBS, SDS, CTMAB, STAB, Triton X-100, betaine
(prepared into 1.0 × 10–2 mol/l solution). HCl, KCl, NaOH, pH 5–8 PBS (phosphate
buffered saline), pH 3.5–5.5 HAc-NaAc buffer solution and concentrations were all
0.1 mol/l. Reagents are all A.R. (analytical reagent) if not specified. Ultrapure water
was used in the experiment (conductivity of 0.0548 μS/cm).
Electrode preparation. 50 mg graphite powder and 20 l paraffin oil were evenly
mixed and filled into the electrode cavity (φ 3 mm) and smoothly polished on the
tracing paper. Next, cyclic voltammetry was applied in the base solution to conduct
10 cycles of scanning for activation, and put into use after the baseline stability15.
After usage, the carbon paste was scooped out and washed in the ultrasonic cleaner
for 2 min through the application of ethanol and ultrapure water.
Experimental method. The determination medium is 10 ml pH 6.5 PBS containing
2.5 × 10–4 mol/l SDBS. The determination is divided into two steps, i.e. enrichment
and oxidation. First of all, it is required to enable the electrode to undergo opened-
loop enrichment in the to-be-measured solution, enable MG as many as possible to
be absorbed on the electrode surface, and conduct anodised scanning from 0.5 to
1.1 V. As a result, MG is oxidised, a sensitive oxidation peak appears at 0.84 V and
this stripping peak is taken as the analytical signal. The determination technologies
include cyclic voltammetry (CV) and linear sweep voltammetry (LSV).
ELECTROCHEMICAL BEHAVIOUR OF MG IN CARBON PASTE ELECTRODE
After CV was applied to determine the existence of nonexistence of SDBS, the out-
comes of electrochemical behaviour of MG in carbon paste electrode were as shown
in Fig. 2. In pH 6.5 PBS containing 1.0 × 10–5 mol/l MG, cyclic voltammetry scan-
ning was conducted from 0.2 to 1.1 V, and there was no existence of SDBS except
for a weak oxidation peak (curve a) emerging at the first loop of 0.84 V. During the
reverse scan, there was no appearance of reduction peak and the oxidation peak
77
current decreased significantly during the subsequent scanning process, indicating
that the behaviour of MG in carbon paste electrode is totally irreversible. After the
addition of 2.5 × 10–4 mol/l SDBS in the determination system, the oxidation peak
current of MG decreased significantly (curve b) and there were almost no changes in
the peak potential, indicating that surfactant played obvious sensitising effect in the
electrochemical response of MG.
10
0 a
b
–10
ip (μA)
–20 c
–30
–40
–50
0.2 0.4 0.6 0.8 1.0 1.2
E (V)
Fig. 2. Cyclic voltammetry curve of carbon paste electrode: pH 6.5 PBS – a; a + 1.0 × 10–5 mol/l MG
– b; b + 2.5 × 10–4 mol/l SDBS – c; scan speed of 100 mV/s
OPTIMISATION OF EXPERIMENTAL CONDITIONS
Effects of surfactants. Through the application of LSV technology, the effects of

different types of surfactants and concentrations in MG oxidation peak current were
studied and the outcomes were as shown in Fig. 3. The investigated surfactants com-
prise anionic surfactants of sodium dodecyl benzene sulphonate (SDBS) and sodium
dodecyl sulphate (SDS), cationic surfactants of cetyl trimethyl ammonium bromide
(CTMAB), sodium triacetoxyborohyride (STAB), and non-ionic surfactant of Triton
X-100, and zwitterionic surfactant of betaine. Among the rest, all anionic surfactants
can play a certain degree of sensitising effects, and SDBS effect is superior to that
of SDS. Cationic surfactant, zwitterionic surfactant and non-ionic surfactant have no
effects or play the inhibition effects.
As shown in the experimental phenomena, the hydrophobic end of anionic
surfactant is adsorbed on the surface of carbon paste electrode, while the negatively-
charged hydrophilic part is extended into the solution. As the electrostatic induction
can absorb the positively-charged MG molecules in solution, more MG concentrated
on the electrode surface and thus increased the oxidation peak current. Furthermore,
the long C-H chain of surfactant was just like a piece of lead wire that played the
role of electron transport. In addition, the more similarities between alkyl structure
of surfactant and MG molecular structure were, the better the sensitising effect would
become. As SDBS shared the same benzene ring structure as that of MG, thus, the
78
sensitising effect of SDBS was superior to that of SDS. Cationic surfactant, zwitterionic
surfactant and non-ionic surfactant played the role of repelling interaction for MG,
hindered the adsorption of MG on electrode surface, and thus exerted the inhibiting
effect on peak current. As seen in the curve a, when the surfactant concentration in-
creased from 0 to 2.5 × 10–4 mol/l, there was a significant increase in MG oxidation
peak current, and if the surfactant concentration kept on increasing, the changes in
MG oxidation peak current became less and presented a declining trend. When the
surfactant concentration was lower than the critical micelle concentration, the sur-
factant molecules presented the single molecule alignment on electrode surface and
absorbed a large amount of MG molecules. When the critical micelle concentration
was reached, the adsorption quantity of MG would reach saturation on the electrode
surface. If the concentration kept on increasing, the adsorption quantity on the electrode
surface decreased contrarily. As the transfer of MG molecules was blocked by a large
amount of surfactant molecules under micelle state, the peak current declined to some
extents. In this experiment, the SDBS concentration was selected as 2.5 × 10–4 mol/l.
80
60
a
ip (μA)
40
b
20
d
c
0
0 10 20 30 40
cS × 10 (mol/l)
–5
Fig. 3. Effects of types and concentrations of surfactant on 1.0 × 10–5 mol/l MG oxidation peak current
Selection of base solution and pH values. A large number of experimental works were
conducted with respect to the selection of base solution for the determination system.
The researches were focused on the effects of pH 5–8 PBS, pH 3.5–5.5 HAC-NaAC
buffer solution (concentration as 0.1 mol/l) and SDBS with different concentrations
on the determination system. As shown in the experimental result, among the strong
acid HCI, strong base NaOH and neutral KCl medium, the colour of solution changed
into milk white or light green and there was significant shift in the peak potential,
indicating that the existential state of MG has changed. The effect of PBS was supe-
rior to that of HAC-NaAC buffer solution, and the oxidation peak current reached
the maximum at pH 6.5, with sharp spike. If pH was greater than or smaller than 6.5,
all peak currents decreased. For this reason, pH 6.5 PBS was taken as the supporting
electrolyte in the determination of MG.
79
Enrichment time. The effects of enrichment time on MG oxidation peak current were
probed, as shown in Fig. 4. There were significant increases in MG peak current,
along with the enrichment time increasing from 0 to 4 min. Due to the increases in
enrichment time, the adsorption quantity of MG on electrode surface increased, and
thus led to the increase of oxidation peak current. After the enrichment time exceeded
4 min, the adsorption quantity of MG on electrode surface reached saturation and the
increases of peak current tended to slow down. In integrated consideration of sensi-
tivity and easy operation of experiment, the enrichment time was selected as 4 min.
20
15
i p (μA)
10
0
0 2 4 6 8
t (min)
Fig. 4. Effect of enrichment on 5.0 × 10–7 mol/l MG oxidation peak current
Scanning speed. SLV was applied in determination and when SDBS concentration
was 2.5 × 10–4 mol/l, the oxidation peak current of 1.0 × 10–5 mol/l MG with the scan-
ning speed varying from 10 to 300 mV was obtained and the result was as shown in
Fig. 5. It was found that the linear relation presented between oxidation peak current
and scanning speed, complying with the equation of ip = 5.814 + 0.26v, r = 0.9962
(unit of ip is μA). It indicated that the MG oxidation on carbon paste electrode was
controlled by adsorption.
100
80
i p (μA)
60
40
20
0
0 50 100 150 200 250 300
v (mV s )
–1
Fig. 5. Effect of scanning speed on 1.0 × 10–5 mol/l MG oxidation peak current
80
Linear range and detection limit. SLV was applied in the exploration of the relations
between MG oxidation peak current and its concentration. It was found that good linear
relationship presented between oxidation peak current and MG concentration within
the range of 1.0×10–8–2.0×10–6 mol/l and the equation ip = (0.106 + 1.16) × 107C, r =
0.9988 (unit of C is mol/l and unit of ip is μA) was obeyed. It indicated that the MG
oxidation on carbon paste electrode was controlled by adsorption. Detection limit was
8.0 × 10–9 mol/l. For 5.0 × 10–7 mol/l MG, parallel determinations were conducted for
ten times and the relative standard deviation was 5.48%, indicating that this method
had good reproducibility and considerable application prospect.
Interference measurement. The effects of other substances on 5.0 × 10–7 mol/l MG
oxidation peak current were determined, and it was found that most substances did
not produce interferences to MG (Table 1). For example, Ca2+, Fe3+, Al3+, Ni2+, Zn2+,
Mn2+ with 100 times of concentration, as well as ascorbic acid (AA), dopamine and
vitamin E with 30 times of concentration almost produced no interferences to the
current response of 5. 0 × 10–7 mol l–1 MG, and the signal changes were smaller than
10%. It indicated that this new method had good selectivity for MG.
Table 1. Effect of interferences on 5.0 × 10–7 mol/l MG oxidation peak current

Interferences Interference level (mol/l)
Ca , Fe , Al , Ni , Zn , Mn2+
2+ 3+ 3+ 2+ 2+
5.0 × 10–5
Ascorbic acid (AA), dopamine (DA) and vitamin E 1.5 × 10–5
Recovery rate. The standard recovery test was conducted for simulated samples. 3.0 ×
10–7 mol/l MG standard solution was continuously added into 5.0 × 10–7 mol/l MG
simulated sample, and the oxidation current was determined (Table 2). For this sample,
an average recovery rate of 97.9% was obtained, and the test results were satisfactory.
Table 2. Determination of recovery rate through the application of standard addition method
CMG × 107 (mol/l) 5 8 11 14
ip (μA) 13.84 17.00 18.42 19.61
Recovery rate 97.9%
CONCLUSIONS
(1) In this experiment, the effects of different types of surfactants on the determina-
tion of MG were studied; SDBS, a kind of anionic surfactant capable of producing
significant sensitising effect in MG determination, was selected, and thus the analyti-
cal sensitivity was enhanced. SDBS concentration selected for experiment was 2.5 ×
10–4 mol/l.
81
(2) The supporting electrolyte, enrichment time and other experimental param-
eters were optimised. In this experiment, pH 6.5 PBS was selected as the supporting
electrolyte in MG determination, and the enrichment time was selected as 4 min.
(3) As shown in the experiment, the peak current of MG and its concentration
presented good linear relationship within the range of 1.0 × 10–8 – 2.0 × 10–6 mol/l,
and the detection limit after 4 min of enrichment reached 8.0 × 10–9 mol/l. As a result,
a fast and sensitive electrochemical method for MG determination was established.
REFERENCES
1. M. A. AHMADI, S. SHADIZADEH: Experimental and Theoretical Study of a New Plant Derived
Surfactant Adsorption on Quartz Surface: Kinetic and Isotherm Methods. J Disper Sci Technol, 36
(3), 441 (2015).
2. V. S. RAO, B. SRIKANTH, P. S. RAO, C. K. SASTRY, G. N. RAO: Impact of Neutral Surfactant on
Protonation Equilibria of L-aspartic, Citric and Succinic Acids. Oxid Commun, 33 (4), 793 (2010).
3. J. H. ZHU, M. Y. QIN, S. P. LIU, Z. F. LIU, J. D. YANG, X. L. HU: Incorporation of Flow Injection
Analysis with Dual-wavelength Overlapping Resonance Rayleigh Scattering for Rapid Determina-
tion of Malachite Green and Its Metabolite in Fish. Spectrochim Acta A, 130, 90 (2014).
4. K. SIVASHANMUGAN, J. D. LIAO, B. H. LIU, C. K. YAO, S. C. LUO: Ag Nanoclusters on ZnO
Nanodome Array as Hybrid SERS-active Substrate for Trace Detection of Malachite Green. Sensor
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5. B. REPEN, E. SCHNEIDER, U. ALEXIEV: Optimization of a Malachite Green Assay for Detection
of ATP Hydrolysis by Solubilized Membrane Proteins. Anal Biochem, 426 (2), 103 (2012).
6. K. MITROWSKA, A. POSYNIAK, J. ZMUDZKI: Determination of Malachite Green and Leuco-
malachite Green Residues in Water Using Liquid Chromatography with Visible and Fluorescence
Detection and Confirmation by Tandem Mass Spectrometry. J Chromatograph A, 1207 (1–2), 94
(2008).
7. L. L. HUANG, F. LUO, Z. L. CHEN: Green Synthesized Conditions Impacting on the Reactivity
of Fe NPs for the Degradation of Malachite Green. Spectrochim Acta A, 137, 154 (2015).
8. Y. HUANG, X. M. XIAO, P. WU: Surface Activator CTAB Synergism Carbon Paste Electrode
Survyes the Research of Ethinyl Estradiol. Zhejiang Chemical Industry, 38 (10), 22 (2007).
9. A. WITEK-KROWIAK: Analysis of Influence of Process Conditions on Kinetics of Malachite Green
Biosorption onto Beech Sawdust. Chem Eng J, 171 (3), 976 (2011).
10. R. M. UDA, T. NISHIKAWA, Y. MORITA: Disruption of Reverse Micelles and Release of Trapped
Ribonuclease. A Photochemically Induced by Malachite Green Leuconitrile Derivative. J Colloid
Interface Sci, 355 (2), 448 (2011).
11. B. SAMIEY, A. R. TOOSI: Adsorption of Malachite Green on Silica Gel: Effects of NaCl, pH and
2-propanol. J Hazard Mater, 184 (1–3), 739 (2010).
12. X. D. LI, Q. Z. ZHAI, M. Q. ZOU: Optical Properties of (Nanometer MCM-41)–(malachite green)
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13. J. W. CHEN, J. W. MAO, X. R. MO, J. Y. HANG, M. M. YANG: Study of Adsorption Behaviour
of Malachite Green on Polyethylene Glycol Micelles in Cloud Point Extraction Procedure. Colloid
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Received 27 April 2015
Revised 9 July 2015
82
Analytical section
STUDY ON DECAY RESISTANCE OF OIL HEAT-TREATED

WOOD WITH CELLULOSE CONTENTS TESTED BY FOURIER
INFRARED SPECTRUM ANALYSIS
WANG YA-MEI*, FAN HUI-QING, DU RUI

College of Forestry Engineering, Inner Mongolia Agricultural University,
010 018 Huhhot, China
E-mail: wym20209@163.com
ABSTRACT
The wood decay is mainly due to the wood including the nutrients that the wood
decay bacteria needed, such as cellulose, hemi-cellulose and lignin. The proportion
of these nutrients has a direct impact on the wood decay resistance. In this paper,
by using FTIR spectroscopy was analysed the change of the cellulose content in the
wood before and after the oil bath and was studied how the change of fibre content
influenced on the wood decay resistance. The results show that: after dealing by the
long time high temperature oil bath, the cellulose content of poplar declines from
47.31297 to 32.79843%, and the weightlessness rate of poplar also declines from
19.4 to 4.99% after the corrosion test. The cellulose content in Mongolia Seoteh Pine
declines from 44.02291 to 36.65213% and the weightlessness rate declines from 8.21
to 3.14% after the corrosion test, and the effect of the high temperature oil bath on
broadleaf wood decay resistance performance is higher than that of needless wood.
The long-time high temperature oil bath can efficiently change the internal structure
of wood and reduce the nutrients that the wood decay bacteria needed and improve
the decay resistance performance of wood efficiently.
Keywords: FTIR, cellulose content, decay resistance, wood.
AIMS AND BACKGROUND

Wood heat treatment technology is an important wood modification technology, be-
cause this method does not add any chemicals, it is very much in line with environ-
mental requirements, and is an environmentally friendly material1,2. The media of heat
treatment technology include steam, vegetable oil, inert gas, etc. Bourgois, Viitaniemi,
Wang, Boonstra et al.3–5 used steam and vegetable oil as the heat medium to treat
*
For correspondence.
83
wood. The results show that the high temperature can improve, to some degree, the
wood dimensional stability, mechanical properties and durability. Windeisen, Callum,
Tunc, etc.6–8 changing the internal components of the heat-treated material analysed
and found that heat treatment causes the degradation of hemicellulose, cellulose and
lignin to a different degree.
Fourier transform infrared spectroscopy is an important means of a study of the
structure and composition of polymer compounds. Using this equipment to analyse the
shape of the measured spectrum bands, location, intensity, and based on the compound
itself has a structure that can reflect the chemical structure and chemical composition
of the real wood. In this paper, we selected Fourier transform infrared spectroscopy
to determine the cellulose content9 of various oil-heat-treated wood rapidly. Such we
could evaluate corrosion resistance of wood by comparing the cellulose content dif-
ferences among various oil-heat-treated woods. Also, the micro-dimension analysis
of the heat treatment technology for wood interior changes in specific substances can
provide a basis for the study of the mechanism of wood decay.
EXPERIMENTAL
Materials Pinus sylvestris (Mongolia Seoteh Pine), Aspen (Poplar) are purchased
in Hohhot, Inner Mongolia. After the specimens were treated by different oil heat
treatment conditions, specifications of 20 × 20 × 10 mm specimens were prepared
for indoor corrosion resistance testing, another part was crushed over 200 mesh sieve
infrared measurement.
White rot fungi using Coriolus versicolor (Coriolus versiolor), brown-rot fungi
using soft rot fungus Poria (Poria plaoenta) were purchased from China Forestry
Culture Collection Centre.
TEST METHODS
Oil heat treatment. Two species specimens were treated at 180, 200 and 220°C for 2 h
and 4 h, respectively. The specific process design is shown in Table 1 (the thermally
conductive medium was soybean oil, the sample was immersed in oil, the sample with
aluminum foil after the end of the experiment surface stains dry).
Table 1. Heat treatment table

Test number Treating temperature (°C) Treating time (h)
1 180 6
2 220 2
3 220 6
4 (untreated group) – –
84
Corrosion test. Test method was according to GB/T 13942.1-2009 – wood durabil-
ity, white rot fungi and brown rot fungus infection time for four weeks, the average
mass loss rate through the sample test to evaluate the durability of natural materials.
Analysis of the infrared absorption spectrum. Fan Huiqing9 and other researchers have
shown that the use of fast Fourier transform infrared spectroscopy methods for wood
cellulose content determination is feasible. Here, we used Fourier transform infrared
spectroscopy under resolution of 4 and a scanning range from 400 cm–1 to 4000 cm–1
conditions to analyse the cellulose content within wood samples. The testing samples
were prepared by mixing 0.0006 g crushed wood powder, 0.0010 g KSCN and 0.0584 g
KBr in the tablet and pressing under a pressure of 10 MPa for 15 s.
Analysis of decay heat oil material. Different oil-heat-treated Aspen and Pinus sylves-
tris samples were treated by white and brown rot fungi for 4 weeks. Then the average
mass loss rate was calculated (Table 2).
Table 2. Mass loss of heat treated samples after decay for four weeks
No Condition of heat Momgolian seoth pine Poplar (%)
treatment (%)
1 untreated group 8.21 19.4
2 180°C – 6 h 4.25 8.11
3 220°C – 2 h 3.14 7.71
4 220°C – 6 h 3.16 4.99
Fig 1. Infrared spectra of the heat-treated poplar
85
Fig. 2. Cellulose content of heat-treated sample
Figure 2 shows the average cellulose content under different heat treatment
conditions of the treated wood. The standard deviation is up to 4.51, and the relative
standard deviation – 13.75%. It is seen that as the temperature rises, the cellulose
content decreases; at the same temperature, prolonged heat treatment time leads also
to degradation of the fibre content. At 200°C, since cellulose structure is stable, it is
difficult to occur, degradation and its change is smaller.
Fig. 3. Change trends of the mass loss after cellulose content changed
Figure 3 shows the corrosion resistance of treated wood in a line graph and the
test material cellulose content, can be obtained from the figure with the decline in
cellulose content, mass loss test materials also decreased, indicating that the oil bath
after handling test material cellulose content decreased, can improve the corrosion
resistance of wood.
86
Profiling Pinus sylvestris. In Figs 4a, b, c are shown the IR spectra of the Mongolica
sample processed in an oil bath at 220°C for 6 h in, processing for 220°C for 2 h, and
processing at 180°C for 6 h.
Fig. 4. IR spectra of the heat-treated poplar
Figure 5 shows the cellulose content differences of Pinus sylvestris under various
oil heat conditions. The standard deviation among different treatments was up to 5.31,
and the relative standard deviation – up to 14.02%. It implied that the temperature was
conductive to the degradation of cellulose, while time extension to 6 h also contrib-
uted to the degradation of cellulose. Comparing with Fig. 4, however, the cellulose
degradation of Pinus sylvestris was less than poplar with increasing temperature. Also,
the treating time for the cellulose degradation of Pinus sylvestris was lower than that
of poplar at high temperature (220°C).
87
Figure 6 shows that the difference of corrosion resistance under different oil-heat
conditions with the difference of cellulose content. The mass loss rate declined with
the decline in cellulose content. It indicates that the corrosion resistance increases
with the decline in cellulose content.
Fig. 6. Change trends of the mass loss after cellulose content changed
Figure 7 shows the same heat treatment conditions in an oil bath, Scotch Pine
and aspen decay test comparison chart. The corrosion resistance of Pinus sylvestris
under different oil heat treatment conditions was all better than that of poplar. The
mass loss rate of poplar decreased as 74.27%, however, the mass loss rate decline of
Pinus sylvestris was only 61.51% after different oil heat treatments.
88
Fig. 7. Mass loss of different heat-treated samples after decay for four weeks
Figure 8 shows the change of Aspen and Pinus sylvestris cellulose content under
different heat treatment conditions. The figure shows that after heat treatment, poplar
internal degradation of cellulose content was significantly higher than that of Pinus
sylvestris. Figures 7 and 8 show that the effect of oil heat treatment technology results
in improvement of the corrosion resistance of poplar better than that of Pinus sylvestris.
89
CONCLUSIONS
(1) With increasing the temperature of oil heat treatment and/or the extension of treat-
ment time, the corrosion resistance of heat-treated wood was gradually increased with
the gradual degradation of cellulose.
(2) FTIR spectroscopy can be used to analyse quantitatively the content of wood
cellulose. By analysing the IR spectra of heat-treated wood, we could find that cel-
lulose content of heat-treated wood was gradually reduced with the increase of oil
heat treatment temperature and the extension of treatment time.
(3) At high temperatures, prolonged oil heat treatment conditions, the amount of
degradation of cellulose hardwood interior is significantly greater than softwood; then,
the effects of increasing heat temperature and time on improving corrosion resistance
of hardwood were more significant.
ACKNOWLEDGEMENTS
This work was supported by the grant (31160142) from the National Natural Science
Foundation of China and Outstanding Youth Foundation (YXQ2014-13) of Inner
Mongolia Agricultural University.
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Revised 25 May 2015
90
Inhibitors, antioxidants
ANTIOXIDANT ACTIVITY OF DIOSGENIN FROM Dioscorea

composita
SHI GAOFENG*, LI FEIYUN, WANG GUOYING, LEI XINMING,

GUO TAICHUN
School of Petrochemical Engineering, Lanzhou University of Technology,
730 050 Lanzhou, China
E-mail: gaofengshi_lzh@163.com
ABSTRACT
We aimed to infer and determine the antioxidant activity of diosgenin from Dioscorea
composita by employing the established in vitro hydroxyl free radical system. In this
paper, the direct hydrolysis-extraction of diosgenin under biphasic reaction conditions
was applied with a 1.5% yield of diosgenin. The antioxidant activity of diosgenin
was initially determined by the Fenton reaction-methylene blue spectrophotometric
method. Results indicate that the hydroxyl radical scavenging activity of diosgenin
was more than that of L-ascorbic acid when the concentration of the scavenger was
higher than 2.5 × 10–3 mmol/l.
Keywords: diosgenin, antioxidant activity, hydroxyl free radical, hydrolysis-extraction.
AIMS AND BACKGROUND

Free radicals and other reactive oxygen species generated in living organisms are
associated with many diseases, including cancer, cardiovascular diseases, cataracts,
asthma, hepatitis, liver injury and immunodeficiency diseases1–3. Meanwhile, increas-
ing amount of attention has been given to alternative natural compounds with potent
antioxidant activity4–7 because of consumers concern on the safety of synthetic anti-
oxidants. For this reason, the antioxidant activities of phytosterols have been studied
and determined8,9. And diosgenin, (25R)-5-spirosten–3β-ol (Fig. 1), is known to
possess anti-hyperlipidemic10, anti-inflammatory11, anti-tumour12,13 and antioxidant
properties14–16. However, pharmacological properties including antioxidant properties
of diosgenin and its mechanism are not very clear. Given the similar structures of
phytosterols and diosgenin, the antioxidant activity of diosgenin from Dioscorea com-
posita was investigated by using the established in vitro hydroxyl free radical system.
*
For correspondence.
91
O
HO
Fig. 1. Structure of diosgenin
The direct hydrolysis-extraction17,18 of diosgenin from D.composita in a single

step and in a two-phase system containing an aqueous mineral acid and an organic
water immiscible solvent was described. The method was applied in this paper, and
the process, which involves the direct acid hydrolysis of dioscin in the plant material
and in situ extraction of diosgenin, was convenient, time-saving and efficient.
Although many hydroxyl free radical detection methods have been developed,
veracity was affected by many factors whether the direct or the indirect method was
used. For instance, the electron spin resonance method has some disadvantages, such
as high equipment cost, low sensitivity and inaccurate quantitative analysis19. Further-
more, the electrochemical activity and UV absorption of diosgenin are not perfect.
For these reasons, the Fenton reaction-methylene blue spectrophotometric method20
was established for the determination of the antioxidant activities of diosgenin and
L-ascorbic acid.
Traditionally, diosgenin is mainly used as an intermediate raw material for the
synthesis of steroid hormones. Additional research on the antioxidant activities of
diosgenin is needed. In this study, we aimed to investigate diosgenin antioxidant ac-
tivity using an in vitro assay system and to provide insight into the medicinal value
of diosgenin.
EXPERIMENTAL
Materials. D. composita was purchased from Chengdu Must Bio-Technology Co.,
Ltd. (Chengdu, China), and diosgenin and L-ascorbic acid were obtained from U.S.
Sigma company. Methylene blue (MB) and other chemical reagents were obtained
from Tianjin Chemical Reagent Company (Tianjin, China). Diosgenin solution was
prepared in methanol and all other aqueous solutions were prepared in distilled water.
Extraction of diosgenin from D. composite. 1 g of D. composita power was weighed
and the hydrolysis-extraction was performed as follows: 10 ml of petroleum ether
(boiling range 90–120°C), 7 ml of methanol, 2 ml of sulphuric acid (18.4 M) and
1 ml of H2O were added, and reflux was performed at 90°C for 6 h under magnetic
stirring. After cooling, the liquid phases were allowed to separate. Diosgenin was
condensed in petroleum ether and recrystallised thrice by petroleum ether. A portion
of the diosgenin methanol solution was injected to the liquid-chromatographic column.
92
Analysis of D. composita extracts for diosgenin. HPLC analysis was carried out on
a Surveyor (Thermo Finnigan, USA) with a UV detector at the wavelength of 201
nm at 25°C. The Hypersil GOLD C18 column was 150 cm long × 2.1 mm i.d. The
mobile-phase was acetonitrile/water with a flow rate of 1 ml min–1 for 10 min. The
melting point (mp) was measured using the Yanagimoto apparatus (uncorrected).
Determination of hydroxyl radical scavenging activity of D. composita extracts for
diosgenin. The Fenton reaction-methylene blue spectrophotometric method was ap-
plied as follows: a suspension of 2.0 ml 0.05 mM MB solution, a certain amount of
diosgenin (> 95%, assay by HPLC), 4.0 ml (pH 2) 76 mM KCl-HCl buffer, 1.6 ml
1 mM FeSO4 solution, 0.8 ml 3% (v/v) H2O2 and distilled water were added to 10 ml
colourimetric tube. The reaction was incubated at 25°C for 30 min in a water bath. The
reaction mixtures without antioxidant and without antioxidant and H2O2 were used as
comparison mixture and as blank reagent, respectively. The absorbance value of the
reaction system was measured at the wavelength of 664 nm against the blank reagent
by UV-2001 spectrophotometer. The percentage of the hydroxyl radical scavenging
activity (HRSA) was calculated using the following formula:
% HRSA = [(Ascavenger – Acomparison)/(Ablank – Acomparison)] × 100
where Ascavenger is the absorbance value of the reaction system after adding the scav-
enger; Acomparison – the absorbance value of the reaction system without the scavenger;
Ablank – the absorbance value of the reaction system without the scavenger and H2O2.

Extraction of diosgenin from D. composite. The direct hydrolysis-extraction was
essentially a ‘one-pot’ process that yielded 1.5% diosgenin. After recrystallisation
by petroleum ether, 95.5% pure diosgenin was obtained by liquid chromatography
determination. The pure diosgenin, (25R)-5-spirosten-3β-ol, white solid had mp:
204–207°C, [α]D20 – 124.7° (c = 0.55; chloroform).
Antioxidant properties of diosgenin. Hydroxyl radical is an extremely reactive free radi-
cal formed in biological systems and has the capacity to join nucleotides in DNA and
cause strand breakage, which leads to carcinogenesis, mutagenesis and cytotoxicity.
The hydroxyl radical scavenging activity was estimated by using MB as the indicator
of the spectral measure study. MB can capture the HO• produced by Fenton reaction,
thereby resulting in a change of colour from blue to achromatic.
Fenton reaction-methylene blue spectrophotometry is simple, sensitive and
needs no expensive instruments. First, the calibration curve of the MB concentration
was established at the wavelength of 664 nm by the UV-2001 Spectrophotometer
(Fig. 2). The linear relationship between the absorbance value of the system and the
concentration of MB was good when the MB concentration ranged from 0 to 2.5
93
× 10–2 mM. Then, the optimum conditions for the Fenton reaction-methylene blue
spectrophotometric method were investigated. The effect of the Fe2+ concentration on
the absorbance value of the reaction system was studied, and the results are shown in
Fig. 3. ΔA (ΔA = Ablank – Acomparison) slightly changed when the Fe2+ concentration was
more than 1.6 × 10–1 Mm (Fig. 3). Thus, 1.6 × 10–1 Mm Fe2+ was used in subsequent
experiments, and the effect of the H2O2 concentration was also investigated. The re-
sults in Fig. 4 show that ΔA decreased with decreasing concentrations of H2O2 from
0.15% (v/v) to 0.75% (v/v) (during the decomposition of H2O2). Thus, the optimum
concentration of H2O2 for the reaction was 0.15% (v/v). Furthermore, the concentration
of MB and the reaction time were crucial. The results suggest that ΔA was maximum
when the concentration of MB was 1.0 × 10–2 mM (Fig. 5) (although ΔA decreased
with increasing concentrations of MB from 1.0 × 10–2 to 1.8 × 10–2 mM because of the
certain amount of hydroxyl radical, ΔA slightly changed when the MB concentration
varied from 1.5 × 10–2 to 1.8 × 10–2 mM) and when the reaction time was 30 min.
The absorbance value of the reaction system remained unchanged when the measur-
ing time was less than 1 h. Under optimum conditions, the antioxidant activities of
diosgenin and L-ascorbic acid were compared. The results in Fig. 6 showed a certain
relationship between the scavenging effects of the hydroxyl free radical and the con-
centration of scavengers. Especially, the %HRSA for diosgenin increased when the
scavenger concentration increased from 0.5 × 10–3 to 5 × 10–3 mM. However, when
the concentration was increased to 1 × 10–2 mM or larger, the %HRSA for diosgenin
slightly changed due to the limited antioxidant activity of diosgenin. And besides, the
%HRSA for diosgenin was not higher than that of L-ascorbic acid when the scaven-
ger concentration was lower than 2.5 × 10–3 mM, but the %HRSA for diosgenin was
higher than that of L-ascorbic acid when the scavenger concentration was higher than
2.5 × 10–3 mM. The %HRSA for diosgenin was 94.1% when its concentration was 5
× 10–3 mM. Although the reason for the difference of %HRSA is still being explored,
the investigation revealed that diosgenin has good hydroxyl scavenging activity and
can be effective when used as a hydroxyl radical scavenger.
94
1.4
1.2
1.0
absorbance
0.8
0.6
0.4
0.2
0.0
0 5 10 15 20 25 30
concentration of MB (10–3 mmol/l)
Fig. 2. Calibration curve of MB

Reaction conditions: 0, 0.5, 1, 2, 3, 4, 5 ml 0.05 mM Methylene blue was respectively added to 10 ml
colorimetric tube, diluted to the mark with distilled water; temperature 25°C
0.55
0.50
0.45
0.40
ΔA
0.35
0.30
0.25
60 80 100 120 140 160 180 200 220 240 260
concentration of Fe (10 mmol/l)

2+ –3
Fig. 3. Effect of Fe2+ concentration in testing solution

Reaction conditions: 0.05 mM Methylene blue, 2 ml; 76 mM KCl-HCl buffer (pH = 2), 4 ml; 3% (v/v)
H2O2, 0.8 ml; 1 mM FeSO4 of various volumes; temperature, 25 °C; reaction time 30 min; blank reaction
conditions: 3% (v/v) H2O2, 0 ml; other reaction conditions were as above
95
0.54
0.52
0.50
0.48
ΔA 0.46
0.44
0.42
0.40
0.38
0.36
0.15 0.3 0.45 0.6 0.75
H2O2 (%)
Fig. 4. Effect of H2O2 conentration in testing solution

Reaction conditions: 0.05 mM Methylene blue, 2 ml; 76 mM KCl-HCl buffer (pH = 2), 4 ml; 1 mM
FeSO4, 1.6 ml; 3% (v/v) H2O2 of various volumes; temperature, 25 °C; reaction time, 30 min; blank
reaction conditions: 3% (v/v) H2O2, 0 ml; other reaction conditions were as above
0.6
0.5
0.4
0.3
ΔA
0.2
0.1
0.0
0 2 4 6 8 10 12 14 16 18 20
concentration of MB (10–3 mmol/l)
Fig. 5. Effect of MB concentration in testing solution

Reaction conditions: 76 mM KCl-HCl buffer (pH = 2), 4 ml; 1 mM FeSO4, 1.6 ml; 3% (v/v) H2O2, 0.8 ml;
0.05 mM Methylene blue of various volumes; temperature, 25°C; reaction time 30 min; blank reaction
conditions: 3% (v/v) H2O2, 0 ml; other reaction conditions were as above
96
96.10 1
hydroxyl radical scavenging activity (%) 96.00
94.10 2
81.10
38.60
0 2 4 6 8 10 12 14 16 18 20 22 24
concentration of scavengers (10–3 mmol/l)
Fig. 6. Hydroxyl scavanging activity of diosgenin and L-ascorbic acid

1 – scavenger reaction conditions: 0.05 mM Methylene blue, 2 ml; 76 mM KCl–HCl buffer (pH = 2),
4 ml; 1 mM FeSO4, 1.6 ml; 3% (v/v) H2O2, 0.8 ml; 0.5 mM diosgenin solution of methanol of various
volumes; temperature, 25°C; reaction time, 30 min. Comparison reaction conditions: 0.5 mM diosgenin
methanol solution, 0 ml; other reaction conditions were as above. Blank reaction conditions: 0.5 mM
diosgenin solution of methanol, 0 ml; 3% (v/v) H2O2, 0 ml; other reaction conditions were as above;
2 – reaction conditions: 0.05 mM Methylene blue, 2 ml; 76 mM KCl–HCl buffer (pH = 2), 4 ml; 1 mM
FeSO4, 1.6 ml; 3% (v/v) H2O2, 0.8 ml; 0.5 mM L-ascorbic acid of various volumes; temperature, 25°C;
reaction time, 30 min. Comparison reaction conditions: 0.5 mM L-ascorbic acid, 0 ml; other reaction
conditions were as above. Blank reaction conditions: 0.5 mM L-ascorbic acid, 0 ml; 3% (v/v) H2O6,
0 ml; other reaction conditions were as above
CONCLUSIONS
Diosgenin from D. composita demonstrated superior hydroxyl radical scavenging
activity. The antioxidant activity of diosgenin was not lower than that of L-ascorbic
acid when the concentration of the scavenger was more than 2.5 × 10–3 mM. Further
study of diosgenin biological effects may provide further information on the medicinal
value of diosgenin.
ACKNOWLEDGEMENTS
The financial support of the National Natural Science Foundation of China (No
21407072) and the Natural Science Foundation of Gansu Province (No 145RJZA109)
is gratefully acknowledged.
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Anti-tumour and Anti-oxidative Potential of Diosgenin against 7,12-Dimethylbenz(a)anthracene
Induced Experimental Oral Carcinogenesis. Pathol Oncol Res, 18 (2), 405 (2012).
14. L. L. ROMERO-HRENANDEZ, P. MERINO-MONTIEL, S. MONTIRL-SMITH, S. MEZA-
REYES, J. LUIS VEGA-BAZE, I. ABASOLO Jr., S. SCHWARTS, O. LOPEZ, J. G. FERNANDEZ-
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98
STUDIES ON ULTRASOUND COMBINED WITH SEMI-BIONIC

EXTRACTION TECHNOLOGY AND ANTIOXIDANT ACTIVITY
OF FLAVONOIDS FROM Mollugo pentaphylla
KEYUE LIUa*, HAIJUN LIUb, YUN LINGa, JIALING QIUa

a
College of Life Sciences, Jiujiang University, 551 Qianjindong Road, Jiujiang,
Jiangxi Province, China
E-mail: liukeyue2011@yeah.net
b
Basic Medical College, Jiujiang University, Jiujiang, China
ABSTRACT
The aim of the research was to explore the optimum condition of ultrasound combined
with semi-bionic extraction of Mollugo pentaphylla flavonoids and investigate the
antioxidant activity of flavonoids. Ultrasound combined with semi-bionic method was
used to extract Mollugo pentaphylla flavonoids. Orthogonal test was applied to find out
the optimum conditions for the following factors: temperature, ethanol concentration,
extraction time and solid-liquid ratio. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method
was used to investigate the antioxidant activity. Orthogonal test results suggested
that the optimum conditions for the extraction of Mollugo pentaphylla flavonoids by
ultrasound combined with semi-bionic method were: temperature 60oC, ethanol con-
centration of 80%, extraction time of 20 min, and solid-liquid ratio of 1:16. Antioxidant
activity experiment results demonstrated that the scavenging rate of DPPH by 12.6
µg/ml Mollugo pentaphylla flavonoids was 94.1%, and the scavenging capacity was
increasing as flavonoids concentration increased within tested range. As a convenient
and economical method, the technology of ultrasound combined with semi-bionic
could be applied to the extraction of Mollugo pentaphylla flavonoids. The antioxidant
activity study demonstrated Mollugo pentaphylla flavonoids with antioxidant capac-
ity, and the antioxidant activity increased as the flavonoids concentration increased.
Keywords: Mollugo pentaphylla L., flavonoids, semi-bionic, antioxidant activity.
AIMS AND BACKGROUND

Mollugo pentaphylla belongs to Aizoaceae family, with whole plant used as medicine.
It is mainly distributed in Jiangxi Province and most parts of southern China, serving
as a traditional herbal medicine in Southeast Asian countries. Commonly known as
*
For correspondence.
99
‘detoxification grass’, it can relieve severe arrhythmia induced by aconite poison-
ing. Modern pharmacological studies have shown that Mollugo pentaphylla extract
has pharmacological effects of dilating coronary arteries and is antihypertensive. It
has also been found that Mollugo pentaphylla extract can be used against aconitine-
induced arrhythmias effectively1. It is reported that Mollugo pentaphylla contains
flavonoids, saponins, terpenes, sterols and alkaloids, among which flavonoids are
abundant. Numerous studies showed that flavonoids demonstrated significant ef-
fects on the diseases that might be caused by oxidation, such as anti-cardiovascular
diseases, anti-cancer, anti-inflammatory and anti-ageing. Therefore, we used Mol-
lugo pentaphylla flavonoids as the main object to explore the ultrasound combined
with semi-bonic optimum technology to extract Mollugo pentaphylla flavonoids and
performed antioxidant activity study on the flavonoids extracted at optimum condi-
tions. Our study provided foundation for the exploration of the antioxidant activity of
Mollugo pentaphylla and the pharmacodynamic material basis treating the diseases
probably caused by oxidation.
EXPERIMENTAL
Materials and equipments. Mollugo pentaphylla was collected from Lushan area
and it was identified as aizoaceae plant Mollugo pentaphylla by Qu Weihong from
Jiujiang University. Rutin was from Sinopharm Chemical Reagent Co.; DPPH was
from Tokyo Chemical Industry Co., Ltd.; sodium hydroxide (AR), 95% ethanol (AR),
monosodium phosphate (AR), and disodium phosphate (AR) were all from Tianjin
Damao Chemical Reagent Factory. Distilled water was from our lab (DZG-303, Aike
Co., Ltd.). Ultrasonic cleaner (SB25-12DTD) was from Ningbo Scientz Biotechnol-
ogy Co., Ltd.; vacuum (AP-9908S) was from Tianjin Aote Saiensi Instrument Co.,
Ltd.; UV-vis. spectrophotometer (722N) was from Shanghai Jinghua Technological
Instrument Co., Ltd.; and electronic analytical balance (BT-224S) was from Sartorius
Scientific Instruments (Beijing) Co., Ltd.
Plot of standard curve. A series of 0.206 mg/ml rutin standard solutions of 1.00, 2.00,
3.00, 4.00, and 5.00 ml were transferred into 10-ml stoppered tubes, respectively. 0.3
ml 5% NaNO2 were added into each tube and stood still for 6 min after shaken well.
Then 0.3 ml 10% Al(NO3)3 were added, shaken well, and stood still for another 6 min.
Further 4 ml 10% NaOH were added and finally 60% ethanol was added to make the
volume exact at 10 ml. After shaken well, the tubes were kept still for 10 min. Finally
the absorbance values were measured at 510 nm. The standard curve was plot with
absorbance as vertical axis and rutin concentrations as horizontal axis2.
Single-factor test. Four factors were investigated in our study, i.e. temperature, solid-
liquid ratio, time, and ethanol concentration3. The suggested optimum conditions were
as follows: ethanol concentration 70%, solid-liquid ratio 1:16, temperature 60oC, and
time 25 min, with power 200 W (Refs 4 and 5).
100
Single factor test – temperature. A sample of 2.0 g Mollugo pentaphylla was added to
32 ml 60% ethanol and the pH was adjusted to 2.0, 7.5, respectively with disodium
phosphate-citric acid buffer. Sonication was performed twice under the condition
of a power of 200 W, a sonication time of 25 min and a temperature of 30, 40, 50,
60, 70, and 80oC, respectively, and the filtrates were combined. The absorbance was
measured to determine the yield.
Single factor test – solid-liquid ratio. A sample of 2.0 g Mollugo pentaphylla L. was
added to 16, 24, 32, 40, 48, 56, and 64 ml 60% ethanol, respectively, and the pH was
adjusted to 2.0, 7.5, respectively, with hydrochloric acid-sodium hydroxide solutions.
Sonication was performed twice under the condition of a power of 200 W, a sonica-
tion time of 25 min and a temperature of 60oC, and the filtrates were combined. The
absorbance was measured to determine the yield.
Single factor test – time. A sample of 2.0 g Mollugo pentaphylla was added to 32 ml
60% ethanol, and the pH was adjusted to 2.0, 7.5, respectively, with hydrochloric
acid – sodium hydroxide solutions. Sonication was performed twice under the condi-
tion of a power of 200 W, a temperature of 60oC and a sonication time of 10, 15, 20,
25, 30, and 35 min, respectively, and the filtrates were combined. The absorbance
was measured to determine the yield.
Single factor test – ethanol concentration. A sample of 2.0 g Mollugo pentaphylla
was added to 32 ml 40, 50, 60, 70, 80 and 90% ethanol, respectively, and the pH was
adjusted to 2.0, 7.5, respectively with hydrochloric acid – sodium hydroxide solu-
tions. Sonication was performed twice under the condition of a power of 200 W, a
temperature of 60oC and a sonication time of 25 min, and the filtrates were combined.
The absorbance was measured to determine the yield.
Orthogonal test. The results from single-factor test demonstrated that there was cer-
tain influence of each of the four factors, temperature, solid-liquid ratio, time, and
ethanol concentration. Therefore, based on the single-factor test, the orthogonal test
L9 (34) was performed to further investigate the interactions among them according
to flavonoids yield. Detailed data are shown in Table 1.
Table 1. Factors and levels in orthogonal array design

Levels Factors
A B C D
extraction tem- ethanol concen- extraction time solid-liquid ratio
perature (oC) tration (%) (min)
1 50 60 20 1:12
2 60 70 25 1:16
3 70 80 30 1:20
101
Antioxidant activity study 6,7. Preparation of sample: a sample of 0.5, 0.7, 1.0, 1.5, 2.0,
3.0, and 4.0 ml extract, respectively, was added to 80% ethanol until a final volume of
25 ml. A sample of 2 ml of the above solutions was added to 2 ml DPPH respectively
and reacted for 30 min in dark at room temperature after shaken well. The absorbance
was measured at 517 nm.
Preparation of interference: the above samples were added to 2 ml 80% ethanol
instead of 2 ml DPPH. The reaction was in dark at room temperature for 30 min after
shaken well. The absorbance was measured at 517 nm.
Preparation of control: a volume of 2 ml 80% ethanol was added to 2 ml DPPH
and reacted for 30 min in dark at room temperature after shaken well. The absorbance
was measured at 517 nm.
Preparation of blank: 4 ml 80% ethanol. DPPH scavenging rate (%) was calcu-
lated for the formulae below:
DPPH scavenging rate (%) = (Acontrol－(Asample－Ainterference))/Acontrol
where Acontrol is the absorbance of control, Asample – the absorbance of sample, Ainterference – the
absorbance of control group.
RESULT AND DISCUSSION

Plot of rutin standard curve. The standard curve was plotted with absorbance (ABS)
as vertical axis and rutin concentrations as horizontal axis.
0.9
0.8 y = 0.008x + 0.0242
0.7
0.6
0.5
ABS
0.4
0.3
0.2
0.1
0
0 20 40 60 80 100 120
concentration of rutin (μg/ml)
Fig. 1. Rutin standard curve
The linear equation is:

y = 0.0080x + 0.0242 r = 0.9991
where y is the ABS value (the absorbance value), x – the concentration of rutin (μg/ml),
r – the correlation coefficient.
Single-factor test results. As shown in Fig. 2, under the conditions of ethanol concen-
tration 70%, solid-liquid ratio 1:16, time 25 min, and power 200 W, the influence of
102
different temperatures on yield demonstrated that the yield was increasing between
temperature 50 and 70oC and decreasing above 70oC. Therefore, 50, 60 and 70oC were
chosen as temperature range for further optimisation.
yield of flavonoids (mg/g) 18

16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90
temperature (°C)
Fig. 2. Effect of extraction temperature on flavonoids yield
As shown in Fig. 3, under the conditions of ethanol concentration 70%, solid-

liquid ratio 1:16, temperature 60oC, and power 200 W, the influence of different time
on yield demonstrated that the yield was increasing between time 10–25 min and
decreasing when time beyond 25 min. Therefore, 20, 25 and 30 min were chosen as
time range for further optimisation.
6
yield of flavonoids (mg/g)
5
4
3
2
1
0
0 5 10 15 20 25 30 35 40
extration time (min)
Fig. 3. Effect of extraction time on flavonoids yield
As shown in Fig. 4, under the conditions of solid-liquid ratio of 1:16, temperature

60oC, time 25 min, and power 200 W, the influence of different ethanol concentration
on yield demonstrated that the yield was increasing between ethanol concentrations
40–70% and decreasing when above 70%. Therefore, 60, 70 and 80% were chosen
as the range of ethanol concentration for further optimisation.
103
6

5
4
3
2
1
0
0 10 20 30 40 50 60 70 80 90 100
concentration of enthanol (%)
Fig. 4. Effect of ethanol concentration on flavonoids yield
As shown in Fig. 5, under the conditions of ethanol concentration 70%, tem-

perature 60oC, time 25 min, and power 200 W, the influence of different solid-liquid
ratio on yield demonstrated that the yield was increasing between solid-liquid ratios
1:12–1:16 and decreasing when below 1:16. Therefore, 1:12, 1:16 and 1:20 were
chosen as solid-liquid ratio range for further optimisation.
6
4
3
2
1
0
0 1:10 1:20 1:30 1:40 1:50 1:60
solid-liquid ratio
Fig. 5. Effect of solid-liquid ratio on flavonoids yield
Orthogonal test results analysis. As shown in Table 2, the optimum conditions within
experimental range were: temperature 60℃, ethanol concentration 80%, time 20 min,
solid-liquid ratio 1:16, with a yield of 15.8 mg/g. The influence order of the factors
was: ethanol concentration > temperature > solid-liquid ratio > time.
104
Table 2. Orthogonal array design matrix and experimental results
No Factors and levels Yield of
A B C D flavonoids
extraction ethanol concen- extraction solid-liquid (mg/g)
temperature tration time ratio
1 1 1 1 1 7.8
2 1 2 2 2 8.4
3 1 3 3 3 11.0
4 2 1 2 3 9.4
5 2 2 3 1 9.9
6 2 3 1 2 15.8
7 3 1 3 2 8.8
8 3 2 1 3 9.3
9 3 3 2 1 10.4
K1 9.067 8.667 10.967 9.367
K2 11.700 9.200 9.400 11.000
K3 9.500 12.400 9.900 9.900
R 2.633 3.733 1.567 1.633
As shown in Table 3, for the factors tested and within the range for each fac-
tor, the influence order on the yield of Mollugo pentaphylla flavonoids was: ethanol
concentration > temperature > solid-liquid ratio > time.
Table 3. Analysis of variance of experimental results

Factors Deviation square Degree of F F0.05
sum freedom
Extraction temperature 11.962 2 3.113 19.000
Ethanol concentration 24.462 2 6.367 19.000
Extraction time 3.842 2 1.000 19.000
Solid-liquid ratio 4.162 2 1.083 19.000
Deviation 3.840 2
Antioxidant activity study. As shown in Fig. 6, the scavenging rate increased as fla-
vonoids increased within certain range. During the flavonoids concentration range
of 0.725–4.1 µg/ml, the increasing of scavenging rate was obvious, whereas within
the range of 8.1–12.6 µg/ml, the increasing was slow. The scavenging rate achieved
maximum value of 94.1% at the flavonoids concentration of 12.6 µg/ml.
105
100
DPPH scavening rate (%)

80
60
40
20
0
0 2 4 6 8 10 12 14
concentration of flavonoids (µg/ml)
Fig. 6. DPPH scavenging rate of flavonoids of Mollugo pentaphylla L.
DISCUSSION
Currently the methods for the extraction of flavonoids compounds include alkali
extraction and acid precipitation, organic solvent extraction, semi-bionic extraction,
ultrasonic extraction, and supercritical fluid extraction. Any single extraction method
abovementioned has some shortcomings, such as long extraction time, destroyed
active ingredient, high impurities in extract, and low yield, etc. Combining several
methods together would be a natural thought to improve the extraction efficiency.
Semi-bionic extraction simulates oral administration and the transport of drug through
the gastrointestinal tract, thus it is a novel extraction method for the design of Chinese
medicines administered through the digestive tract2. Ultrasonic extraction method ac-
celerates the dissolution of effective medicinal ingredients in a solvent, and does not
change the structure of the active ingredient; besides it also reduces extraction time
to improve yield at the same time3. Therefore, we used the combination method of
ultrasound and semi-bionic to extract Mollugo pentaphylla L. flavonoids with high
yield and short time.
Studies have shown that cardiovascular diseases, inflammation and cancer are
all associated with oxidation. Therefore, in our study we investigated the antioxi-
dant activity of Mollugo pentaphylla flavonoids. Using DPPH method, our results
have shown that flavonoids have antioxidant capacity, and the antioxidant capacity
increased as the flavonoids concentration increased within a certain concentration
range. Considering the factors and the range for each factor studied, the scavenging
rate maximised with a value of 94.1% at a flavonoids concentration of 12.6 µg/ml.
CONCLUSIONS
Optimum conditions for the extraction of Mollugo pentaphylla flavonoids by ultra-
sound combined with semi-bionic method were: temperature 60oC, ethanol concen-
tration of 80%, extraction time of 20 min, and solid-liquid ratio of 1:16. Antioxidant
106
activity experiment results demonstrated that the scavenging rate of DPPH by 12.6
µg/ml Mollugo pentaphylla flavonoids was 94.1%, and the scavenging capacity in-
creased as flavonoids concentration increased within tested range. As a convenient
and economical method, the technology of ultrasound combined with semi-bionic
could be applied to the extraction of Mollugo pentaphylla flavonoids. The antioxidant
activity study demonstrated Mollugo pentaphylla flavonoids with antioxidant capac-
ity, and the antioxidant activity increased as the flavonoids concentration increased.
The above results provided both theoretical and experimental foundation for further
development of Mollugo pentaphylla flavonoids.
ACKNOWLEDGEMENTS
This project received support from National Natural Science Foundation of China
(No 81260606), Natural Science Foundation of Jiangxi Province of China (No 2012
2BAB205074), Educational Department of Jiangxi Province of China (No GJJ14740)
and Health Department of Jiangxi Province of China (No 2010A148), Science and
Technology Support Program of Jiangxi Province of China (No 20141BBG70084).
REFERENCES
1. K. Y. LIU: The Research Progress of the Plant Resources, Ingredients and the Pharmacological
Effects of Mollugo pentaphylla L. Lishizhen. Medicine and Materia Medica Research, 20 (2), 397
(2009).
2. X. X. HUANG, H. L. WEI, Y. F. PAN: Ultrasound Combined with Semi-bionic Extraction of Lobelia
Flavonoids and the Anti-oxidative Activity Study. Sci Technol Food Industry, 3 (3), 140 (2013).
3. X. X. HUANG, H. F. LAI: Ultrasound-assisted Semi-bionic Extraction of Polyphenols from Emilia
sonchifolia. Guangdong Agricultural Sciences, 6 (4), 110 (2013).
4. X. M. SUN, Z. W. ZHANG: The Establishment of the Semi-bionic Extraction Technology Platform
in Traditional Chinese Medicine. Chinese Med, 28 (4), 61 (2006).
5. F. JIANG, Y. Y. ZHAO, M. L. JIANG, L. XIU: Application of Power Ultrasonic Technique to Ex-
traction of Traditional Chinese Medicine．Chem Ind Eng Progress, 26 (7), 944 (2007).
6. N. GOUGOULIAS: Comparative Studies on Polyphenols Profile and Antioxidative Activity of
Some Berry Fruits. Oxid Commun, 37 (3), 713 (2014).
7. N. GOUGOULIAS, N. MASHEV: Antioxidant Activity and Polyphenols Content of Some Herbal
Teas of Lamiaceae Family from Greece and Bulgaria. Oxid Commun, 38 (1), 25 (2015).
107
ALPHA-AMYLASE INHIBITORY ACTIVITY AND BLOOD

GLUCOSE AND LIPID-LOWERING POTENTIAL OF
Heliotropium strigosum
A. MAHMOODa*, R. A. SARFRAZa,b, I. A. BHATTIA, F. HUSSAINc

a
Department of Chemistry, Faculty of Sciences, University of Agriculture,
Faisalabad, Pakistan
E-mail: ayesha_ph.d244@yahoo.com
b
Central High-tech laboratory, University of Agriculture, Faisalabad, Pakistan
c
Department of Biochemistry, Faculty of Sciences, University of Agriculture,
ABSTRACT
Heliotropium strigosum, a traditional medicinal plant was investigated to determine
antioxidant activity, phenolic profile and in vitro inhibitory potential against enzyme
responsible for hyperglycemia. Aqueous extract showed potential hypoglycemic ef-
fect via alpha-amylase inhibition (54.56%) than ethanolic extract (44.11%). Efficacy
study was conducted, providing aqueous and ethanolic extracts along with basal diet
for a period of 21 days to alloxan induced diabetic rabbits. Both extracts containing
active components caused significant reduction in total cholesterol, triglycerides and
low density lipoprotein level indicating their potential against hypercholesterolemic
perspectives and allied disorders. HPLC analysis confirmed higher concentration of
phenolic acids in aqueous extract than ethanolic extract. Treatment with the glibencla-
mide, aqueous and ethanolic extracts of Heliotropium strigosum significantly reduced
the blood glucose levels of diabetic rabbits with mean values of 131.6 ± 12.9, 109.4 ±
14.1 and 107 ± 6.9 mg/dl, respectively, at the end of treatment. We found that aqueous
extract is 7.42% more effective in lowering blood glucose level than glibenclamide.
From the present investigation, it is deduced that Heliotropium strigosum is effective
against hypercholesterolemia, hyperglycemia and allied disorders.
Keywords: Heliotropium strigosum, glibenclamide, hypercholesterolemia and hyper-
glycemia.
*
For correspondence.
108
AIMS AND BACKGROUND
Diabetes mellitus (DM) is an endocrine syndrome caused by an absolute or relative
deficit of insulin and/or reduced insulin proficiency that results in hyperglycemia and
fluctuations in carbohydrate, fat and protein metabolic rate. The severity of diabetes
mellitus has ever been increasing. Main causes of problem are aging, urbanisation
and increasing privilege leading to corpulence, physical inactivity and depression or
mental stress1.
Plants have been playing a key role in the cure of diabetes, particularly in de-
veloping nations where majority of people belongs to poor background and cannot
afford modern and expensive management of this syndrome. There has been observed
growing interest in developed world to adopt herbal medications as alternative rem-
edies to cure diabetes because of side effects related to consumption of insulin and
oral antidiabetic medicines2.
One of the tactics to treat postprandial hyperglycemia in diabetic patients is
to avert absorption of sugar after meal intake. Oligosaccharides, disaccharides and
composite starches should be converted into discrete units before getting absorbed in
the duodenum and upper jejunum. Abdominal distention, flatulence, meteorism and
diarrhea are side effects of synthetic α-amylase inhibitors (acarbose and miglitol).
Previous studies have shown that such effects are probably produced by the extreme
inhibition of pancreatic α-amylase causing irregular bacterial fermentation of undi-
gested sugars in the colon. α-amylase enzyme from plants have been revealed to show
lower inhibitory potential against α-amylase effect and so can be recommended as
operative remedy for postprandial hyperglycaemia with negligible lateral effects4.
Experiments carried out on rabbits revealed the facts that serum triglycerides,
total cholesterol and low density lipoproteins levels increased in rabbits after the
induction of diabetes mellitus5. Plant rich in polyphenols have been shown to lower
blood sugar levels6,7. Polyphenolic present in plant food have been testified to act
as insulin in glucose utilisation, inhibitors of α-amylase and α-glucosidase enzymes
linked to non-insulin dependent diabetes mellitus and lipid peroxidation in tissues8.
Heliotropium strigosum is an important medicinal plant of family Boraginaceae.
Traditionally, this plant was widely used as diuretic and laxative. The juice extract
of this plant helps to treat sore eyes and gum boils, in addition to cure for stings of
nettles, snake and insects bite9. This present study was designed to investigate the
alpha-amylase inhibitory potential of Heliotropium strigosum coupled with its anti-
oxidant properties. Antihyperglycemic and antihyperlipidemic potential of aqueous
and ethanolic extracts of Heliotropium strigosum were also evaluated in alloxan
induced diabetic rabbits.
EXPERIMENTAL
Sample preparation. Extraction was carried out on a magnetic stirrer using distilled
water and ethanol10.
109
DPPH radical scavenging activity assay. Antioxidant activity was measured by per-
forming DPPH radical scavenging assay11.
Determination of total phenolic content. The total phenolic content was determined
using the Folin–Ciocalteu reagent with analytical grade gallic acid as standard12.
1.4
y = 0.0057x + 0.0856
1.2 R² = 0.9836
1
absorbance
0.8
0.6
0.4
0.2
0
0 50 100 150 200 250
concentration (µg/ml)
Fig. 1. Standard curve of gallic acid for total phenolic contents determination in plant sample
Alpha-amylase inhibition assay. In vitro antihyperglycemic activity was assessed with

the help of alpha amylase inhibition assay4.
EVALUATION OF ANTIHYPERGLYCEMIC AND ANTIHYPERLIPIDEMIC ACTIVITIES

OF PLANT EXTRACT
Animals. Adult rabbits of either sex, weighing 1–1.5 kg were used for experiments.
Animals were kept in individual cages located in the animal house of Department of
Clinical Medicine and Surgery, Faculty of Veterinary Sciences, University of Agri-
culture, Faisalabad. They were kept at room temperature of 25–35°C and at 45–55%
relative humidity for 12 h, each of dark and light cycle. They were fed with a standard
diet and water ad libitum.
Induction of diabetes. Rabbits were made diabetic by a single intravenously (marginal
ear vein) injection of alloxan monohydrate (Sigma Chemicals, USA) 5% (w/v) in
normal saline at a dose of 120 mg/kg, body weight. Then 5 days later, rabbits with
blood glucose level > 250 mg/dl were considered to be diabetic and were used in the
experiment13.
Treatment. The rabbits were divided into groups containing healthy and diabetic
animals. Group I served as normal control (healthy rabbits, n = 3). The diabetic rab-
bits were divided into four groups (II–V) of three animals each. Group II served as
diabetic control. I and II groups remain untreated. Group III received glibenclamide
as reference drug (5 mg/kg, p.o.) suspended in water (10 ml/kg) for 21 days. The
aqueous and ethanolic extracts of Heliotropium strigosum, suspended in water were
administered at the dose of 100 mg/kg orally in a volume of 10 ml/kg for 21 days to
110
the animals of groups IV and V, respectively. Blood samples were collected from the
marginal ear vein at 1st, 8th, 15th and 22nd day of treatment.
Biochemical analysis. The plasma glucose level, plasma total cholesterol, triglyceride
and HDLs were evaluated by enzymatic test kits (Sigma Co.). The LDLs level was
calculated by using the formula: LDL = total cholesterol – HDL – (triglyceride/5).
High performance liquid chromatography. For HPLC analysis sample preparation was
done according to the method described earlier14. The separation of plant samples on
gradient HPLC was performed using shim-pack CLC-ODS (C18), 25 cm × 4.6 mm,
5 µm column. The chromatographic separation was carried out using a mobile phase
gradient: A (H2O:acetic acid 94:6, pH 6), B (acetonitrile 100%) 0–15 min = 15% B,
15–30 min = 45% B, 30–45 min = 100% B with 1 ml/min flow rate using UV-vis.
detector at 280 nm wavelength at room temperature. The identification of each com-
pound was established by comparing the retention time and UV-vis. spectra of the
peaks with those previously obtained by the injection of standards. The quantification
was performed by external calibration with standards.
Statistical analysis. Three samples of each plant material were assayed. Each sample
was analysed individually in triplicate for its total phenolic contents, antioxidant and
in vitro antidiabetic activities. Data was reported as mean ± standard deviation. Data
were analysed by analysis of variance (ANOVA) using Minitab 2000 version 13.2
statistical software (Minitab Inc. Pennysalvania, USA) at 5% significance level15.

% Yield. The percentage yield of aqueous extract was found to be higher (21.32%)
while it was 7.88% for ethanolic extract (Table 1). In another study, only 1% yield was
obtained by macerating coarse powder in methanol for 15 days9. Variation in our and
previous findings might be attributed to difference of solvent and extraction technique.
Antioxidant activity and total phenolic contents. Antioxidant activity based on DPPH
radical inhibition assay varied from 42.39 ± 0.04 to 71.43 ± 0.1% in ethanolic and aque-
ous extract, respectively (Table 1). Three flavonoids, naringenin, 3-O-methylgalangin
and 7-O-methyleriodictiol have been isolated from the plant, Heliotropium taltalense
and flavonoids isolated exhibited antioxidant activity which suggests that H. strigosum
may possess flavonoids responsible for antioxidant activity16.
Table 1. Yield, DPPH scavenging activity and TPC of plant extracts

Plant extracts Yield DPPH TPC (mg GAE /1g
(%) (%) of plant extract)
Heliotropium strigosum (aq.) 21.32 71.43±0.1 52.4±0.09
Heliotropium strigosum (eth.) 7.88 42.39±0.04 21.02±0.37
aq. – aqueous extract, eth. – ethanolic extract, TPC – total phenolic contents, GAE – gallic acid equivalent.
111
Total phenolic contents of Heliotropium strigosum ethanolic and aqueous extracts
were determined using standard curve of gallic acid (Fig. 1). TPC of ethanolic and
aqueous extracts were found to be 21.02 ± 0.37 and 52.4 ± 0.09 mg GAE/g plant extract
(Table 1). In another study17 TPC of Heliotropium indicum methanolic and aqueous
extracts and contents were found to be 6.01 ± 0.23 and 4.34 ± 0.37 mg GAE/g plant
extract17. Variations in the both findings could be due to difference in polarity of the
solvents, geographical location of the plant, its genotype, technological measures,
year and so on18.
Effect of solvents on α-amylase inhibition activity (%) of plant extracts. In an array to
explore the in vitro anti-diabetic activity, various concentrations (1, 10, 100 and 1000
ppm) of aqueous and ethanolic extracts of Heliotropium strigosum were screened for
the α-amylase inhibition property (Fig. 2). Initial screening of Heliotropium strigosum
extracts showed that the aqueous extract had significantly higher α-amylase inhibition
activity at all observed concentrations than that of ethanolic extract. It is clear from
Fig. 2 that acarbose, aqueous and ethanolic extracts showed maximum inhibition 52.47,
54.56 and 44.11% and at 100 ppm. The ability of Heliotropium strigosum aqueous
extract to inhibit enzyme might be due to higher phenolic contents.
Fig. 2. α-Amylase inhibitory activities of acarbose, aqueous and ethanolic Heliotropium strigosum
extracts (n = 3)
High performance liquid chromatography. Table 2 shows the presence of phenolic

compounds in both extracts of Heliotropium strigosum. Higher amounts of phenolic
acid and flavonoids were found in aqueous extract than ethanolic extract. Aqueous
solvents resulted in the extraction of more phenolic glycosides, which are more water
soluble19. Phenolic acids and flavonoids identified in these extracts have been proven as
potent antioxidant agents in plants. The antidiabetic activity of Heliotropium strigosum
aqueous extract to inhibit enzyme may be attributed to phenolic acids.
112
Table 2. Phenolic compounds (mg/g) of Heliotropium strigosum extracts
Phenolic compound Aqueous extract Ethanolic extract
Quercetin 0.16 8.17
Gallic acid 3.78 3.72
Caffeic acid 11.40 1.71
Chlorogenic acid 9.69 n.d.
m-coumeric acid 5.73 0.57
Trans-4-hydroxy-3-methoxy cinnamic acid 20.20 n.d.
Syringic acid n.d. 2.31
Vanillic acid n.d. 2.31
Sinaptic acid n.d. 3.74
n.d. – not detected.
Effect of Heliotropium strigosum on blood glucose level (mg dl–1) in alloxan-induced

diabetic rabbits. Treatment of diabetic rabbits with the glibenclamide (5 mg/kg body
weight (b.w.)), aqueous (100 mg/kg b.w.) and ethanolic (100 mg/kg b.w.) extracts
of Heliotropium strigosum caused significant (p < 0.05) reduction in blood glucose
levels of diabetic rabbits at the end of treatment. As it is evident from Fig. 3 that plant
extracts are significantly effective than the standard drug, glibenclamide. Glibencla-
mide is long acting sulphonylurea drug, which augments pancreas in the secretion of
insulin20. Continued administration of glibenclamide, aqueous and ethanolic extracts of
Heliotropium strigosum to diabetic rabbits normalised blood glucose levels. Treatment
with aqueous extract at a dose of 100 mg/kg b.w caused 64.18% reduction in blood
glucose level at the end of treatment period whereas it was 50.39 and 56.76% with
ethanolic extract (100 mg/kg b.w.) and glibenclamide (5 mg/kg b.w.), respectively.
Phytochemical investigation of Heliotropium strigosum revealed the presence
of flavonoids and other phenolic acids (Table 2). Most of these phenolics have been
reported as active antidiabetic compounds. Hypoglycemic effect of these natural
compounds might be attributed to their separate or synergistic actions21. It is well es-
tablished that tannins and polyphenols have antidiabetic effects7,22. As it is clear from
HPLC profile that both plant extracts are rich in phenolic acids. Heliotropium strigo-
sum extracts exhibited an antidiabetic effect because of the phenolic acids present.
113
350
300
blood glucose level (mg/dl)

250 0 day
200 7 day
14 day
150
21 day
100
50
0
NC DC GT HS 1 HS 2
groups
Fig. 3. Effect of Heliotropium strigosum on blood glucose level in alloxan-induced diabetic rabbits over
a period of 21 days
Normal control – NC, diabetic control – DC, glibenclamide treated – GT, Heliotropium strigosum aque-
ous exract (HS1) and Heliotropium strigosum ethanolic exract (HS2) treated groups. Each value shown
is mean ± S.E.M. (n = 3), means in the same group not sharing a common superscript letter (a, b, c) are
significantly different (P < 0.05)
Effect of Heliotropium strigosum on cholesterol, triglyceride, LDLs and HDLs level

(mg dl–1) in alloxan-induced diabetic rabbits. Levels of cholesterol, triglyceride and
low density lipoprotein increased significantly in diabetic rabbits, whereas HDL values
showed a reduction. Heliotropium strigosum treatment displayed a reduction in cho-
lesterol, triglyceride and LDL values and increase in HDL level in the diabetic rabbits.
The lipid profile of normal, diabetic and treated diabetic rabbits is shown in Table 3.
Table 3. Effect of Heliotropium strigosum on plasma cholesterol, triglyceride, LDLs and HDLs level
(mg dl–1) in alloxan-induced diabetic rabbits
Treated Total cholesterol Triglyceride LDLs HDLs
groups (mg/dl) (mg/dl) (mg/dl) (mg/dl)
NC 50±2.64b 71±8.9c 14.3±0.71c 21.5±0.51a
DC 80.3±1.52a 173.3±10.69a 30.44±1.52a 15.2±0.7b
GT 51.5±0.57b 78.8±15.8bc 13.24±0.66c 22.5±1.55a
HS1 54.6±1.56b 72±11c 20.7±1.03b 22.5±2.29a
HS2 51.3±3.51b 107.3±11.15b 7.84±0.39d 22±2.21a
Effect of Heliotropium strigosum on lipid level in alloxan induced diabetic rabbits over a period of 21
days; normal control – NC, diabetic control – DC, glibenclamide treated – GT, Heliotropium strigosum
aqueous exract (HS1) and Heliotropium strigosum ethanolic exract (HS2) treated groups; a, b, c, d values
with different superscript letters in the same column indicate significant difference among the groups
(p < 0.05); number of rabbits in each group = 3; values are expressed as mean ± SD.
There are reports on artificial hypercholesterolemia and hypertriglyceridemia

in rabbits after the induction of diabetes by alloxan5,23 and a similar increase was
witnessed in our study. Changes in the level of serum lipids in control and diabetic
114
treated rabbits are illustrated in Table 3. Total cholesterol, LDL and triacylglycerol
increased and HDL decreased significantly in alloxan induced diabetic rabbits (p <
0.05) in comparison to normal rabbits. Mobilisation of free fatty acids from the pe-
ripheral fat depots caused abnormal high concentration of serum lipids in the diabetic
rabbits24. High levels of total cholesterol and LDL cholesterol are major risk factors
for cardiovascular diseases, whereas increased HDL cholesterol is associated with a
decrease in cardiovascular disease risk. However, most drugs used in the management
of hypercholesterolemia decrease both total cholesterol and HDL-cholesterol. Reduc-
tion in HDL level is observed in NIDDM and that eventually causes atheromatous
disease. It has been reported that elevation of triglycerides in diabetes might be at-
tributed to deficiency of lipoprotein lipase activity25. It is clear from Table 3 that oral
administration of aqueous and ethanolic leaves extracts of Heliotropium strigosum
exhibited hypotriglyceridemic and hypocholesterolemic effects while at the same time
increase in HDL was noticed which might be attributed to the hydrolysis of certain
lipoproteins and selective uptake and metabolism by different tissues.
CONCLUSIONS
This study provided useful information about phytochemical contents, antioxidant
and alpha-amylase inhibitory activity of Heliotropium strigosum. Aqueous extract
exhibited higher contents of phytochemicals and α-amylase inhibitory activity. Pre-
sent research work also reported antihyperglycemic and antihyperlipidemic effects of
Heliotropium strigosum in alloxan induced diabetic rabbits, a finding never mentioned
earlier in literature. The utilisation of this indigenous plant as potential source of
natural antioxidant and antidiabetic agent is encouraged. Moreover, the isolation and
characterisation of antidiabetic compounds from Heliotropium strigosum, is highly
suggested for more effective drug development.
ACKNOWLEDGEMENTS
The authors thankfully acknowledge the financial support extended by Higher Edu-
cation Commission (HEC) of Pakistan and technical support provided by Central
Hi-Tech laboratory staff and Department of Clinical Medicine and Surgery, Faculty
of Veterinary Sciences, University of Agriculture, Faisalabad, Pakistan to carry out
this work.
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117
USE OF PRINCIPAL COMPONENT AND HIERARCHICAL

CLUSTER ANALYSIS TO CHARACTERISE STRAWBERRIES
LI LIa, BIN LIa, QI ZHANGa, LIYAN GONGb, XIANJUN MENGa*

a
Department of Food Science, Shenyang Agricultural University,
110 866 Shenyang, China
E-mail: mengxjsy@126.com
b
College of Grain Science and Technology, Shenyang Normal University,
110 034 Shenyang, China
ABSTRACT
This study was designed to investigate the differences in physical and chemical
properties between five strawberry varieties by using pattern recognition tools, to
provide a theoretical basis for quality variation among samples. The properties
measured were fruit weight, firmness, vitamin C content, total anthocyanin content,
total phenols content, pH, crude protein, water, L value, a*, b*, total sugar/total acid,
and total soluble solid/total acid. The data were subjected to principal component
analysis (PCA), and the results were used to classify each strawberry variety on the
basis of quality. The first principle component (PC1) explained 51.017% of the total
variance in the data set, PC2 explained 18.136%, and PC3 explained 15.853%. The
accumulative variance contribution of the first three main compositions extracted by
principal component analysis was 85.006%. Thirteen properties could be reduced to
three main components, and the five strawberry cultivars were distinguished based
on PC scores. Hierarchical cluster analysis classified samples into two main groups
on the basis of the measured parameters; the results derived using this approach cor-
related well with the PC score chart. The results of this study provide guidance for
the evaluation of strawberry quality.
Keywords: strawberry, physicochemical properties, principal component analysis,
hierarchical cluster analysis.
AIMS AND BACKGROUND

Strawberry (Fragaria ananassa D u c h.) is one of the most commonly consumed ber-
ries in the world, largely due to its appealing taste and attractive surface colour. Among
the fruit characteristics that consumers prioritise are that they should be of high quality,
*
For correspondence.
118
have a typical taste and texture, be free of contaminants, and contain nutritional and
health promoting substances; indeed, in addition to the external appearance, the internal
quality of fruit is becoming increasingly important. The strawberry has a varied and
well-balanced composition. It is rich in micronutrients and phytochemical compounds,
particularly anthocyanins, vitamin C, sugar, acid, and phenolic compounds. The levels
of these phytochemicals strongly influence sensorial-organoleptic attributes and nu-
tritional value1–9. Sugar is the main soluble component of ripe strawberry fruit, with
glucose, fructose, and sucrose accounting for almost 99% of total sugar content10,11.
Sugars and organic acids play a key role in taste perception of strawberries. In recent
years, in addition to taste and quality attributes, the selection of strawberry cultivars
had focused on their content of health-promoting compounds, as these might play a
significant role in the prevention of chronic diseases6. The level of anthocyanins is an
important indicator of strawberry maturity, as they provide the red colour. Obtaining
a red colour over half or three-fourths of the fruit yields an index of maturity that
signals harvesting is appropriate. Strawberry leaves contain high amounts of diverse
phenolic compounds that potentially possess defensive activities against microbial
pathogens and other beneficial properties with regard to human health12.
To describe the qualitative characteristics comprehensively, chemical and physical
analyses are needed. The chemical composition of strawberries varies significantly
with genotype13. Statistical processing of such a large amount of heterogeneous
chemical data according to classical methods provides a rich source of information
on every single variable12. However, this approach does not provide a global knowl-
edge on the relationships between these different chemicals, nor does it allow the
grouping of samples with homogeneous characteristics. Therefore, reducing the data
to a few discrete elements may facilitate the extraction of trends of some particular
phenomenon. Evaluation of production variability requires a large amount of data to
be collected, and the results often are hard to analyse. In such situations, multivari-
ate statistical methods are often employed which allow for variable reduction and
facilitate the presentation of results in a clear, graphical manner14. One such example
is principal component analysis (PCA), which permits the identification of the most
important directions of variability in a multivariate data matrix and allows the results
to be shown in a graphical plot.
PCA is a mathematical tool that reduces the dimensionality of data and allows the
visualisation of underlying structures in experimental data and relationships between
data and samples. Standardisation is an essential first step in order to ensure each
parameter contributes equally to data set variance, and that each parameter carries
equal weight in principal component calculation; this can eliminate the influence of
different magnitudes of the analyte values. PCA has been applied to the characteri-
sation of textural properties of selected ready-to-eat meat products15. Keenan et al.
used multivariate analyses of physicochemical properties to select apple cultivars for
use in ready-to-eat desserts16. However, the PCA and hierarchical clustering analysis
(HCA) methods are less commonly used for evaluating the quality of strawberries.
119
The present study was designed to use chemometrics tools to analyse differences
in fruit quality based on the physical and chemical properties of several strawberry
varieties. Fruit weight, firmness, vitamin C, total anthocyanin content, total phenols
content, pH, crude protein, water, L value, a*, b*, total sugar/total acid, and total
soluble solid/total acid were all considered. The results of this approach provide a
solid theoretical basis than can be applied during the evaluation of strawberry quality
and eventual selection for culture, harvesting, and consumption.
EXPERIMENTAL
Strawberry samples. Five strawberry cultivars were harvested at commercial ripeness
(red ripe) (Table 1) in two regions (Liaoning and Shenyang) in Liaoning Province,
China. Ripe fruits of strawberry varieties were harvested from an experimental site
in China. All strawberry plants were cultivated in the open field, which coincided
with the commercial harvesting period for the five varieties. All fruits were harvested
at full mature stage (completely red); strawberries were picked for each cultivar at
physiological maturity and stored at 4°C. The fruit samples were removed from cold
storage and held at room temperature before measurement, and some strawberries
were selected randomly to assess each cultivar characteristics.
Table 1. Sampling date and growing sites of the strawberry cultivars

Number Cultivar Sampling date Growing sites
1 Akihime May 15, 2014 Liaoyang
2 All-star May 16, 2014 Shenyang
3 99 May 17, 2014 Shenyang
4 R7 June 10, 2014 Liaoyang
5 Ever bearers June 15, 2014 Shenyang
Total soluble solids (TSS). The soluble solids content was obtained by measuring the
refractive index of the strawberry juice using a digital hand-held refractometer (Haibin
equipment Co., Ltd, Shenzhen, China). A drop of the juice was placed on the lens
and the reading was taken in degree Brix (Bx). This reading gives the % of soluble
solids content (% SSC) in the fruit. Calibration was made with deionised water and
the lens was carefully rinsed between samples16.
Fruit weight. Twenty fruits were selected randomly to measure fruit weight. Mean
fruit weight was estimated from triplicate measurements on an analytical balance
(accuracy ± 0.01 g).
Firmness. Firmness was measured as the puncture force (g) (Ref. 17). Puncture was
made with a 5-mm diameter probe in the equator of strawberries using a FT-001Texture
Analyser. Two determinations on opposite paired sides of each strawberry were carried
120
out. The speed of penetration was set at 5 mm/s, and the test was stopped after penetra-
tion to 10 mm depth. The mean puncture force was used as an indicator of firmness.
Vitamin C. Aliquots of 1 g of the lyophilised samples were weighed into Erlenmeyer
flasks, and 25 ml of meta-phosphoric acid (1.5%, w/v; pH 3.5) were added. The
suspension was homogenised for 20 s using an Ultra-Turrax T18 (IKA, Germany)
blender and centrifuged at 1600 rpm (573 × g) for 5 min. The supernatant was sub-
jected to ascorbic acid analyses using a commercial enzyme test kit (R-Biopharm,
Darmstadt, Germany).
Total sugar. The anthrone colourimetric method was adopted to determine the content
of carbohydrate total sugar; mean values were derived from triplicate measurements.
Colour. Twenty-five fruits were sampled from each treatment, and two readings were
taken on opposite sides of each fruit for the surface and flesh colour18. The readings
were always performed in the middle of the fruit, both externally and internally. The
CIE Lab colour was evaluated by a CIE 108 observer using an X-Rite 968 reflection
spectrophotometer (range 400–770 nm, spectral sampling interval 20 nm), a D65 il-
luminant, and a ceramic white calibration standard (Part No 968-62; L* = 94.69, a* =
−1.19, b* = 1.42). L* (lightness), a* (greenness [–] to redness [+]) and b* (blueness
[–] to yellowness [+]) were measured.
Total anthocyanin content (TAC). The total anthocyanin content in the fruits was
determined as described previously14,15. Anthocyanins were extracted from 5 g of
strawberries with methanol acidified with 0.1 M HCl. The berries were ground with
quartz sand and extracted with 20 ml volumes of solvent until the sample became
colourless. The extract was diluted with acidified methanol, and its absorption was
measured using a TU1810 UV/vis. spectrophotometer (PERSEE Scientific Equipment,
Beijing, China) at 535 nm. The concentration of anthocyanins was determined from a
calibration curve and expressed in mg of cyanidin-3-glucoside in 100 g of berries (f.w.).
Total phenols content (TPC). Samples for the TPC assay were obtained by extracting
5 g of homogenised strawberries with 50 ml of methanol at ambient temperature for
1 h with constant shaking. The solution was filtered, and the residue was repeatedly
extracted with 50 ml of methanol for 1 h. Finally, the extracts were combined and
diluted to 500 ml with methanol3. The TPC in berry extracts was determined using
the Folin–Ciocalteu reagent according to the method of Slinkard and Singleton us-
ing gallic acid as a reference19. The reagent was prepared by diluting a stock solution
(Sigma-Aldrich Chemie, Shanghai, China) with distilled water (1/10, v/v). The samples
(1.0 ml, three replicates) were introduced into the test cuvettes and mixed with 5.0
ml Folin–Ciocalteu phenol reagent and 4.0 ml Na2CO3 (7.5%). The absorbance was
measured at 765 nm in a TU1810 UV/vis. spectrophotometer (PERSEE Scientific
Equipment, China) after incubation at ambient temperature for 1 h. The TPC was
determined from the calibration curve and expressed in mg of gallic acid equivalents
in 100 g of berries (f.w.).
121
pH and titratable acids (TA). The pH of the purée was measured with a calibrated
pH meter. The pH was determined at 20°C with a pH meter (Bricem Science and
Technology Co., Ltd, Beijing, China). For determination of TA, samples (30 g) were
further homogenised for 45 s using an Ultra-Turrax T18 homogeniser (IKA, Germany),
and centrifuged at 12 000 rpm for 10 min at 4°C by a centrifuge 3-16KL (Sigma,
Germany). The supernatant (5 ml) was diluted 1:10 with distilled water followed by
titration to pH 8.1 with 0.1 M NaOH. The content of TA was calculated as citric acid
(mg/100 g f.w.).
Crude protein (CP). According to the Association of Official Analytical Chemists
(AOAC, Ref. 4), samples were weighed and transferred into the Kjeldahl digestion
flask containing 5 g of catalyst (96% K2SO4/4% CuSO4.5H2O) and 20 ml of concen-
trated H2SO4. After 2 h of digestion in a unit with electrical heat and fume removal
and cooling to room temperature, 40 ml of distilled water, 80 ml of 40% NaOH and
0.25 g zinc (Zn) were added to each flask. By distillation, ammonium hydroxide was
trapped as ammonium borate in a 4% boric acid solution (4 g of boric acid in 100 ml
deionised water (w/v)). Total nitrogen was determined by titration with standardised
HCl to a mixed indicator endpoint (0.1 g/100 ml bromocresol green and 0.1 g/100
ml methyl red in 95% ethanol).
Water. Water content was determined according to AOAC regulations4.
DATA ANALYSIS
All measurements were performed three times. Pattern recognition methods were
applied to the data collection as previously reported6; these included PCA as an unsu-
pervised classification method and HCA as an unsupervised learning method. PCA and
HCA were performed using SPSS19.0 software package. PCA transforms the original,
measured variables into new uncorrelated variables called principal components7, 8,13.
The first principal component covers as much of the variation in the data as possible,
whereas the second principal component is orthogonal to the first and covers as much
of the remaining variation as possible, and so on. HCA calculates the distances (or
correlation) between all samples using a defined metric such as Euclidean distance
and Manhattan distance7. HCA is the most common approach wherein clusters are
formed sequentially. The most similar objects are first grouped, and these initial groups
are merged according to their similarities. Eventually, as the similarity decreases, all
subgroups are fused into a single cluster. PCA has previously been used to characterise
quince fruit10. PCA and HCA have also been used to classify fruits, vegetables, and
spices based on their in vitro antioxidant activities17.

Physical and chemical characteristics of five strawberry varieties. The composi-
tion of five strawberry cultivars is presented in Table 2. The coefficient of variance
122
(CV) was used to weight each variable. The CVs of 13 properties ranged from 1.31
to 48.67%. The TPC, pH, CP, water and TSS/TA showed little variation among the
different strawberry varieties (≤10%), with respective CVs of 9.43, 2.59, 4.48, 1.30,
and 9.81%. Strikingly, however, variation in other parameters, including fruit weight,
and a* and b* was higher than 30%. The properties of fruit are affected by cultivar,
growing region, climate, cultivar practices, and harvest maturity20. In this study, all
samples had the same cultivation, management, and picking conditions; thus, all dif-
ferences were entirely due to genetic diversity.
In order to determine the variables that are most suitable for the evaluation of
fruit quality, we performed correlation analysis using pooled data. Table 3 shows
the correlation coefficients between the physical and chemical variables for the five
samples. Most mechanical variables were significantly correlated (R = 0.98, P < 0.01)
with fruit weight and crude protein. Additionally, a higher fruit weight was positively
correlated with the amount of crude protein. Meanwhile, crude protein and L value
were also significantly positively correlated (R = 0.96, P < 0.01). The firmness and L
value were negatively correlated (R = −0.93, P < 0.05); this variable was also signifi-
cantly negatively correlated with TS/TA. These results show that firmness influences
the colour and taste of strawberries. Total anthocyanin and L value were negatively
correlated (R = −0.95, P < 0.05). Specifically, a higher total anthocyanin content was
correlated with a darker fruit colour. This variable was also significantly negatively
correlated with fruit weight. The results of the correlation analysis indicate that crude
protein, total anthocyanin content, and fruit firmness are the main variables that are
associated with nutritional properties of strawberries.
Table 2. Descriptive statistics of strawberry physicochemical characteristics

Indicator Mean Coefficient of varia- Standard deviation
tion (%)
Weight (g) 11.48 30.49 3.50
Firmness (g/cm2) 78.16 22.98 17.96
VC (mg/100 g) 62.45 11.10 6.93
TAC (mg/100 g) 117.77 19.70 23.19
TPC (mg/100 g) 182.93 9.43 17.26
pH 3.48 2.59 0.09
CP (%) 0.67 4.48 0.03
Water (%) 90.55 1.30 1.18
L value, L* 49.67 11.44 5.68
a value, a* 21.53 30.14 6.49
b value, b* –15.93 48.65 7.75
TS/TA 10.55 18.48 1.95
TSS/TA 13.86 9.81 1.36
123
124
Table 3. Correlation coefficients between the strawberry quality variablesa
W F VC TAC TPC PH CP WC L a* b* TS/TA TSS/TA
W 1
F –0.85 1
VC –0.83 0.45 1
TAC –0.92* 0.78 0.66 1
TPC –0.37 0.03 0.76 0.06 1
pH 0.77 –0.76 –0.46 –0.85 0.20 1
CP 0.98** –0.92* –0.74 –0.87 –0.34 0.72 1
WC 0.27 0.06 –0.28 –0.55 0.08 0.26 0.14 1
L 0.95* –0.93* –0.64 –0.95* –0.12 0.83 0.96** 0.30 1
a* –0.59 0.41 0.54 0.63 0.15 –0.85 –0.46 –0.16 –0.51 1
b* –0.09 0.07 –0.23 0.05 –0.39 0.45 –0.05 –0.12 –0.04 –0.84 1
TS/TA 0.81 –0.96* –0.43 –0.72 0.02 0.84 0.86 –0.17 0.86 –0.59 –0.20 1
TSS/TA 0.35 –0.60 –0.17 –0.07 –0.36 –0.03 0.52 –0.61 0.36 0.27 –0.44 0.52 1
*significant at P < 0.05; **significant at P < 0.01; a total anthocyanin content (TAC); total phenols content (TPC); crude protein (CP); total sugar (TS); total
soluble solids (TSS); titratable acids (TA).
Principal component analysis (PCA). PCA was applied to the standardised values
of analysed parameters of the five strawberry cultivars. In order to define the most
appropriate descriptors of quality of materials, by the cross-validation technique it
was established that three principal components are significant for explanation of
total variability of physicochemical properties measures. The results of calculations
are given in Tables 4 and 5.
PC1 explained 51.017% of the total variance in the data set, PC2 explained
18.136%, and PC3 explained 15.853%. The accumulative variance contribution of
these three main components extracted by PCA was 85.006%, In other words, 85.006%
of the total variance in the 13 considered variables can be condensed into three new
variables (PCs).
Screen plots can be used to determine the optimum number of principal com-
ponents. In the screen plot, the steepest part of the eigenvalue slope corresponds to
the number of principal components. Figure 1 shows the screen plot of evaluation
indexes upon PCA of samples; this reveals that the first three principal components
yielded a greater coefficient of variation while attachment is relatively steep. The first
three principal components (λ > 1) are shown in Table 4, it was the most appropriate
extraction for 3 principal components. The accumulative variance contribution by
PCA was 85.006%, and thus the analysis accounted for most of the information that
explains strawberry quality.
Table 4. Results from the principal component analysis for the first three principal components
Component Eigenvalues % of variance Cumulative %
1 6.632 51.017 51.017
2 2.358 18.136 69.152
3 2.061 15.853 85.006
6
eigenvalue
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
component number
Fig. 1. Screen plot of evaluation indices upon PCA of samples
The component score coefficient matrix of the 3 main principle components are
shown in Table 5, and the loading plots for different properties, in Fig. 2. The com-
125
ponent score matrix (Table 5) shows that the most important variables for the first
PC were eating characteristics (with the exception of L value), fruit weight, firmness,
pH, crude protein, and TS/TA. In fact, in the loading plot (Fig. 2) these variables are
located far from the origin of the first PC. In particular, the eating characteristics placed
to the right in the loading plot are close together and, therefore, positively correlated.
PC2 mainly accounted for colour (a* and b*) in which the ‘a’ value indicates the
position between red and green and ‘b’ indicates the position between yellow and blue.
These two variables, which are located on the left of the loading plot, are positively
correlated. Finally, it can be seen that VC and total phenols are important for PC3,
which reflects the nutritional information that they contain.
Fig. 2. Principal component analysis (PCA) plots describing fruit properties

a – PCA loading plots for different properties on PC1 and PC2; b – PCA loading plots for different
properties on PC1 and PC3; fruit weight is referred to as weight, vitamin C as VC, total sugar as TS,
total anthocyanin content as TAC, total phenols as TPC, total soluble solid as TSS, titratable acid as TA,
crude protein as CP, and water as W
126
Table 5. Component score coefficient matrix of PCA
Properties Component
PC1 PC2 PC3
Fruit weight 0.90 0.12 –0.41
Firmness –0.97 0.03 0.01
Vitamin C –0.50 –0.24 0.82
Total anthocyanin –0.90 –0.11 0.14
Total phenols 0.01 0.07 0.99
pH 0.83 0.51 0.13
Crude protein 0.94 –0.02 –0.35
Water content 0.17 –0.07 –0.05
L 0.98 0.01 –0.15
a* –0.48 –0.86 0.09
b* –0.05 0.99 –0.04
TS/TA 0.92 0.23 0.03
TSS/TA 0.43 –0.46 –0.24
The relationships in the PCA score plot can be used as variety and quality indica-
tors . From the sample score plots of PC1 versus PC2 and PC1 versus PC3 (Figs
5,18,21
3a,b), a number of conclusions can be drawn. First, cultivar 1 has a higher score on
PC1 and is located in the first interval, indicating that it has higher fruit weight, TS/
TA and L value, and lower firmness. Cultivar 2 is located on the left side of PC1 in
the second interval, indicating that it has lower fruit weight, TS/TA and L value, and
higher firmness. Cultivars 4 and 5 are located in the third interval, indicating that they
have lower fruit weight, TS/TA and L value, and higher firmness. In the fourth inter-
val, cultivar 3 had higher weight, TS/TA and L value, and lower firmness. Cultivars
2, 4 and 5 had higher acidity, firmness, and darker colour. From the sensory tasting
standpoint, these features are not suitable for fresh, maybe these varieties suitable
for processing. The greater the strawberry firmness is more suitable for storage and
transportation. Cultivar 1 and 3 had higher weight, TS/TA and L value, and lower
firmness. This indicates that cultivar 1 and 3 fruits with good taste and appearance
were from the freshest varieties.
127
a
Fig. 3. PCA plots describing strawberry cultivars

a – PCA loading plots for different strawberry cultivars on PC1 and PC2; b – PCA loading plots for dif-
ferent strawberry cultivars on PC1 and PC3 Akihime, cultivar 1; All-star, cultivar 2; cultivar 99, cultivar
3; R7, cultivar 4; Ever bearers, cultivar 5
Hierarchical cluster analysis (HCA). A hierarchical agglomerative procedure was

employed to establish clusters. Samples were grouped on the basis of similarities in
hierarchical cluster analysis, without taking into account the information about the
class membership21. The results obtained following HCA are shown as a dendrogram
(Fig. 4) in which two well-defined clusters are visible. Samples are grouped in clusters
in terms of their nearness or similarity. Cluster analysis (CA) uses less information
(distances only) than PCA. The first group of samples (A) is clearly discernible and is
composed of the cultivar All-star, R7 and Ever bearers. These species are associated
with high firmness and lower pH, TS/TA, and fruit weight. This is in agreement with
the results of the PCA. The second cluster (B) consists of the cultivars Akihime and
99 because of the higher TS/TA, weight, L value, and lower firmness. The tendencies
to form natural sample groupings arising from common analytical characteristics are
128
clear in this data analysis procedure. Our clustering data thus have similarities with
those of Patras et al.21, who also successfully used cluster analysis to classify fruits
and vegetables based on in vitro antioxidant activity.
sample cluster
Fig. 4. Dendrogram of hierarchical cluster analysis

A: 1 – cultivar Akihime; 2 – cultivar All-star; 3 – cultivar 99
B: 4 – cultivar R7; 5 – cultivar Ever bearers
CONCLUSIONS
Considerable variations were observed between different strawberry cultivars in terms
of physical and chemical properties. The unsupervised pattern recognition techniques
of PCA enabled visualisation of this complex dataset and unmasked underlying rela-
tionships responsible for clustering observed. Thirteen properties were reduced to three
main components. Five strawberry cultivars clustered significantly on PC1, PC2, and
PC3. The combination of characterisation and multivariate data analysis facilitated
the inference of similarities and differences between strawberry cultivars based on
their physical and chemical properties. This data analysis technique provides powerful
insights into the variations of properties between different strawberry cultivars, and
thus could be applied to the selection of optimum hybrid strawberry cultivars tailored
for different purposes in strawberry breeding programs. We suggest that approaches
such as this could be integrated into commercial agricultural programs in order to
maximise freshness, harvest timing, and appeal to consumers.
ACKNOWLEDGEMENTS
This project was funded by the scientific and technological projects of national public
sectors of China (grant No 2012BAD31B05-3).
129
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131
EVALUATION OF THE ANTIOXIDANT EFFICIENCY OF

BERRY EXTRACTS IN SUNFLOWER OIL
S. OANCEAa*, O. DRAGHICIa, C. GROSUb

a
Department of Agricultural Sciences and Food Engineering, ‘Lucian Blaga’
University of Sibiu, 7–9 Ion Ratiu Street, 550 012 Sibiu, Romania
E-mail: simona.oancea@ulbsibiu.ro
b
Department of Environmental Sciences, ‘Lucian Blaga’ University of Sibiu,
5–7 Ion Ratiu Street, 550 012 Sibiu, Romania
ABSTRACT
The efficiency of anthocyanin extracts of blackberries, red raspberries, sweet cherries
and synthetic α-tocopherol on delaying lipid oxidation of sunflower oil during 14-
day storage at 30 and 60oC was comparatively studied. The oxidative stability of oil
samples was monitored in terms of peroxide value (PV), thiobarbituric acid‐reactive
substances (TBARS) and differential scanning calorimetry (DSC). PV results showed
improved oxidative stability of sunflower oil supplemented with natural extracts
compared to control and oil containing α-tocopherol, with better abilities for red
raspberry. Among natural extracts, blackberry showed improved antioxidant activity
as measured by TBARS, compared to control, and significantly higher compared to oil
supplemented with synthetic α-tocopherol after 14 days of storage at 60oC. Similarly,
DSC investigation revealed good protective effects of blackberry extract on sunflower
oil during storage at both temperatures. These results may contribute to the future
consideration of anthocyanin fruit extracts as potential additives to enhance oxidative
stability of sunflower oil.
Keywords: anthocyanins, sunflower oil, peroxide value, TBARS, DSC.
AIMS AND BACKGROUND

Sunflower oil is a very popular and health-promoting vegetable oil. Unfortunately,
similar to other edible oils, it becomes highly susceptible to auto-oxidation and ther-
mal oxidation under air, light, temperature or enzymes, due to the content of (poly)
unsaturated fatty acids. The oxidative deterioration generates primary oxidation
products (hydroperoxides) which decompose under elevated temperatures, light and
*
For correspondence.
132
presence of pro-oxidants to secondary oxidation products (aldehydes, ketones, alco-
hols, furans, hydrocarbons and acids)1. This process affects both the quality of the
product in terms of flavour, colour and texture, and the nutritional value leading to
potential toxic products2. For this reason, antioxidant protection is required. Effective
synthetic antioxidants such as t-butylhydroquinone (TBHQ), t-butylhydroxytoluene
(BHT), t-butylhydroxyanisole (BHA) and propyl gallate (PG) have been frequently
used to delay lipid oxidation in oils, fats and foods. Some food regulation constraints
of synthetic additives moved the research toward the identification and development
of new natural antioxidants as promising ways to achieve current consumers demand
on product safety. Natural antioxidants are safer than synthetic ones and provide vari-
ous health benefits.
Highly distributed in higher plants, natural antioxidant compounds have been
intensively studied for their antioxidant capacity and phenolic content, particularly
in medicinal plants3.
Promising natural fat-soluble antioxidants, such as essential oils of aromatic
plants4,5 and carotenoid extracts of different herbal origin6 have been reported to en-
hance oxidative protection of sunflower oil. Little has been reported on research on
fruits as alternative sources of water-soluble antioxidants used to delay lipid oxidation
in fat-containing foods7,8. Fruits of red and blue colour are rich in anthocyanins, which
are water-soluble pigments with demonstrated beneficial human health effects based
on their free‑radical scavenging and antioxidant activities9.
Previous work of us described the efficiency of adding various wild fruits extracts
to rapeseed oil to improve its oxidative stability10, and the requirement of natural lipid
peroxidation inhibitors to be used in sunflower oil11. Considering this, the present
study was performed to investigate the antioxidant activity of anthocyanin crude
hydroethanolic extracts obtained from blackberries, red raspberries and wild sweet
cherries to stabilize cold-pressed sunflower oil during 14-day storage at two differ-
ent temperatures (30 and 60oC). The objectives were: (i) to determine the content of
total anthocyanins and total phenolics as main antioxidants in selected fruits; (ii) to
evaluate the oxidative stability of sunflower oils with added crude extracts by assess-
ing peroxide values (PV), thiobarbituric acid reactive substances (TBARS) and by
differential scanning calorimetry (DSC) analysis; (iii) to compare their antioxidant
abilities to those of synthetic α-tocopherol in sunflower oil.
EXPERIMENTAL
Plant materials and reagents. Frozen fruits grown and harvested in Romania were
used: wild-grown blackberries, red raspberries cv. Heritage, and wild-grown sweet
cherries. Chemical reagents of analytical grade without further purification were used.
Extraction and assay of total anthocyanins. Anthocyanins were extracted using 70%
ethanol (v/v), overnight, at 4oC. The extract was filtered and centrifuged at 4oC at 8000
rpm for 10 min. The refrigerated centrifuge (Hettich Universal 320R, Germany) was
133
used. The total anthocyanins content was spectrophotometrically determined by the
pH differential method12. Specord 200Plus UV-vis. spectrophotometer (Analytik Jena,
Germany) was used. Total anthocyanins were expressed as cyanidin-3-O-glucoside
(Cyn-3-O-G) in mg/100 g fresh mass (mg/100 g FM).
Extraction and assay of total phenolics. Phenolics were extracted using 90% methanol
(v/v). The total phenolics content was determined spectrophotometrically according
to the Folin-Ciocalteu method13. Gallic acid was used as standard for the calibration
curve. Total phenolics were expressed in mg of gallic acid equivalents 100 g–1 fresh
mass (mg GAE/100 g FM).
Reverse micelles preparation. Locally purchased cold-pressed sunflower oil was used
for the purpose of addition of aqueous ethanolic plant extracts to it. The stabilisation
of such dispersion was achieved by adding a phospholipid surfactant, process that
facilitates the formation of thermodynamically long stable reverse micelles, also called
microemulsions14,15. As phospholipid surfactant, commercial liquid soy lecithin was
used, which is a low cost and food grade surfactant. Reverse micelles were prepared
by dissolving liquid soy lecithin in sunflower oil to obtain 0.5% concentration. Then,
300 ml of anthocyanin extract were added to the mixture, under vigourous stirring
conditions (mechanical homogenisation). Aliquots from the obtained emulsions were
used for PV and TBARS analysis. All emulsions were stored in duplicate in Petri
dishes in dark at 30 and 60oC over a 14-day period. Two aliquots of each were removed
periodically for PV and TBARS analysis. Similar preparation was done when using
α-tocopherol (Alfa-Aesar, Germany) as fat-soluble antioxidant added to sunflower
oil in order to give 0.05% final concentration.
Lipid hydroperoxides determination by peroxide value (PV). Peroxide value was
determined by iodometric standard procedure and expressed as meq kg–1 (Ref. 16).
Data from the PV measurements were plotted against time.
Thiobarbituric acid reactive substances (TBARS) determination. The TBARS method17
with some modifications was used to measure the antioxidant activity of anthocyanin
extracts related to lipid oxidation in sunflower oil. Malonaldehyde (MDA) standard
curve was prepared using 1,1,3,3-tetramethoxypropane (Acros Organics, Belgium).
TBARS values were expressed as mg of malonaldehyde kg –1 oil sample (meq
MDA kg–1).
Differential scanning calorimetric (DSC) analysis. Lipid oxidation was monitored by
the standard test method (ASTM E2009-02) using the DSC calorimeter (SDT Q600,
TA Instruments) calibrated with pure indium. Sunflower oil samples of 2.0 ± 0.5 mg
were heated in aluminum pan at a rate of 10oC min–1, from 35 to 300oC. Experiments
were performed under an oxygen flow rate of 100 ml min–1. Evaluation of oil oxidative
stability is based on calculation of the protection factor (PF). In previous works, this
value was defined as the ratio of induction time of treated oil samples to induction
time of control using Rancimat apparatus18. Hereby we have calculated PF as ratio of
134
the oxidation onset temperature of treated oil sample to the oxidation onset tempera-
ture of control (untreated oil sample) using DSC. PF values higher than 1 indicate a
protective effect of added extracts on the oil oxidative stability. The oxidation onset
temperature is defined as the temperature at which exothermic processes between oil
and oxygen occur. The extrapolation of the tangents of the curve taken at the steepest
slope and at extended baseline permits the determination of the onset temperature at
their intersection point.

Total anthocyanins and total phenolics content of crude extracts. Table 1 shows the
content of total anthocyanins and total phenolics of selected fruits.
Table 1. Total anthocyanins and total phenolics content of blackberry, red raspberry and wild sweet
cherry samples
Sample Total anthocyanins Total phenolics
(mg/100 g FM) (mg GAE/100 g FM)
Wild blackberry (Rubus fruticosus L.) 167.34 564.39
Red raspberry cv. Heritage (Rubus idaeus L.) 41.56 154.87
Wild sweet cherry (Prunus avium L.) 23.35 122.30
These plant bioactive compounds are well-known for their strong antioxidant
properties19. The in vitro evaluation of antioxidant activity is broadly based on hy-
drogen atom transfer reactions (oxygen radical absorbance capacity ORAC, total
reactive antioxidant potential TRAP) and on single-electron transfer reactions (total
phenolics assay, ferric‑reducing antioxidant power FRAP, Trolox equivalent antioxi-
dant capacity TEAC, thiobarbituric acid reactive substances TBARS, DPPH radical
scavenging activity)20.
In the present study, apart the total phenolics assay used to evaluate the anti-
oxidant activity of extracts, other simple assays based on the evaluation of primary
and secondary oxidation products generated during storage of treated sunflower oil,
such as PV and TBARS, were performed. The applied methodologies provide both
the antioxidant potential of fruit extracts and their potential application as natural
oxidative stabilisers of edible oils. The efficiency of the antioxidant effect was also
monitored by the DSC technique.
Monitoring the antioxidant effect of crude extracts by PV and TBARS. We investi-
gated the oxidative stability of sunflower oil in a reverse micelle system formed of
sunflower oil and anthocyanin crude extracts in the presence of soy lecithin. The
oxidative stability monitored over a 14-day period at 30 and 60°C was compared to
control and to oil containing synthetic α-tocopherol.
PV measurement is a well-established method (AOCS Official Method) for the
determination of primary oxidation products in fats and related compounds, while
135
TBARS is widely used for measuring the level of secondary oxidation products (e.g.
aldehydes).
The PV results indicate lower levels of lipid peroxidation in all sunflower oil
samples containing anthocyanin extracts compared to those in control and sample
containing a-tocopherol during 14 days of storage at 30 and 60°C, as shown in Figs
1 and 2.
Fig. 1. Evolution of PV of sunflower oils (control and treated samples with anthocyanin extracts and
α-tocopherol) during storage at 30°C
Fig. 2. Evolution of PV of sunflower oils (control and treated samples with anthocyanin extracts and
α-tocopherol) during storage at 60°C
136
The average PV values of the 3 samples containing natural extracts were about
10% lower than those of control after 14 days of storage at 30°C, and about 12%
lower after same storage period at 60°C, respectively. Better results were obtained
with sample containing red raspberry anthocyanin extract (0.062 mg anthocyanins
100 g–1 oil). This extract showed an improvement of oxidative stability of sunflower
oil during the first five days of storage at 60°C, with PV values which decreased by
34% compared to control and by 45% compared to sample containing a-tocopherol.
After 14 days of storage, the decrease of PV values of this sample was 14% compared
to control and 17.5% compared to sample containing a-tocopherol. The anthocyanin
extracts of blackberry and sweet cherry in sunflower oil determined a decrease of 13
and 9% of the PV, respectively, compared to control after 14 days of storage at 60°C.
After this storage period, PV values of sample containing synthetic a-tocopherol
registered higher values than control and than all samples containing anthocyanin
extracts. The time-related decrease of the inhibition of oxidative reactions by toco-
pherols added to edible oils and their pro-oxidant action at higher concentration was
previously shown21.
Despite the lower content of total phenolics and anthocyanins in red raspberry
compared to blackberry, this extract showed better antioxidant effect when monitor-
ing the primary oxidation products. This is probably due to the presence of other
non-phenolic antioxidant compounds, which may act synergistically. The presence
of phospholipids of soy lecithin may also contribute to the enhancement of the an-
tioxidant effect of anthocyanins in oils, despite that lecithin on its own determined
higher PVs in oil compared to anthocyanin extracts, as shown in our previous work22.
Regarding the evaluation of secondary oxidation products by TBARS assay,
results showed a better antioxidant effect for the sample containing blackberry an-
thocyanin extract compared to control and to samples containing the other extracts
and a-tocopherol, as presented in Figs 3–4. TBARS value of the sample containing
blackberry anthocyanin extract (0.251 mg anthocyanins/100 g oil) was 13.33% lower
than that of control, and slightly lower than that of sample with a-tocopherol (50
mg/100 g oil).
137
Fig. 3. Changes in the TBARS values of sunflower oils (control and treated samples with anthocyanin
extracts and α-tocopherol) during storage at 30°C
Fig. 4. Changes in the TBARS values of sunflower oils (control and treated samples with anthocyanin
extracts and α-tocopherol) during storage at 60°C
We determined the TBARS values during storage at 60 and 30oC for 7 and 14
days, as well. The obtained results indicate an efficient antioxidant effect for samples
containing natural extracts stored at 60oC compared to that for samples stored at 30oC
until the end of the experiment, when a pro-oxidant effect seems to occur. TBARS
values of samples containing natural extracts were found lower than of control, but
higher than those of sample containing a-tocopherol after 14 days of storage at
60oC. It seems that inhibition of the generation of secondary oxidation products by
the natural extracts is not as efficient as for the a-tocopherol added to sunflower oil
when stored at 60oC for long periods, probably due to the thermal lability of these
bioactive compounds.
Previously reported studies have shown that antioxidant compounds of rose-
mary, sage, basil, black pepper and garlic appear to be relatively stable when used to
stabilise edible oils23.
138
To our knowledge, literature is scarce in such studies on anthocyanins, but suc-
cessful attempts were done by our group by using bilberry anthocyanin extract which
exhibited remarkable improvement of oxidative stability of cod liver oil24.
Monitoring the antioxidant effect of natural extracts by DSC analysis. DSC at non-
isothermal mode was used to measure the stability of sunflower oil containing antho-
cyanin extracts and compared to control and sample containing α-tocopherol. The oil
samples were stored at 30 and 60oC, for 0, 7 and 14 days, respectively.
The obtained DSC curves, except of samples stored at 60oC for 14 days, when
only a curve for a-tocopherol was conclusive and therefore comparison could not be
obtained, are shown in Figs 5–9.
Fig. 5. DSC oxidation curves of sunflower oils before storage at 30°C

1 – control; 2 – sunflower oil containing blackberry extract; 3 – sunflower oil containing red raspberry
extract; 4 – sunflower oil containing sweet cherry extract; 5 – sunflower oil containing α-tocopherol
Fig. 6. DSC oxidation curves of sunflower oils after 7 days storage at 30°C
139
Fig. 8. DSC oxidation curves of sunflower oils tocopherol before storage at 60°C
140
In non-isothermal DSC analysis, the heat flow curve deviates from baseline and
dramatically increase in the exothermic signal during the heating of the oil sample
under oxygen environment. The extrapolation of the tangents of the thermal curve
taken at this steepest slope and at baseline gives the onset temperature. The oxida-
tion onset temperature is defined as the temperature at which exothermic processes
occur; the higher these values the more stable the oil sample. Values of the oxidation
onset temperatures are given in Table 2. As noticed, higher values were registered for
samples containing anthocyanin extracts while the lowest temperature was registered
for sample containing a-tocopherol.
Table 2. Oxidation onset temperature (oC) of sunflower oil samples stored at 30 and 60oC
Storage temperature (oC)
Sample
30 60
Storage time (days) 0 7 14 0 7 14
Control 164.27 163.25 159.93 158.49 150.77 –
Oil with blackberry anthocyanin
166.61 168.61 163.24 169.78 153.28 –
extract
Oil with raspberry anthocyanin
166.62 167.57 167.22 166.62 149.76 –
extract
Oil with sweet cherry anthocya-
161.91 166.66 167.29 163.73 148.55 –
nin extract
Oil with α-tocopherol 159.26 165.01 165.41 157.47 148.77 130.72
The calculation of the protection factor (PF) as the ratio of the oxidation onset
temperature of treated oil to the oxidation onset temperature of control (untreated oil)
allows the comparison of the obtained results and the estimation of the antioxidant
efficiency. Results of PFs calculated based on values of onset temperature given in
Table 2 are shown in Table 3. PF values higher than 1 indicate a protective effect of
added extracts on sunflower oil oxidative stability.
Table 3. Protection factor (PF) values of sunflower oil samples stored at 30 and 60oC
Storage temperature (oC) 30 60
Storage time (days) 0 7 14 0 7
Sam- control 1 1 1 1 1
ple oil with blackberry anthocyanin 1.014 1.032 1.020 1.071 1.016
extract
oil with raspberry anthocyanin 1.014 1.026 1.045 1.051 0.993
extract
oil with sweet cherry anthocyanin 0.985 1.020 1.046 1.033 0.985
extract
oil with α-tocopherol 0.969 1.010 1.034 0.993 0.986
141
As noticed, the addition of sweet cherry anthocyanin extract and a-tocopherol
to sunflower oil before storage at 30oC produced a slight pro-oxidant effect, with PF
values of 0.985 and 0.969, respectively. However, during storage at 30oC, protective
effects occur in both cases. It seems that the addition of each anthocyanin extract
determines good protective effects on sunflower oil during 14-day period of storage
at 30oC. When stored at 60oC for 7 days, oil sample containing blackberry extract
showed higher PFs, while oil samples containing the other natural extracts and a-to
copherol registered lower PFs.
Other authors have shown that monitoring oxidative stability of oils by non-
isothermal DSC allows the registration of two stages of oxidation, the first one deal-
ing with oxidative changes, and the second one, with quantitative decomposition of
oils25. Considering this, we have determined the temperatures of maximum heat flow
(Tp1 and Tp2) using the DSC scan, as shown in Fig. 10. We found good correlation
(0.914224) between Tp1 and PV of sunflower oil containing blackberry anthocyanin
extract stored at 60oC for 7 days.
Fig. 10. DSC oxidation onset temperature and temperatures of maximum heat flow (Tp1 and Tp2) for
sunflower oil sample containing blackberry anthocyanin extract, stored at 60oC for 7 days
Our results highlight the great potential of fruits anthocyanin extracts to stabilise
sunflower oil when added in small amounts without much affecting colour character-
istics, as measured by common tests of oxidative stability.
CONCLUSIONS
The antioxidant effect of anthocyanin extracts of blackberries, red raspberries and
sweet cherries added to cold-pressed sunflower oil was evaluated during storage at 30
and 60oC and compared to that of synthetic a-tocopherol. Evaluation was performed
by measurements of primary oxidation products by peroxide value (PV), of second-
142
ary oxidation products by thiobarbituric acid‐reactive substances (TBARS) and by
differential scanning calorimetric (DSC) analysis.
The results showed that addition of small amounts of anthocyanin extracts
produced a decrease of primary and secondary oxidation products of sunflower oil
during 14-day storage at 30 and 60oC with respect to control and sample containing
α-tocopherol. The results of DSC scans showed higher values of protection factor (PF)
produced by anthocyanin extracts on oil samples stored at 30oC, which means greater
oxidative stability compared to α-tocopherol. It seems that higher temperatures (60oC)
for longer periods determine a negative effect on both oil and anthocyanins, except
the oil sample with added blackberry extract for which PF was > 1.
These results may contribute to the future consideration of anthocyanins extracted
from fruits for obtaining stable sunflower oil without addition of synthetic additives.
ACKNOWLEDGEMENTS
This work was supported by a grant of the Romanian National Authority for Scientific
Research CNCS–UEFISCDI, project number PN-II-ID-PCE-2011-3-0474.
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Received 27 March 2015
144
CHEMICAL COMPOSITION, CYTOTOXICITY AND

ANTIOXIDANT ACTIVITY OF ESSENTIAL OIL FROM Vitex
agnus-castus FRUITS, GROWING IN BULGARIA
I. ZHELEVa*, T. BATSALOVAb, L. GEORGIEVAc, B. DZHAMBAZOVb,

A. STOYANOVAd, I. DIMITROVA-DYULGEROVAe
a
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Medical University
of Varna, 55 ‘Marin Drinov’ Street, 9000 Varna, Bulgaria
E-mail: ijelev80@abv.bg
b
Department of Developmental Biology, Faculty of Biology, Plovdiv University
‘Paisii Hilendarski’, 24 ‘Tzar Assen’ Street, 4000 Plovdiv, Bulgaria
c
Department of Organic Chemistry, Technological Faculty, University of Food
Technologies, 26 Maritza Blvd., 4000 Plovdiv, Bulgaria
d
Department of Essential Oils, Technological Faculty, University of Food
Technologies, 4000 Plovdiv, Bulgaria
e
Department of Botany, Faculty of Biology, Plovdiv University ‘Paisii Hilendarski’,
24 ‘Tzar Assen’ Street, 4000 Plovdiv, Bulgaria
ABSTRACT
Fruit essential oils from Vitex agnus-castus L. growing in Bulgaria were obtained by
water distillation and were analysed by gas chromatography. Thirty-five components
were identified in a sample isolated from fruit material from Southern Bulgaria, with
1% oil content and main constituents (up to 3%): 1.8-cineole (20.39%), a-pinene
(15.12%), b-pinene (9.40%), (Z)-β-farnesene (6.88%), bicyclogermacrene (6.08%),
b-caryophyllene (5.27%) and terpinyl acetate (4.13%). Thirty-three components
were identified in the essential oil sample from Northern Bulgaria, with 0.5% oil
and main constituents: (Z)-β-farnesene (16.38%), bicyclogermacrene (12.26%),
limonene (7.51%), a-pinene (6.24%), germacrene (4.12%), eicosane (4.03%), he-
neicosane (3.97%), b-pinene (3.99 %) and nonadecane (3.66%). The cytotoxicity of
the essential oil from Southern Bulgaria against three human adenocarcinoma cell
line LS180, cervical adenocarcinoma cells, lung adenocarcinoma cell lines (LS180,
HeLa, A549) as well as normal amniotic cell line were investigated. V. agnus-castus
fruit essential oil exhibited cytotoxic effects against all four tested cell lines. HeLa
cell line showed the strongest sensitivity, suggesting a potential use of Vitex agnus-
*
For correspondence.
145
castus fruit essential oil as a chemotherapy agent for cervical cancer. The essential
oil antioxidant activity measured by ABTS assay showed better values than those
obtained with the DPPH assay.
Keywords: Vitex agnus-castus fruits, essential oil, chemical composition, antioxidant
activity, cytotoxicity.
AIMS AND BACKGROUND

Vitex agnus-castus L. (Verbenaceae) is a deciduous shrub widely distributed in the
Mediterranean region of Europe, Crimea, Caucasus and Western Asia. The spike-like
inflorescence of the plant contains blue or pink coloured and aromatic flowers. The
fruit is spherical and elongated drupe, and varies in colour from reddish to black1,2.
V. agnus-castus fruit extracts have been used in traditional medicine as a remedy for
menstrual disorders, premenstrual syndrome, menopausal and perimenopausal symp-
toms, corpus luteum insufficiency, acne, infertility and other female conditions3–6.
Phytochemical studies of the V. agnus-castus fruits showed the presence of polyphe-
nols7,8, diterpenoids9,10, essential and fatty oils11,12, iridoid glycosides13, ecdysteroids14.
There is a growing interest to investigations on different natural antioxidants
aiming to prevent free radical damage and improve human health15. Essential oils
similar to polyphenols have been studied for their potential and ability to prevent
oxidative decay of easily oxidised metabolites16–19. As a source of these compounds
V. agnus-castus fruits were investigated for antioxidant properties10,12. Antioxidant
properties of natural compounds have another important pharmacological role in
human health – anticarcinogenic effect20,21. The cytotoxicity of the V. agnus-castus
fruit extracts against different cancer cell lines were investigated from many research
groups22–25. However, the antitumor activity of essential oil from these fruits has not
been intensively studied. Thus, the aim of this work was to evaluate the cytotoxicity
of essential oil from V. agnus-castus fruits against different cancer cell lines (HeLa,
LS180, A549), as well as to investigate the antioxidative properties of the essential
oil by DPPH and ABTS assay. In addition, the chemical composition of essential oils
from two regions of Bulgaria has been determined.
EXPERIMENTAL
Plant material. Vitex agnus-castus L. fruits were collected from the cities of Plovdiv
and Varna, Bulgaria in September 2013. The plant materials were identified at the
Department of Botany, Plovdiv University ‘Paisii Hilendarski’, by Ass. Prof. Dr.
I. Dimitrova-Dyulgerova, according to Tutin et al.1 and Gramatikov2. The moisture of
the fruits was estimated by drying to 105°C, according to Russian Pharmacopoeia26.
Voucher specimens (No 060437, 060438) were deposited in the Herbarium of the
Agricultural University, Plovdiv, Bulgaria (Herbarium SOA).
146
Isolation of essential oil. The air-dried and grinded (up to 0.5 mm) V. agnus-castus
fruits were submitted to water-distillation for 4 h using a British-type Clevenger ap-
paratus. The obtained oil was dried over anhydrous sodium sulphate and stored in
tightly closed dark vials at 4°C until analysis.
Gas chromatography (GC) analysis. The GC analysis was performed using Agilent
7890A apparatus with mass detector 5975 C; column HP-5 MS (30 m × 250 μm; film
0.25 mm); temperature: 40°C for 3 min then 5°C/min to 300°C for 5 min, run time
60 min; split ratio 60:1; gas flow 1.0 ml/min; MS source: 230°C; MS quad: 150°C.
The relative percentages of separated compounds were determined from the total ion
current by computerised integrator. The RIs of the compounds were recorded with a
standard n-hydrocarbon calibration mixture (C9-C36) (Restek, Cat No 31614, sup-
plied by Teknokroma – Spain) using AMDIS 3.6 software.
Cytotoxicity assays and evaluation of antitumor activity. The cytotoxic effects and
potential antitumor activity of V. agnus-castus fruit essential oil were evaluated by
MTT assay using four cell lines: A549 (human lung adenocarcinoma epithelial-like cell
line; ATCC CCL 185, NBIMCC 2404), HeLa (human cervical adenocarcinoma cell
line; ATCC CCL 2), LS180 (human Caucasian colon adenocarcinoma cell line; ATCC
CL-187) and FL cell line (derived from normal human amniotic cells, ATCC CCL
62, NBIMCC 94). The cells were seeded in 25 cm2 flasks in the Dulbecco Modified
Eagle Medium (Gibco™, Invitrogen, USA) supplemented with 10% heat-inactivated
fetal calf serum (PAA Laboratories GmbH, Linz, Austria), 100 U/ml penicillin and
100 mg/ml streptomycin (Sigma, Germany). The cells were grown at 37°C, 5% CO2
and high humidity.
Prior exposure to different concentrations of V. agnus-castus fruit essential oil,
the cultures were trypsinised and the number of viable cells was evaluated by the
Trypan blue exclusion assay. Then, the cells were plated on 96-well tissue culture
plates at a density of 4.5 × 104 cells/well in 200 ml total volume per well. After 24 h,
the cultures were treated with different concentrations of V. agnus-castus fruit es-
sential oil – 0.5, 0.25, 0.125, 0.06, 0.03, 0.015, 0.008, 0.004, 0.002 and 0.001%. The
essential oil was dissolved in DMSO (Sigma, Germany) yielding 50% solution and
further dilutions were done using cell culture medium. Thus, all tests included con-
trols treated with equivalent concentrations of DMSO (0.5, 0.25, 0.125, 0.06, 0.03,
0.015, 0.008, 0.004, 0.002 and 0.001%) in addition to the untreated control grown in
standard culture medium. The cells were exposed to the test-compounds for 24, 48
and 72 h. All experiments were performed in triplicates.
MTT assay. This assay is based on the ability of mitochondrial dehydrogenases in
viable cells to convert the soluble tetrazolium salt MTT [3-(4′,5′-dimethylthiazol-
2′-yl)-2,5-diphenyltetrazolium bromide] (Sigma, Germany) into an insoluble purple
formazan product. In our experiments, 20 ml MTT solution (5 mg/ml, dissolved in
phosphate-buffered saline – PBS) was added to the cells at the end of the exposure
period. The culture plates were then incubated for 3 h at 37°C, 5% CO2 and high
147
humidity. After that, the supernatant was discarded and the cells were washed with
PBS. The formazan product accumulated in viable cells was solubilised with 100 ml/
well DMSO. The plates were incubated with DMSO for 15 min at 37°C and then
absorbance (570 nm) was detected using an ELx800TM Absorbance Microplate Reader
(BioTek, USA).
METHODS FOR ANTIOXIDANT ACTIVITY
DPPH assay. The electron donation ability of V. agnus-castus extracts and some pure
compounds was measured by DPPH assay. This assay is based on the bleaching of
purple coloured methanol solution of DPPH. 0.15 ml extract was mixed with 2.85 ml
freshly prepared 0.1 mM methanol solution of 1,1-diphenyl-2-picrylhydrazyl radical
(DPPH, Sigma). The reaction was performed at 37°C in a dark place. After 15 min,
absorbance at 517 nm was recorded against methanol. The antioxidant activity was
expressed as % inhibition27.
I % = (Acontol – Asample)/Acontrol × 100,
where Acontrol is the absorbance of the control (containing all reagents except the test
compound), and Asample – the absorbance of the test compound.
ABTS assay. The ABTS assay was performed as described by Thaipong et al.28, with
some modifications. Briefly, ABTS radical was generated by mixing aliquot parts of
7.0 mM 2,2′-azinobis (3)-ethylbenzthiazoline-6-sulphonic acid (ABTS, Sigma) in
doubly distilled H2O (dd H2O) and 2.45 mM potassium persulphate (Merck) in dd H2O.
The reaction was performed for 16 h at room temperature in the dark. The gen-
erated ABTS radical (ABTS•+) is stable for several days. Before analyses, 2.0 ml of
ABTS•+ solution was diluted with methanol at proportions 1:30 (v/v), so the obtained
final absorbance of the working solution was about 1.0–1.1 at 734 nm. For the assay,
2.85 ml of ABTS•+ solution were mixed with 0.15 ml of obtained extracts. After 15
min at 37°C in the dark absorbance was measured at 734 nm against methanol. The
antioxidant activity was expressed as % inhibition (I %):
I = (Acontol – Asample)/Acontrol × 100,
where Acontrol is the absorbance of the control (containing all reagents except the test
compound), and Asample – the absorbance of the test compound.
Statistics. Statistical analyses of the cytotoxicity assays data were performed using
the Mann Whitney U test (Statview software program, 5.0.1 version). P values lower
than 0.05 were regarded as significant. All results were compared to those from the
control group if not otherwise stated.

Chemical composition of Vitex agnus-castus essential oils. The essential oils were
yellow oil-like liquids with specific pungent odour. The essential oil content was 1%
148
in sample 1 (V. agnus-castus sample from Southern Bulgaria) and 0.5% in sample 2
(V. agnus-castus sample from Northern Bulgaria). The distribution of major groups
of aroma substances in the oils is shown in Fig. 1.
50
45
40
35
30
% 25
20
15
10
5
0
1 2 3 4 5 6
sample 1 sample 2
Fig. 1. Groups of components in the oils (%)

1 – monoterpene hydrocarbons; 2 – oxygenated monoterpenes; 3 – sesquiterpene hydrocarbons; 4 –
hydrocarbons; 5 – oxygenated sesquiterpenes; 6 – aromatic compounds; sample 1 – essential oil from
V. agnus-castus growing in Southern Bulgaria; sample 2 – essential oil from V. agnus-castus growing
in Northern Bulgaria
Monoterpene hydrocarbons are the dominant group in sample 1 – 36.04%,

followed by oxygenated monoterpenes – 30.74% and sesquiterpene hydrocarbons –
22.68%. Sesquiterpene hydrocarbons dominate in sample 2 – 45.67%, followed by
monoterpene hydrocarbons – 23.82% and hydrocarbons – 13.57%. Our results differ
from the findings of Sarikurkcu et al. (2009) who showed that the essential oil was
rich in oxygenated monoterpenes (45.38%).
Thirty-five components were identified in sample 1, representing 88.33% of all
oil compounds (Table 1).
The main components (up to 3 %) were 1,8-cineole (20.39%), a-pinene (15.12%),
b-pinene (9.40%), (Z)-β-farnesene (6.88%), bicyclogermacrene (6.08%), b-caryo-
phyllene (5.27%) and terpinyl acetate (4.13%). Thirty-three components represent-
ing 85.92% of the total oil were identified in sample 2. The main components were:
(Z)-β-farnesene (16.38%), bicyclogermacrene (12.26%), limonene (7.51%), a-pinene
(6.24%), germacrene (4.12%), eicosane (4.03%), heneicosane (3.97%), b-pinene (3.99
%) and nonadecane (3.66%). Camphen, p-cymen and cis-sabinene hydrates were not
present in sample 2, while γ-eudesmol was not present in sample 1.
149
Table 1. Phytoconstituents of essential oil from V. agnus-castus
No Compound RT RI Composition (% of TIC)
sample 1 sample 2
1 tricyclene 9.29 927 0.28 0.15
2 α-pinene 9.61 939 15.12 6.24
3 camphene 9.92 942 0.24 nd
4 β-pinene 10.87 979 9.40 3.99
5 β-myrcene 11.42 991 2.56 0.67
6 α-phellandrene 11.82 1002 2.27 0.56
7 α-terpinene 12.17 1017 0.22 0.31
8 p-cumene 12.44 1026 0.41 nd
9 d-limonene 12.64 1030 1.12 7.51
10 1,8-cineole (eucalyptol) 12.75 1034 20.39 2.96
11 γ-terpinene 13.51 1060 0.35 0.48
12 cis-sabinene hydrate 13.78 1069 0.13 nd
13 α-terpinolene 14.42 1088 0.27 0.56
14 β-linalool 14.81 1097 0.31 0.36
15 trans-sabinene hydrate 16.83 1109 0.25 0.26
16 terpinen-4-ol 17.14 1177 0.68 0.85
17 α-terpineol 17.49 1188 0.73 2.29
18 isobornyl acetate 20.20 1289 0.54 0.48
19 terpinyl acetate 21.99 1355 4.13 0.74
20 β-elemene 23.06 1395 0.25 0.27
21 α-gurjunene 23.54 1404 0.62 1.68
22 β-caryophyllene 23.88 1420 5.27 3.56
23 β-gurjunene 24.09 1432 0.46 0.39
24 (Z)-β-farnesene 24.76 1457 6.88 16.38
25 germacrene D 25.48 1485 0.15 4.12
26 bicyclogermacrene 25.82 1499 6.08 12.26
27 δ-cadinene 26.33 1514 0.32 0.58
28 ±-trans-nerolidol 27.42 1563 0.22 0.74
29 spathulenol 27.67 1578 0.68 1.55
30 caryophyllene oxide 27.80 1583 0.53 0.99
31 viridiflorol 27.99 1593 0.24 0.52
32 γ-rudesmol 28.33 1630 nd 1.73
33 α-cadinol 29.11 1654 1.55 1.08
34 nonadecane 35.53 1900 2.15 3.66
35 eicosane 36.30 2000 2.21 4.03
36 heneicosane 37.97 2100 1.32 3.97
Sample 1 – essential oil from Plovdiv town, Southern Bulgaria; sample 2 – essential oil from Varna
town, Northern Bulgaria; RT – retention time; RI – relative indices; % of TIC – % of total ion current;
nd – not detected.
150
Several previous studies on the composition of essential oil from V. agnus-castus
have been reported11,12,29,31 . The oils obtained from Southern Italy and Northern Brazil
were of 1,8-cineole chemotypes29,30. 1,8-cineole is the major oil compound together
with a-pinene and sabinene in V. agnus-castus from Turkey12. Sørensen and Katsio-
tis11 reported the following main components for mature fruits: sabinen, 1,8-cineole,
trans-β-farnesene and b-caryophyllene. Similarly, 1,8-cineole is the most abundant
constituent in the oil from Southern Bulgaria (sample 1 in the present study) in
combination with a-pinene. Nowak et al.31 reported two different chemotypes for V.
agnus-castus oils – a-pinene and a-terpinyl acetate types. In our study, terpinyl acetate
was detected in a small quantity (4.13% in sample 1 and 0.74% in sample 2). In the
oil from Northern Bulgaria (sample 2) (Z)-β-farnesene and bicyclogermacrene were
the major components. These results demonstrate the role of environmental condi-
tions for the accumulation of biologically active substances and for the formation of
essential oil specific chemotypes.
Antioxidant activity. The antioxidant potential of plant essential oils and their pos-
sible application as natural additives is of interest for both biomedicine and industry.
Therefore, we evaluated the antioxidant capability of the essential oil obtained from
V. agnus-castus from Southern Bulgaria. The sample was diluted in 7% solution of
cyclodextrins and tested in four different concentrations – 1.0, 5.0, 10.0 and 100 mg/ml.
There are various methods described in the literature for determination of in vitro
antioxidant activity and usually differences between them are due to the mechanism
of action. In the present study the antioxidant activity of V. agnus-castus essential
oil was determined by DPPH and ABTS radical scavenging assays. The degree of
decolorisation of the reaction mixture is an indicator for the free radical scavenging
potential of the sample.
As can be seen from Table 2, the free radical scavenging effect of the sample
shows a dose-dependent increase.
Table 2. Scavenging effect (%) on DPPH and ABTS of essential oil* of V. agnus-castus at different
concentrations
Sample Sample (mg/ml)
1 5 10 100
% inhibition
DPPH ABTS DPPH ABTS DPPH ABTS DPPH ABTS
Essential 0.25±0.07 1.99±0.37 0.85±0.07 4.73±0.43 1.05±0.07 10.32±0.37 6.66±0.66 72.36±0.45
oil*
*Sample from Southern Bulgaria.
The weakest radical scavenging activity according to the DPPH assay was 0.25 ±
0.07% at 1.0 mg/ml essential oil concentration. The weakest radical scavenging
activity determined by the ABTS assay was 1.99 ± 0.37% at 1.0 mg/ml essential oil
concentration. The highest inhibition of free radicals was achieved by the sample
151
with 100 mg/ml concentration of essential oil – 6.66 ± 0.66% for DPPH assay and
72.36 ± 0.45% for ABTS assay. The antioxidant activity, determined by both methods,
increases with increasing the essential oil ratio in each sample. Still, the ABTS values
are higher than those obtained from the DPPH assay.
The relatively low results for the antioxidant activity of the analysed samples could
be explained with the established correlation between radical scavenging capacity and
polarity of the samples. Saricurkcu et al.12 investigated different solvent extracts and
essential oil scavenging effect on DPPH radical. They established that the antioxidant
activity of water extract from the same plant is higher than those of essential oil.
Citotoxicity and anticancer activity. The essential oil from V. agnus-castus growing
in Southern Bulgaria was further examined for its cytotoxic effects and potential an-
ticancer activity using 4 cell lines: three cancer cell lines derived from lung (A549),
cervical (HeLa) and colorectal (LS180) carcinoma and a cell line derived from normal
human amniotic cells (FL). Only sample 1 was used in these experiments because of
its higher yield and rich composition (35 ingredients, major components – 1,8-cineole
and a-pinene). The results from the cytotoxicity assays are shown in Fig. 2. They
demonstrate dose- and time-dependent cytotoxic effects against all 4 cell lines. Treat-
ment with 0.5% essential oil led to 100% inhibition of the cell growth and viability in
both normal and cancer derived cell lines. Thus, these data suggest a common cyto-
toxic effect of the tested essential oil sample. However, the IC50 value for HeLa cells
(Table 3) was significantly lower compared to the values for the other three cell lines.
Table 3. Antitumor and cytotoxic activity of V. agnus-castus essential oil expressed as IC50 values*
Cell lines IC50
FL 0.213±0.011
HeLa 0.118±0.002 †
A549 0.212±0.012
LS180 0.252±0.032
* IC50 values represent % essential oil concentration in the culture medium that inhibits the growth of
50% of the cells; † p < 0.05, compared to IC50 values for FL cell line.
This demonstrates increased sensitivity of human cervical cancer cells to V.

agnus-castus essential oil compared with human non-small lung adenocarcinoma cells
(A549), colon adenocarcinoma cells (LS180) and normal amniotic FL cells. Hence,
the essential oil from V. agnus-castus growing in Southern Bulgaria could be further
investigated as a potential chemotherapy agent for cervical cancer.
152
Fig. 2. Cytotoxic effects of V. agnus-castus essential oil against different human cell lines
A549, FL, HeLa and LS180 cells were exposed to different concentrations of V. agnus-castus fruit essential oil for 24, 48 and 72 h. The cytotoxic effects
were evaluated by MTT assay. The essential oil sample was dissolved in DMSO and therefore, a control treated only with corresponding concentrations
of DMSO was included in all experiments. Graphs show the mean % inhibition (±SEM). Statistical analysis was performed using Mann Whitney U test:
*<0.05; **<0.01. All experiments were done in triplicates
A, B, C – cytotoxic effects of V. agnus-castus essential oil on A549 cells treated for 24 h, 48 h and 72 h, respectively; D, E, F – V. agnus-castus essential
oil cytotoxicity on FL cell line; D – shows cytotoxic effect after 24 h exposure to the oil; E – cytotoxic effect after 48 h exposure, and F – after 72 h
exposure; G, H, I – cytotoxic effects of V. agnus-castus essential oil on HeLa cell line treated for 24, 48 and 72 h, respectively; J, K, L – represent V.
153
agnus-castus essential oil cytotoxicity on LS180 cells treated for 24, 48 and 72 h, respectively.
To date, the cytotoxic effects of V. agnus-castus fruit extracts have been exten-
sively studied using different cell lines23,32,33. Ohyama et al.24 reported cytotoxicity
of V. agnus-castus extract against human embryofibroblast (HE-21), human uterine
cervical canal fibroblast (HCF), cervical carcinoma (SKG-3a), breast carcinoma
(SKOV-3), colon carcinoma (COLO 201), gastric signet ringcarcinoma (KATO-III),
and small cell lung carcinoma (Lu-134-A-H) cells. Imai et al.25 extended further these
investigations with a thorough study on the growth inhibition of human colon carci-
noma cell line COLO 201 both in vitro and in vivo). V. agnus-castus extract inhibited
proliferation of different human prostate epithelial cell lines (BPH-1, LNCaP, PC-3)31.
Kikuchi et al.32 showed that V. agnus-castus fruit extracts exhibit cytotoxic effects
towards leucemic cell lines and that these effects depend on the diferentiation status
of the cells. Nevertheless, there is a lack of sufficient information on the cytotoxicity
and anticancer properties of V. agnus-castus essential oils. Recently, Duymus et al.34
reported cytotoxic effects of essential oil from V. agnus-castus fruits (collected from
Izmir, Turkey) against three cancer cell lines – A549, MCF 7 (breast cancer cell line)
and C9 rat glioma cells. They demonstrated that V. agnus-castus fruit essential oil
induced apoptosis in A549 and MCF 7 cells. Similar to our results, Duymus et al.34
detected slightly weaker effect on A549 cells while MCF 7 cells showed the strongest
sensitivity. In the present report, we show for the first time significant cytotoxic effect
of V. agnus-castus fruit essential oil against human cervical adenocarcinoma cells.
CONCLUSIONS
The chemical composition of fruit essential oils of V. agnus-castus growing in South-
ern Bulgaria and Northern Bulgaria was found to be different. The essential oil from
Southern Bulgarian V. agnus-castus was with higher yield and richer composition. The
dominant components in this sample were 1,8-cineole and α-pinene, while the most
important constituents in the essential oil of fruits from Northern Bulgaria were (Z)-β-
farnesene and bicyclogermacrene. These differences are the basis for further research
in order to confirm possible two chemotypes of V. agnus-castus in Bulgaria. Essential
oil from V. agnus-castus growing in Southern Bulgaria exhibited cytotoxic effects
against human colon adenocarcinoma cell line LS180, cervical adenocarcinoma cell
line HeLa, lung adenocarcinoma cell line A549 as well as normal amniotic cell line.
Among the four cell lines, HeLa showed the strongest sensitivity to V. agnus-castus
fruit essential oil suggesting a potential use of this compound as a chemotherapy agent
for cervical cancer. The essential oil antioxidant activity showed better values when
measured by the ABTS radical scavenging assay.
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Revised 12 May 2015
156
Biological and biochemical oxidation processes
EFFECT OF ARECA-NUT AQUEOUS WATER ON BMD

AND OXIDATIVE STRESS STATUS OF MICE WITH
OSTEOPOROSIS
ZHAO MINGa, WANG ZHIWEIb, ZHAO MINGa*

a
Department of Upper Limb Injury, Luoyang Orthopedics and Traumatology
Hospital of Henan Province, 82 Qiminnan Road, 471 000 Luoyang City, Henan
Province, China
E-mail: zhaomingluoyang@163.com
b
Department of Sports Medicine, Luoyang Orthopedics and Traumatology Hospital
of Henan Province, Luoyang, China
ABSTRACT
The aim of the research was to observe the effect of areca-nut aqueous water on BMD
(bone mineral density) and oxidative stress status of mice with osteoporosis, and made
a preliminary discussion on its mechanism. 24 KunMing mice were divided into 3
groups randomly with 8 each group. With the removal of ovary, the osteoporosis model
was built up for the osteoporosis group and the areca-nut group. 6 months later, mice
weight and BMD were measured, the content of H2O2, MDA (malondialdehyde),
GSH (glutathione) and CAT (catalase) were determined in the blood, as well as the
content of OPG (osteoprotegerin) and RANKL (Receptor activator of NF-κB ligand).
Compared with the osteoporosis group, the areca-nut group witnesses that weight (p <
0.05), BMD (p < 0.05), GSH (p < 0.05) increases significantly while MDA decreases
(p < 0.05) significantly 6 months after surgery. Compared with the areca-nut group,
the content of CAT in the osteoporosis group drops significantly (p < 0.05). However,
the difference between CAT content in the areca-nut group and the control group has
no statistical significance (p > 0.05). It was concluded that areca-nuts can inhibit the
worsening of mice osteoporosis effectively, whose mechanism may be related to the
effective inhibition of OPG decrease, RANKL increase and oxidative stress status.
Keywords: osteoporosis, oxidative stress, areca-nut, mice.
AIMS AND BACKGROUND

Osteoporosis is a type of systemic skeletal disease, characterised by reduced bone
mineral, degraded bone microstructure, weakened bone strength, increased bone
*
For correspondence.
157
brittleness, and higher risk of fracture1. It is one of the diseases with high morbidity
(from 2 to 7% during the acute phase and up to 45% at 12 months) and healthcare
costs2. According to one research, the cost per process during hospitalisation ranged
from 10 590 to 15 573 euros3. As early as in the 1998, estrogen deficiency has been
identified as the cause of post-menopause osteoporosis women and contributes to the
development of osteoporosis in elderly men4. Some study hints that has close relation-
ship with osteoporosis, ROS (reactive oxygen species) increase is an important factor
of senile osteoporosis5, post-menopause osteoporosis and other primary osteoporosis,
which can improve the activity of osteoclasts, decrease the number of osteoblasts6,
and directly involve in the degradation of bone matrix, moreover, estrogen deficiency
can lead to ROS (reactive oxygen species) increase, while ROS can regulate the se-
cretion of cytokines. Areca-nuts are abundant in all kinds of antioxidants, and have
high antioxidant activities7,8. From April 2014 to February 2015, this study built up
the osteoporosis model by removing the ovary, so as to observe the effect of areca-nut
aqueous water on BMD and oxidative stress status of mice with osteoporosis.
EXPERIMENTAL
EXPERIMENTAL ANIMALS
24 km mice, 8-year old, female, non-pregnant, weight 21.70 ± 0.86 g, offered by

Centre of Laboratory Animal Science of Guangdong. 24 KM mice were divided into
3 groups randomly: the control group, the osteoporosis group and the areca-nut group,
with 8 each group.
METHODS
Building up the osteoporosis model. Intraperitoneal anesthesia of 0.1 l/g sodium

pentobarbital, after 20 min, mice in the osteoporosis group and the areca-nut group
had the operation of bilateral ovary removal operation with ventral approach to build
up the osteoporosis model, while mice in the control group were only excised with
little fat around the ovary. After the operation, they were raised in different cages.
Administration methods. After the operation, add osteoporosis lixivium into the drink-
ing water in the areca-nut group (add 30 g areca-nuts in every 100 ml of water), with
self-help feeding and drinking, change it every 2 days; no osteoporosis lixivium in
the control group and the osteoporosis group, with self-help feeding and drinking.
6 months later, detect all indexes of the mice. Measure weight 6 months before and
after the operation. 6 months before and after the operation, scan mice spinal bones
with Spiral CT, collect the data of 4 parts and take an average. The content of H2O2,
GSH, MDA and CAT in the plasma should be measured by kits produced by Nanjing
Institute of Biological Engineering, and the operation should follow the manual.
The content of OPG and NF-KB RANKL should be measured by kits produced by
Shanghai JiNing industrial Co., LTD and the operation should follow the manual.
158
Statistical methods. Statistical methods should apply to the SAS12 statistical software,
group comparison should apply to one-way Anova analysis, pair wise comparison
should apply to LSD detection, intra-group comparison should apply to paired t-test.
RESULTS
Weight comparison among mice in all groups: before the operation, weight differ-
ence among 3 groups has no statistical significance (p > 0.05). 6 months after the
operation, weight gains a lot compared with preoperative weight (p < 0.05), while
the osteoporosis group witnesses the most weight gain, whose weight is far heavier
than that of the areca-nut group (Table 1).
Table 1. Weight comparison among mice in all groups `x

( ± s) g
Groups NNT Pre-operation Post-operation
Areca-nut group 8 21.94±0.97 32.50±2.11*∆
Osteoporosis group 8 21.47±1.20 35.59±2.56*
Control group 8 21.73±0.79 33.65±1.26*
* Compared with preoperative weight p < 0.05; ∆ – compared with postoperative weight of the osteo-
porosis group p < 0.05.
BMD comparison among all groups of mice: before the operation, BMD difference
among 3 groups of mice has no statistical significance (p > 0.05). 6 months after the
operation, BMD all fall down greatly compared with pre-operation (p < 0.05), while
BMD of mice in the osteoporosis group witnesses the biggest drop, which is drasti-
cally lower than that of the areca-nut group (p < 0.05), BMD of mice in the control
group witnesses the smallest drop (Table 2).
Table 2. BMD comparison among mice in all groups `x

( ± s) g/cm2
Groups Sample size Pre-operation Post-operation
Areca-nut group 8 366.9±39.1 284.0±34.6*∆
Osteoporosis group 8 368.2±46.6 259.1±43.2*
Control group 8 360.2±40.4 317.0±21.1*
* Compared with preoperative weight p < 0.05; ∆ – compared with postoperative weight of the osteo-
porosis group p < 0.05.
Comparison of the content of H2O2, GSH, MDA and CAT in all mice. After the opera-
tion, the content of H2O2 and MDA of mice in the areca-nut group is the lowest (p <
0.05). Although the content of H2O2 and MDA of mice in the control group is lower
than that in the osteoporosis group, it has no statistical significance (p > 0.05). The
content of GSH of mice in the areca-nut group is higher than that in the osteoporo-
sis group (p < 0.05). The content of CAT of mice in the osteoporosis group drops
compared with that in the areca-nut group (p < 0.05), the difference between CAT
159
content of mice in the areca-nut group and that in the control group has no statistical
significance (p > 0.05) (Table 3).
Table 3. Comparison of H2O2, MDA, GSH, CAT among mice

Groups Sample size H2O2 MDA GSH CAT
(mmol/l) (mmol/l) (mmol/l) (U/ml)
Areca-nut group 8 27.5±0.8* 74.6±13.5* 1.11±0.12* 9.36±2.21*
Osteoporosis group 8 32.4±4.1 99.8±17.9 0.65±0.06 7.41±1.25
Control group 8 31.2±1.9 86.0±12.6 0.66±0.13 9.65±2.09
* Compared with the osteoporosis group p < 0.05.
Comparison of OPG, RANKL content of mice in all groups. After the operation, OPG
content of mice in the osteoporosis group is the lowest (p < 0.05), OPG content of
mice in the areca-nut group drops compared with that in the control group (p < 0.05).
RANKL content of mice in the osteoporosis group is the highest, and the difference
between RANKL content of mice in the areca-nut group and that in the control group
has no statistical significance (p > 0.05) (Table 4).
Table 4. Comparison of OPG and RANKL among post-operation mice in all groups `x
( ± s) pmol/l
Groups Sample size Pre-operation Post-operation
Areca-nut group 8 132.0±16.22*∆ 21.69±3.01*
Osteoporosis group 8 121.6±25.40 23.75±2.91
Control group 8 144.8±21.03* 21.91±1.70*
* Compared with the osteoporosis group p < 0.05; ∆ – compared with control group p < 0.05.
DISCUSSION
As a traditional Chinese medicine, areca-nuts are abundant in all kinds of antioxidants,
and have high antioxidant activities7,8. We speculate that the antioxidants in mice can
affect their oxidative stress status, and slow down the development of osteoporosis.
The study has proven that mice without ovary can effectively imitate postmenopausal
osteoporosis9. Offering mice with areca-nut aqueous water can imitate how the human
body gets active materials from areca-nuts, so as to analyse if areca-nuts can effec-
tively postpone osteoporosis. BMD is an important index to diagnose osteoporosis
and judge the severity of fracture, also seen as ‘The Golden Standard’ to diagnose
osteoporosis10. This study measures the lumbar vertebrae bone mineral density of
mice with CT, figures up BMD with the average of 4 points. The result shows that
areca-nuts can effectively alleviate the osteoporosis process after removing the ovary.
In recent years, study shows that OPG/RANKL system has played an important
role in osteoporosis. OPG is a kind of secreted protein, can prevent osteoclasts from
differentiation and activation, increase BMD, so it is also called ‘osteoprotegerin’.
160
OPG can negatively regulate the differentiation of OC, so it plays a significant role
in the development process of OC. RANKL is a key factor to stimulate OC differ-
entiation, after combining with RANK, leads to the signal transduction of mononu-
clear phagocyte system in the bone marrow, causes specific gene expression, so as
to result in cell differentiation and maturation11. However, lack of post-menopause
estrogen will accelerate bone loss in the early period, lower OPG expression, so cy-
tokine expression that leads to bone resorption increase, with biological function of
RANKL strengthening, active OC differentiates and activates, so as to increase bone
resorption12. At the same time, some study shows that: osteosclerosis protein level
has positive relation with OPG and OPG/RANKL level. In the study, OPG content of
mice in the areca-nut group is obviously higher than that in the osteoporosis group,
while the difference between RANKL content and that in the control group has no
statistical significance, far lower than that in the osteoporosis group, which explains
that areca-nut extract can reduce osteoporosis by regulating OPG/RANK/RANKL
cell stimulating factor system.
ROS plays an important role in post-menopause osteoporosis13. ROS inhibition
of osteoblasts and acceleration of OC will generate that bone resorption is bigger
than bone formation in the BRU, and form negative bone remodelling. Clinical
studies show that serum MDA in patients with postmenopausal osteoporosis rises,
while antioxidants like SOD drops, implying that because oxidative damage hap-
pens at the same time with postmenopausal osteoporosis, the body balancing status
of oxidation and antioxidation is destroyed14. ROS inhibits mesenchymal stem cells
and osteogenic precursor cells from proliferating, prevents them from differentiating
into osteoblasts, moreover, it restrains cells secreting bone matrix and mineralising
matrix, even induces its apoptosis15. ROS brings negative effects to osteoblasts in
many ways. Wnt3/beta-catenin pathway can affect osteoblasts differentiation and
maturation, but B-catenin and ROS can work together to activate FoxO1, so as to
inhibit osteoblasts activity16,17. ROS is an important regulatory factor to a series of
signal pathway mediated by RANKL. By means of TRAF6, Rac1, NADPH oxidase
l and other key factors of signal, RANKL increases ROS concentration in the cells,
activates key factors: JNK (c-Jun N-terminal kinase) and P38MAPK (38 kD mitogen
activated protein kinase), induces OC to differentiate and maturate18, inhibits ROS
production and the differentiation of osteoclast precursor cells19.
CONCLUSIONS
The study result shows that after removing the ovary, the content of ROS in the mice
rises drastically, after taking water extract of areca-nuts, the content of MDA and
H2O2 drops, while the content of antioxidant GSH and antioxidase CAT increases. It
means that areca-nuts can inhibit the development of mice osteoporosis by mediating
ROS level.
161
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Revised 25 January 2016
162
HEAVY METALS (COPPER, CHROMIUM AND CADMIUM)

INDUCED OXIDATIVE STRESS BIOMARKERS ON
HAEMATOLOGICAL PARAMETERS OF Labeo rohita
M. U. KHALIDa*, N. A. QURESHIb, M. S. MUBARIKa, S. A. BUKHARIc

a
Department of Zoology, Government College University, Faisalabad, Pakistan
E-mail: drusmankhalid86@gmail.com; sameech2002@yahoo.com
b
Government College Women University, Faisalabad, Pakistan
E-mail: naureenaziz.qureshi@gmail.com
c
Department of Biochemistry, Government College University, Faisalabad,
Pakistan
E-mail: bukhari.shazia@yahoo.com
ABSTRACT
The analysis of serum to study the various oxidative stress parameters in Labeo rohita
during the 96 h (acute) and two months (chronic) experiment was the best tool to
evaluate the fish under stress. For this purpose treatment of different heavy metals in
controlled conditions was carried out. The different concentration of each heavy metal
viz. chromium, copper and cadmium showed significant differences of ALT (alanine
aminotransferase), AST (aspartate aminotransferase), TOS (total oxidant status) and
TAS (total antioxidant status) in the serum of the fish for both the studies. However,
there is less stress showed by the Labeo rohita for the different heavy metals. High
stress on Labeo rohita was observed as the concentration of the heavy metals increased
in various treatments. Significant differences were observed as the concentration of
the heavy metals increased or decreased in various treatments. The results revealed the
fact that of all the three heavy metal pollutants the ALT was found in order Cd > Cu >
Cr in case of the long-term (chronic study) and short- term exposure (acute study),
whereas the order of the AST was Cd > Cu > Cr for the acute study and Cu > Cd >
Cr for the chronic study. Similarly the TOS was present in order Cr > Cd > Cu for
acute and chronic study. In case of the TAS the order observed was Cd > Cu > Cr for
the acute study while Cu > Cd > Cr was observed for the chronic study. The overall
results showed that heavy metal pollutants cause oxidative stress on fish.
Keywords: fish, heavy metals, oxidation, serum.
*
For correspondence.
163
AIMS AND BACKGROUND
Pollution of natural water bodies is increasing day by day as the population of hu-
man beings increased to many folds in the last few decades1. This pollution is not
only concern to the natural water bodies but also includes land and air pollution. It
is much difficult to assess the aquatic pollution as compared to land and air pollu-
tion2. This contamination of the natural water bodies is due to the agricultural runoff,
industrial effluents and man-made activities. Long-term persistence of these highly
toxic chemicals into the aquatic environment results in absorption into the lower
trophic level, then to the higher trophic level3. As few chemicals are biodegradable
whereas a large amount of these chemicals are non-biodegradable and remains in
the aquatic environment through a long period of time. Therefore, the effect of these
chemicals and their persistent in the natural water bodies has attracted the attention
of the world population.
The chemicals reaching in the various water bodies consist of a wide range of
pollutants including pesticides, metals, fertiliser wastes, metalloids, dyes. The chemi-
cals which are non-biodegradable are more dangerous for the aquatic life and for the
humans who use these fleshes of the aquatic animals. As these non-biodegradable
chemicals remain in the flesh of the aquatic animals and ultimately absorbed in the
human being using them as their diets. In these chemicals the metals are also known
as dangerous contaminants enter into the body in the form of ions4. These pollutants
increased so many folds with the increase in industry, mining, welding, smelting and
agricultural wastes along with the excessive use of vehicles. These increasing pol-
lutants with the passage of time accumulate in the water bodies and also piles in the
form of the sediments in the large water bodies5.
The heavy metals (copper, potassium, zinc) to some extent are useful for the
normal functioning of life, as these are helpful in carrying of various metabolic
functions in living organisms. However, their excessive amounts disrupt the normal
functioning of life causing stress on the living organism. The same happened with
the aquatic organism such as fish6. The fish due to a good profile of protein content
is considered as a healthy meat for the human beings. The humans consuming such
type of fish having a heavy load of heavy metal pollutants is dangerous for human
health. The fish is a very helpful tool used as bioindicator of various pollutants. The
heavy metal pollutant causes oxidative stress on fish, so indicating that the fish is
under stress or not7.
The process in which these metals imposed stress on the fish is the result of
formation of ROS (reactive oxygen species). The process followed through the two
different mechanisms. The first one is the redox cycling in which the heavy metal
pollutants directly bind with the active site of the biomolecules and cause oxidative
stress, while in the other case the metals having not any redox potential impair with
the antioxidant defense system and resulting in the increased stress on the fish8. So,
164
for the stress on the aquatic organism including the fish is useful to determine the
toxicity level in fish exposed to the different concentrations of heavy metal salts.
Fish is used as heavy metal induced bio-indicator aquatic organism, and differ-
ent heavy metal pollutants at different concentrations are tested against Labeo rohita
for poor health conditions. The purpose of the study was to evaluate t how much the
heavy metals increased stress on the Labeo rohita rather than the fish which grows
under normal conditions.
EXPERIMENTAL
The experiment was divided into two parts and runs in triplicate. In the initial step
the LC50 values for the juvenile fish was calculated for 96 h. Acute toxicity (96 h)
experiment of water borne heavy metals viz. chromium, copper and cadmium was
performed on Labeo rohita in static water system in glass aquaria with controlled
conditions of water physiochemical parameters. For this purpose, the salts of copper
chloride, chromium nitrate and cadmium chloride were used to calculate the LC50
(lethal concentration at which 50% dies) values for acute toxicity of the heavy metal
pollutants. The concentrations of each heavy metal (on metallic ion concentration
base) initially started from zero (control) and then with gradual increase for high
concentrations to calculate the LC50 value. Fresh air was pumped by capillary action
system into each aquarium. After preparation of stock solution, six dilutions of the
heavy metal salts were formed separately for the calculation of LC50 value. On the
basis of LC50 value, the four dilutions were made to check the oxidative stress on the
Labeo rohita in long term studies in the chronic toxicity experiment, whereas the stress
was calculated after 24 h in acute toxicity experiment. The experiment was carried
out in triplicate in order to minimise the chances of errors.
COLLECTION OF SAMPLES
For the collection of samples, the fish was captured by hand net and 3 ml of blood
was drawn into the heparinised tube in order to check the oxidative stress on the fish.
In case of the acute toxicity experiment the samples were collected after each 24 h,
whereas after one week collection was made in the chronic toxicity experiment. Fresh
blood was then obtained from the fish centrifuged in order to check the level of ALT
(alanine aminotransferase), AST (aspartate aminotransferase), TOS (total oxidant
status) and TAS (total antioxidant status) in the serum of the fish.
PHYSIOCHEMICAL PARAMETERS
During the whole study the water related physiochemical parameters such as tem-
perature (28°C), total hardness (200 mg/l) and pH (8) were kept constant in order to
avoid any other stress.
165
ALANINE AMINOTRANSFERASE
The serum samples of the fish were analysed by using the BioVission ALT assay kit.
In this kit of ALT, the reaction catalyses the transfer of an amino group from alanine
to α-ketoglutarate. The resulting products of this reaction are pyruvate and glutamate.
The pyruvate is detected in this reaction which is converted from a colourless probe to
coloured one. The variations in absorbance at 365 nm are proportional to the pyruvate
activity. The following formula was used to calculate the ALT value:
ALT (U/L) = ∆A/min × 3235
ASPARTATE AMINOTRANSFERASE
The serum samples of the fish after collection were analysed by using the BioVis-
sion AST assay kit. In this kit of AST, the reaction followed the transfer of an amino
group from aspartate to α-ketoglutarate. In this reaction the resulting products are
oxaloacetate and glutamate. The glutamate in this reaction is detected which is con-
verted from nearly a colourless probe to coloured one. The variations in absorbance
at 365 nm are proportional to the AST activity. The following formula was used to
calculate the AST activity:
AST (U/L) = ∆A/min × 3235
TOTAL OXIDANT STATUS
The total oxidant status in serum samples of the Labeo rohita was determined by
using the method of Erel9.
ASSAY REAGENTS
Reagent 1. Reagent 1 comprised of 8.18 g of NaCl and 114 mg of xylenol orange

dissolved in 900 ml of sulphuric acid (25 mM) solution. In the above solution 100 ml
of glycerol were added. Finally, this reagent consisted of 140 mM NaCl, 150 µM
xylenol orange and 1.35 M glycerol. The pH was maintained at 1.75 and the solution
was stable for 6 months at a temperature of 4°C.
Reagent 2. Reagent 2 was prepared by dissolving 3.17 g of o-dianisidine dihydro-
chloride and 1.96 g of ferrous ammonium sulphate in l l of sulphuric acid (25 mM)
solution. This reagent consisted of 10 mM o-dianisidine dihydrochloride and 5 mM
ferrous ammonium sulphate. Reagent 2 was also stable for 6 months keeping at a
temperature of 4°C.
PROCEDURE
The Eppendorfs were taken in two rows by the addition of 35 µl of fish serum sample
in each. Then 225 µl of reagent 1 were added in each eppendorf. The absorbance of
the first row was taken without reagent 2, whereas a volume of 11 µl of reagent 2 was
166
added in second row eppendorf and the absorbance was taken after the incubation of
4 min at 37°C. The absorbance was taken with the help of a spectrophotometer with
bichromatic wavelength of 560 and 800 nm.
The total oxidant status was then calculated by the following formula:
TOS = ∆Abs/0.0815–0.22, whereas ∆Abs = Reagent-2 – Reagent-1.
TOTAL ANTIOXIDANT STATUS
The antioxidant status in the serum samples of the Labeo rohita was checked by the
method of Erel10.
ASSAY REAGENTS
Reagent 1. Reagent 1 was prepared with 0.4 mol/l acetate buffer solution with a pH
of 5.8; in 1 l of deionised water 32.8 g of sodium acetate were dissolved. Then in
another flask 22.8 ml of glacial acetic acid were diluted with 1 l of deionised water.
Further 940 ml of sodium acetate solution were mixed with 60 ml of acetic acid and
the pH was maintained at 5.8 with the help of a pH meter.
Reagent 2. Reagent 2 consisted of 30 mmol/l buffer solution of sodium acetate and
was prepared as follows: 2.46 g of sodium acetate were dissolved in 1 l of deionised
water. Then 1.705 ml glacial acetic acid were diluted with 1 l deionised water. The
sodium acetate buffer was prepared by mixing 75 ml of sodium acetate solution with
925 ml acetic acid solution and the pH was maintained at 3.6. Then 278 µl of hydrogen
peroxide solution were diluted with 1000 ml of buffer solution (final concentration:
2 mmol/l). After that 0.549 g of ABTS were dissolved in 100 ml of the prepared
solution. After at least 1 h incubating at the room temperature, the characteristic
ABTS colour produced. The solution was also stable with the colour for 6 months at
a temperature of 4°C.
PROCEDURE
The antioxidant status was calculated with the help of a spectrophotometer after adjust-
ing at a wavelength of 660 nm. In 5 µl of the serum sample 200 µl of reagent 1 were
added in eppendorf. The first sample reading was taken as blank. Then the reading
with reagent 1 was noted. Then 10 µl of reagent 2 were mixed up with the solution
and again reading was noted. This reading was noted after the incubation of 3–4 min
with reagent 2.
The following formula was applied to calculate the values of TAS in the serum
samples of the Labeo rohita:
TAS = (Abs – 0.8366)/(–0.4991)
167
STATISTICAL ANALYSIS
The LC50 values were determined on the basis of probit analysis (Minitab version
15). The parameters of oxidative stress, i.e. ALT, AST, TOS and TAS were analysed
by the implementation of ANOVA (three-way completely randomised data analysis
in the study).
RESULTS
The present study showed that the level of serum enzymes increased with the increase
in heavy metal salts concentration. Similarly when the exposure time period increased
for the treated fishes the level of ALT also increased too many times in the blood stream
of the fish. In case of the control fishes with zero concentration of heavy metal salts
non-significant results were obtained in the present study (Figs 1a, b, c).
Fig. 1. Graph showing alanine aminotransferase level of Labeo rohita during different time intervals in
the treatment of: chromium – a; copper – b, and cadmium – c in acute toxicity experiment
The treatment of heavy metals salts in the chronic study showed that the ALT
level was present in order Cd > Cu > Cr (Table 1) in the serum samples of the fish.
The cadmium results in more production of ALT in the blood stream of the fish as
compared to copper and chromium. The analysis of variance for ALT showed highly
significant differences for metals, treatments and time intervals during the study pe-
riod. The interactions of metal × treatments, metal × time interval, treatment × time
interval and metal × treatment × time interval showed highly significant differences
p < 0.001 (Table 1).
168
Table 1. Analysis of variance of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total
oxidants status (TOS) and total antioxidant status (TAS) during the acute toxicity experiment
Source of variation DF ALT AST TOS TAS
Metal 2 77.53*** 173.03*** 420.61*** 276.92***
Treatments 6 1878.11*** 14829.16*** 1093.82*** 832.57***
Time interval 4 2063.06*** 1612.77*** 1687.24*** 689.03***
Metal × treatments 12 6.46*** 12.9*** 51.38*** 6.05***
Metal × time interval 8 8.90*** 37.34*** 27.05*** 35.75***
Treatments × time interval 24 229.07*** 1683.97*** 939.33*** 64.65***
Metal × treatments × time interval 48 1.96*** 6.98*** 26.12*** 5.22***
Error 210
Total 314
***
Highly significant (p < 0.001).
Similar results were obtained in case of the two months study period (chronic
study). The ALT in the control fishes observed in the range of 25 U/L, whereas its
concentration increased in the Labeo rohita treated with different concentrations of
heavy metal salts. Maximum value was 47.49 U/L in case of the copper metal with
a minimum value of 44.18 U/L in the chromium treatment. The results cleared the
fact that in the long time period study the copper showed more toxicity towards the
fish rather than the cadmium, which was more toxic for the 96 h study (Figs 2a–c).
Fig. 2. Graph showing alanine aminotransferase level of Labeo rohita during different time intervals
in the treatment of: chromium – a; copper – b, and cadmium – c in acute chronic toxicity experiment
The level of the AST was also studied for three heavy metals, chromium, cop-
per and cadmium in the Labeo rohita after an interval of 24 h for a time period of
96 h and two months. The results showed that the AST also increased in the serum
samples of the fish. However, the AST production was not as higher as the ALT in
the study (Figs 3a–c).
169
Fig. 3. Graph showing aspartate aminotransferase level of Labeo rohita during different time intervals
in the treatment of: chromium – a; copper – b, and cadmium – c in acute toxicity experiment
The highest production of AST was observed in the cadmium treatment and lowest
in the chromium treatment (Table 2). For the AST the significant results obtained in
the study period as both the concentration as well as time period enhances the enzy-
matic activity in the blood stream of the Labeo rohita for chronic study (Figs 4a–c).
Table 2. Analysis of variance of alanine aminotransferase (ALT), aspartate aminotransferase (AST),

total oxidants status (TOS) and total antioxidant status (TAS) during the chronic toxicity experiment
Source of variation DF ALT AST TOS TAS
Metal 2 1356.03*** 794.58*** 903.63*** 874.49***
Treatments 3 1303.93*** 1541.41*** 2370.06*** 3325.13***
Time interval 7 7368.8*** 9745.46*** 1413.37*** 2651.16***
Metal × treatments 6 799.21*** 969.68*** 120.56*** 120.13***
Metal × time interval 14 173.31*** 105.22*** 16.62*** 8.79***
Treatments × time interval 21 1583.96*** 1803.26*** 291.78 ***
319.14***
Metal × treatments × time interval 42 153.88*** 92.92*** 8.73*** 14.97***
Error 192
Total 287
***
Highly significant (p < 0.001).
170
Fig. 4. Graph showing aspartate aminotransferase level of Labeo rohita during different time intervals
in the treatment of: chromium – a; copper – b, and cadmium – c in chronic toxicity experiment
Total oxidants level in the serum of the Labeo rohita was also observed while
treated with the different concentrations of heavy metal salts for acute study (Figs
5a–c). The AST in case of the two months study with diluted heavy metals salts also
increased as for as the level of toxicity maintained in the control treatment for the two
months study (Figs 6a–c). From the result it was cleared that copper proved itself more
toxic for the fish for two months study as its concentration observed was 36.99 U/L
(Table 2). The statistical analysis of the obtained data showed significant differences
for the study as p < 0.001 (Table 2).
The more TOS level was seen in the samples treated with different concentra-
tions of the heavy metals, however less increase was observed in the fishes kept in
the ordinary tap water. The maximum value of TOS observed during the study was
for the chromium (Figs 5a–c and 6a–c). The value was 19.93 during the acute study
of 96 h and 18.73 for the two months (Tables 1 and 2).
171
Fig. 5. Graph showing total oxidant status of Labeo rohita during different time intervals in the treatment
of: chromium – a; copper – b, and cadmium – c in acute toxicity experiment
Fig. 6. Graph showing total oxidant status of Labeo rohita during different time intervals in the treatment
of: chromium – a; copper – b, and cadmium – c in chronic toxicity experiment
172
The ions of heavy metal salts as for as with increasing the oxidative stress also
decreasing the antioxidant activity in the Labeo rohita. The TAS level of the fish de-
creases with the increased concentration of heavy metal ions and similarly when the
exposure time period also increased the level decreased too much extent in the two
months study as well as 96 h study (Figs 7a–c and 8a–c). In case of the chromium
heavy metal salts it was 6.64 and 7.07 for the acute and the chronic study, respectively
(Tables 1 and 2). The cadmium heavy metal ions resulted 8.20a in the 96 h study and
decreases the level less as compared to the cadmium, whereas a value of 7.57 was
observed for the copper treatment during the two months study period.
Fig. 7. Graph showing total antioxidant status of Labeo rohita during different time intervals in the
treatment of: chromium – a; copper – b, and cadmium – c in acute toxicity experiment
173
Fig. 8. Graph showing total antioxidant status of Labeo rohita during different time intervals in the
treatment of: chromium – a; copper – b, and cadmium – c in chronic toxicity experiment
DISCUSSION
Polluted water bodies contained elevated levels of different heavy metal ions. These
heavy metal ions enter into the body of the fish and accumulate in various tissues
depending upon the concentration of heavy metals and exposure period. The heavy
metals exert oxidative stress on the fish along with decreasing the antioxidant status
that combat to reduce the oxidative stress on the fish8. In the present study the level
of oxidative enzymes too much increased in the 96-hour study as well as two-month
study. The high production of ALT, AST, and TOS along with the decreasing efficiency
of TAS is indication of the stress11. In a study it was reported that the increased level
of ALT and AST in the blood stream of the fish is due to the cell injury; this injury
of cell is due to the high absorption of metallic ions into the tissues12. The authors
explained that hepatic injury is due to the oxidative stress caused by the free radical
scavengers. In the present study the values noted for ALT and AST showed that the
level was gradually increased into the treated water medium of fish with the heavy
metals as compared to the fish kept under the ordinary tap water.
It was reviewed that several pesticides (pyrethroids and cypermethrin) result in
hematological as well as biochemical changes in birds, mammals and fish13. It was
discussed increased AST and ALT in the serum of the animals faced with the pesti-
cides, meaning that animals under oxidative stress resulting in the outflow of ALT
174
and AST into the blood stream. The use of insecticides and pesticides increased too
many times for the high agricultural production in Pakistan. These pesticides during
formulation treated with different metallic ions, also cause stress on the animals includ-
ing the fish14. This study also showed that the level of ALT in chronic experiment was
25.28 for control, whereas 70.60 maximum was observed in the highest concentration
during the two months study. Similarly, the level of AST in chronic experiment was
15.81 for control, whereas 60.82 maximum was observed in the highest concentration
during the two months study. The same was observed in case of the TOS and TAS
which was low in control fishes as compared to the treated ones. The overall results
explained that the concentration of the enzymes increased in the blood stream of the
fish with the increase in heavy metal pollutants.
CONCLUSIONS
The fishes under the treatment of heavy metal salts showed high stress as compared
to the fishes kept under the ordinary tap water, normal conditions. The stress was
shown by the fishes in the form of increased level of various enzymes in the serum.
The values increased too much which showed that the fishes are under stress.
REFERENCES
1. WHO: Environmental Health Criteria, Chromium. WHO, Geneva, 1988.
2. S. NAZ, M. JAVED: Growth Responses of Fish during Chronic Exposure of Metal Mixtures under
Laboratory Conditions. Pak Vet J, 33, 354 (2013).
3. A. BEGUM, S. HARIKISHNA, K. IRFANULLA: Analysis of Heavy Metals in Water, Sediments
and Fish Samples of Madivala Lakes of Bangalore, Karnataka. Int J ChemTech Re, 1, 245 (2009).
4. K. TODOROVA, I. VELCHEVA, S. PETROVA, V. YANCHEVA, S. STOYANOVA, E. GEOR-
GIEVA: Effects of Heavy Metals on Survival and Oxygen Consumption in Common Carp. Oxid
Commun, 37 (2), 563 (2014).
5. S. N. LUOMA, P. S. RAINBOW: Sources and Cycles of Trace Metals, Metal Contamination in
Aquatic Environment: Sciences and Lateral Management. Cambridge University Press, Cambridge,
2008.
6. I. VELCHEVA, E. GEORGIEVA, K. TODOROVA, S. PETROVA: Study on the Survival and Oxygen
Consumption of Carp after 96-hour Intoxication with Heavy Metals. Oxid Commun, 36 (4), 1127
(2013).
7. D. R. LIVINGSTONE: Oxidative Stress in Aquatic Organism in Relation to Pollution and Agriculture.
Revue de Med Veter, 154, 427 (2003).
8. M. SEVCIKOVA, H. MODRA, A. SLANINOVA, Z. SVOBODOVA: Metals as a Cause of Oxidative
Stress in Fish: A Review. Veterinarni Medicina, 56 (11), 537 (2011).
9. O. EREL: A New Automated Colorimetric Method for Measuring Total Oxidant Status. Clin Biochem,
38, 1103 (2005).
10. O. EREL: A Novel Automated Method to Measure Total Antioxidant Response against Potent Free
Radical Reactions. Clin Biochem, 37, 112 (2004).
11. M. I. YOUSEF, T. I. AWAD, E. H. MOHAMMAD: Deltamethrin-induced Oxidative Damage and
Biochemical Alterations in Rat and Its Attenuation by Vitamin E. Toxicology, 227, 240 (2006).
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12. P. R. MANNA, D. W. EUBANK, E. LALLI, P. SASSONE-CORSI, D. M. STOCCO: Transcriptional
Regulation of the Mouse Steroidogenic Acute Regulatory Protein Gene by the cAMP Response Ele-
ment Binding Protein and Steriodogenic Factor 1. J Mol Endocrinol, 30, 381 (2003).
13. K. AHRAR, L. AHMED, M. Z. KHAN: Hemato-biochemical Changes Induced by Pyrethroid In-
secticides in Avian, Fish and Mammalian Species. Int J Agr Biol, 14, 834 (2012).
14. A. R. AHMED, A. N. JHA, S. J. DAVIES: The Efficacy of Chromium as a Growth Enhancer for
Mirror Carp (Cyprinus carpio L.): An Integrated Study Using Biochemical, Genetic and Histological
Resposes. Biol Trace Elem Res, 148, 187 (2012).
Received 7 May 2015
176
STUDY ON THE ECOLOGICAL EFFECT OF PEAT ON THE

REMEDIATION OF Cd-CONTAMINATED VEGETABLE PLOT
DU RUIYINGa,b,c, WANG YANHONGa,b, TANG MINGDENGa,b,

LI MENGJUNa,b, AI SHAOYINGa,b*
a
Key Laboratory of Plant Nutrition and Fertilisers in South Region,
510 640 Guangzhou, China
E-mail: aishaoying209@163.com
b
Institute of Agricultural Resources and Environment, Guangdong Academy of
Agricultural Sciences, 510 640 Guangzhou, China
c
Laboratory of Quality and Safety Risk Assessment for Agro-product (Guangzhou),
510 640 Guangzhou, China
ABSTRACT
Effect of peat on cadmium content in soil morphology, cabbage growth and cadmium
content and ecological characteristics of the microbial using Cd-contaminated veg-
etable plot is investigated. The results showed that the pH of soil can be significantly
improved and effective Cd content in soil can be reduced after peat was added. Lasting
effect remains significant although three crops of cabbage were continuously planted.
The application of peat, continuous planting cabbage compared with the control can
significantly increase the biomass and decrease the Cd content in shoots. Continuous
planting of three crop of cabbage and the content of Cd increased, but were lower than
the limited value in GB2762-2012. Peat can increase the activity of microorganisms
in soil, peat and plant with more obvious effect on the increase in microbial activity
in soil. The ability to increase can promote Chinese cabbage growth and inhibit the
absorption of Cd by polymers, carbohydrates, carboxylic acids, amino acids and other
carbon sources.
Keywords: vegetable soil, cadmium pollution, microbial metabolic characteristics.
AIMS AND BACKGROUND

The term ‘trace elements’ is frequently used to refer to heavy metals (like Cd, Pb,
and Zn) and metalloids (such as As, Sb, and Se) that can cause environmental and
toxicological problems1–4. As the data available regarding the concentrations of these
*
For correspondence.
177
elements in soils and plants increase, it is gradually more apparent that considerable
areas of soil in many parts of the world are contaminated and pose toxicity problems
and that trace element pollution of land is extremely widespread. Soil pollution exists
where, due to human activities, a substance is present at a concentration above the
natural (background) level and has a negative impact on some or all of the constituents
of the environment5-7. The main anthropogenic sources of pollution can be divided into
two main groups: extensive contamination over large areas (atmospheric, flooding and
sediment deposition) and localised contamination (e.g. agriculture and horticulture,
urban soils and industrial, mining and smelting contamination)8.
The polluted area and the degree of the heavy metal polluted by the agricultural
soil are increasing, and soil remediation technologies become a research hot spot in
recent years. Chemical remediation, phytoremediation, microbial remediation meas-
ures have the characteristics of low cost, quick effect and so on, but less studies on
the effect of soil and crop growth. In this case, damage modifier, repair plant put into
production practice it is likely to bring the soil ‘heavy metal pollution’8–10. Cadmium
is a heavy metal element of biological toxicity, very strong, not only can cause serious
soil pollution, lead to a decline in crop yield and quality, but also through the migration
of soil plant food intake by human way, thereby endangering human health. Because
the soil composition is complex, Cd transformation in the soil and migration process
are also extremely complex, the research results reported are very different11–15. The
study on the Pearl River Delta farmland soil pollution being were mild, provides
more reasonable scientific basis for rational use of peat, but also provides a theoreti-
cal support for the development of ecological remediation of soil contaminated with
heavy metals in the field.
EXPERIMENTAL
Design of experiment. Continuous pot experiment was carried out with the acquisition
of representative soil (total Cd content of 10.54 mg/kg, pH 5.8). Modifier gradient peat
dosage was set for: 0, 7.5, 15, 22.5, 30 g/kg, 5 kg of soil in each pot, they are marked
for CK, T1, T2, T3 and T4, repeating 3 times. The vegetables were transplantted after
peat was applied into soil for balance 2 weeks, and the next crop of vegetables was
transplantted after harvest within 15 days. Three stubbles were continuous cropping.
Determination of the content of soil and plant metal. Determination of Cd in soil and
various forms of content was optimised by using European Community Bureau of
Reference (BCR) method. Acetic acid (HOAC) was used to change the solution extrac-
tion (1:40), Fe–Mn oxide bound was extracted by NH2OH HCl (0.5 mol/l) solution,
organic matter bound was extracted by H2O2 (8.8 mol/l) solution. The Cd content in
Chinese cabbage was determined by graphite furnace atomic absorption spectrometry
with wet digestion method referring to GB 5009.12-2010.
178
Analysis of microbial function parameters. Microbial functional diversity was analysed
by the 96 holes ecology (ECO) micro-plate made in America BIOLOG Company.
ECO micro-plate was divided into 3 districts and there were 32 holes in each district.
In addition to 1 hole without carbon source (blank) in each region and the remain-
ing 31 holes contained 31 kinds of carbon sources. 31 kinds of carbon sources in
ECO board for each region were divided into 6 classes of compounds according to
the chemical composition: carbohydrates, carboxylic acids, polymers, amino acids,
amines and miscellaneous. Absorbance value of each hole in 72 h was normalised.
The total average well-coloured development (AWCD) value was calculated according
to every kind of compounds including carbon source, and the principal component
analysis (PCA) was carried out.
Data analysis. Through single factor analysis of variance of the data measured by
statistical product and service solutions (SPSS) 16 statistical software was used to
analyse the correlation of 6 kinds of carbon source utilisation and availability, soil
metal plant metal content, the degree of correlation with the Pearson correlation coef-
ficient, correlation with the two tailed test.

Effect of peat on forms of Cd in soil. As shown in Table 1, 15 days after adding peat,
the peat amount on soil exchange state Cd content compared to the control, and no
significant difference is present. Continuous planting of three crop of cabbage, ex-
changeable Cd contents in all treatments were significantly lower than those of the
control, and exchangeable Cd content significantly decreased with increasing dosage
of peat and (p < 0.05). Cd content of Fe–Mn oxide bound in soil has no significant
difference compared to adding peat after 15 days in the control group, but in the cab-
bage planted after three consecutive crops was observed that iron-manganese oxide
bound Cd content significantly increased with increasing amount of peat, and with the
increase of the number of different cropping and control processing is more significant
(p < 0.05). In the whole process, the peat treatment has no significant difference on
organic Cd content in soil compared to the control group.
179
Table 1. Effect of peat on forms of Cd in soil (mg/kg)
Morphology Treat- Adding amend- First stubble Second stubble Third stubble
ment ments for 15
days
Exchange- CK 0.150±0.009a 0.167±0.003a 0.178±0.006a 0.164±0.002a
able form T1 0.245±0.043a 0.159±0.003b 0.165±0.003b 0.156±0.002b
T2 0.245±0.064a 0.142±0.002c 0.148±0.002c 0.146±0.001c
T3 0.221±0.038a 0.130±0.002d 0.143±0.006cd 0.139±0.002d
T4 0.173±0.017a 0.120±0.000e 0.132±0.002d 0.132±0.002e
Iron manga- CK 0.036±0.001a 0.143±0.002c 0.176±0.004c 0.145±0.008d
nese oxide T1 0.074±0.022a 0.144±0.001c 0.189±0.001b 0.156±0.002cd
combination T2 0.051±0.006a 0.150±0.002b 0.200±0.004a 0.160±0.003bc
state T3 0.060±0.013a 0.154±0.001b 0.205±0.002a 0.171±0.001ab
T4 0.038±0.002a 0.160±0.002a 0.203±0.003a 0.176±0.002a
Organic CK 0.068±0.003a 0.073±0.008a 0.064±0.006a 0.053±0.002a
combination T1 0.170±0.047a 0.061±0.001a 0.075±0.015a 0.062±0.012a
state T2 0.132±0.031a 0.066±0.003a 0.098±0.017a 0.053±0.002a
T3 0.151±0.055a 0.084±0.013a 0.077±0.012a 0.052±0.001a
T4 0.077±0.007a 0.071±0.002a 0.057±0.008a 0.051±0.002a
Note: data in the table are the average ± standard deviation (n = 4); CK – control group.
Effect of peat on the growth and Cd content of Chinese cabbage. As shown in Table 2,
the effect of peat on Pakchoi biomass is related to crop stubble number, and above-
ground biomass is present in the following order: second stubble > first stubble >
third stubble. Second crop biomass peat treatments were significantly higher than the
control treatment, but the third type T3 and T4 treatment significantly reduced the
biomass than the control treatment. First, the two batch of peat on Chinese cabbage
root biomass had no significant effect, but the third crop root biomass increased with
peat content decrease, T3 and T4 were significantly lower than the control, the change
trend is consistent with the ground.
Table 2. Effect of peat on Pakchoi biomass

Treat- Fresh mass of the part on the ground (g) Fresh mas of the part under the ground
ment (g)
first stubble second stubble third stubble first stubble second third stubble
stubble
CK 116.7±7.8b 156.9±4.1b 155.3±15.2ab 2.7±0.3a 3.9±0.3a 4.6±0.4a
T1 131.1±7.1b 205.1±5.3a 131.8±14.1ab 3.0±0.2a 4.6±0.2a 3.7±0.1ab
T2 160.5±15.2ab 189.1±35.9a 144.5±33.6ab 3.2±0.4a 4.6±0.7a 3.8±0.8ab
T3 174.1±20.1ab 201.3±18.6a 80.9±18.6b 3.4±0.8a 4.7±0.4a 2.4±0.2bc
T4 160.5±18.5ab 195.2±12.8a 82.6±32.1b 3.3±0.3a 4.8±0.4a 2.0±1.0c
Note: data in the table are the average ± standard deviation (n = 4).
180
Effect of peat on Cd content in Chinese cabbage. As shown in Table 3, adding peat can
significantly reduce the Cd content in Chinese cabbage. The Cd content in Chinese
cabbage increases with increasing planting time, peat treatment with three stubbles
could significantly decrease the content of Cd in Chinese cabbage compared with the
control group. There is no significant difference between first and second crop plant-
ings. There is significant difference about T3 and T4 on the reduction of Cd content
in Chinese cabbage compared with the other two treatments (p < 0.05). Effect of peat
on Cd content in Chinese cabbage has no significant difference compared with the
control group (p < 0.05).
Table 3. Effect of peat on Cd content in Chinese cabbage

Treat- Content of Cd of the part on the ground Content of Cd of the part under the ground
ment (μg/kg) (μg/kg)
first stubble second stubble third stubble first stubble second stubble third stubble
CK 3.82±0.20a 73.86±6.92a 142.09±7.85a 1.50±0.15abc 258.35±63.08a 65.74±3.53b
T1 2.25±0.16bc 53.69±4.02b 102.61±6.72bc 1.41±0.17abc 306.75±23.17a 61.68±3.61b
T2 2.13±0.15bc 45.18±2.23b 118.89±6.02ab 1.34±0.14abc 324.87±62.32a 196.70±52.00a
T3 2.00±0.22bc 41.82±3.36b 77.04±9.82cd 1.06±0.20bc 226.82±32.12a 201.40±25.57a
T4 1.73±0.22c 43.34±3.18b 58.50±10.83d 0.91±0.12c 244.01±16.11a 131.57±60.84ab
Note: data in the table are the average ± standard deviation (n = 4).
Effect of peat and cabbage on soil microbial community AWCD. Microbial activity of
soil is expressed by AWCD value in BIOLOG ECO board, which is shown in Fig. 1.
The AWCD value showed S curve along with the culture time extended. Microbial
activity of soil significantly was improved after administration improver compared
with the control group, and the AWCD value is about 2 times of the control group. The
AWCD value (Fig. 1a) increased rapidly in 72 h. The value of AWCD after planting
Chinese cabbage (Figs 1b, c and d) began to rise rapidly from 24 h and began to flatten
at 96 h. Microbial activity in soil increased by 1.5–3 times in the process of continuous
planting cabbage than before planting Chinese cabbage. Microbial activity achieves
maximum with the second stubble, and increase of AWCD value with T2 is the most
obvious. But AWCD value with T2 is lower than T3 and T1 with the third stubble.
181
Fig. 1. Effect of modifier – cabbage on soil microbial activity: a – planting cabbage; b – the first stubble;
c – the second stubble; d – the third stubble
Effect of modifiers and Chinese cabbage on the soil microbial carbon utilisation abil-
ity. Based on understanding the microbial utilisation of 31 kinds of carbon sources,
31 kinds of carbon sources were divided into 6 classes of compounds according to
the chemical composition: carbohydrates, carboxylic acids, polymers, amino acids,
amines and miscellaneous. Which kind of carbon source is with increased microbial
activity contribution is further to understand. The classification of 6 kinds of carbon
sources was carried on the AWCD value at 72 h and the result is shown in Fig. 2. The
ability of using 6 kinds of carbon source for all of microbial community in soil after
planting Chinese cabbage significantly increased, and the ability of using different
carbon sources was 4–5 times than no planting.
Correlation analysis among carbon species, effective state of metal in soil and each
indicator of the plants. The results of the correlation analysis among 6 carbon sources,
metal content in plants and available metal content in soil are shown in Table 4. There
is no significant correlation between 6 carbon sources and content of exchangeable Cd
in the soil. In addition to the amine class, it showed a significant positive correlation
between 5 class carbon source and Cd, the oxidation state of iron and manganese,
organic bound and aboveground biomass, amino acids and cabbage belowground
carbon content of cadmium in soil Cd content of iron and manganese oxidation states
and underground fresh weight and Cd content, soil organic bound Cd content in shoot
fresh weight of cabbage, cabbage root fresh weight and cadmium content (P < 0.05).
182
It showed a significantly negative correlation between sugars carbon and Cd content
of cabbage shoots (P < 0.01).
Fig. 2. Effect of planting Chinese cabbage on utilisation ability of different carbon sources by micro-
organisms in soil: a – planting Chinese cabbage; b – the first stubble; c – the second stubble; d – the
third stubble
Analysis of the rate of change of different carbon sources on the PC axis contribu-
tion. Table 5 shows the carbon source for the contribution rate of the absolute value
greater than 0.2 in PC1 and PC2 axes. The use of microorganisms to carbon source
has only 4 kinds before growing vegetables, and using microorganisms to carbon
source increased after planting vegetable. Increase of the number of microorganisms
on different carbon sources also increased utilisation capacity, and it increased to 9,
11, 15 after planting 3 stubbles of vegetables.
183
184
Table 4. Correlation analysis among carbon species, effective state of metal in the soil and each indicator of the plants
Exchange- Iron manga- Organic Fresh mass Fresh mass Content of Content of
able specia- nese oxide combination of the part on of the part Cd of the Cd of the
tion combination state the ground under the part on the part under
state ground ground the ground
Polymer –0.362 0.669** 0.609* 0.518* 0.387 –0.378 0.448
Carbohydrate –0.047 0.343 0.704** 0.603* 0.329 –0.662** 0.184
Carboxylic acid 0.082 0.557* 0.739** 0.641** 0.433 –0.473 0.469
Amines –0.387 0.306 0.396 0.280 0.069 –0.370 0.143
Amino acids 0.031 0.700** 0.577* 0.697** 0.496 –0.422 0.578*
Others 0.075 0.516* 0.603* 0.600* 0.423 –0.459 0.454
Exchangeable speciation –0.181 –0.017 0.020 0.218 0.327 0.179
Iron manganese oxide combination state 0.299 0.502 0.527* 0.045 0.853**
Organic combination state 0.587* 0.345 –0.495 0.156
Fresh mass of the part on the ground 0.877** –0.146 0.369
Fresh mass of the part under the ground 0.293 0.531*
The content of Cd of the part on the ground 0.371
* Means significant at 0.05 level; ** means significant at 0.01 level.
Table 5. PC axis on the contribution rate of absolute value greater than 0.2 carbon source
Carbon source Preplant First stubble Second stubble Third stubble
PC1 PC2 PC1 PC2 PC1 PC2 PC1 PC2
Methyl pyruvate –0.112 0.881
Twain 40 –0.085 0.399
Alpha-cyclodextrin 0.114 0.479 0.250 –0.242
Glycogen 0.388 –0.004
D-fibre two sugar –0.204 0.217 0.216 0.310
Alpha-D-galactose 0.235 0.002 0.289 –0.3240.043 –0.217
Beta-methyl-D-glucoside –0.166 0.245
D-xylose 0.545 0.009 –0.450 0.557 0.344 0.536 0.081 0.227
D-mannitol 0.346 0.029
N-acetyl-D-glucosamine 0.277 0.369
D-glucosamine 0.232 0.055
Glucose-1-phosphate 0.187 0.213
D-galactose acid gamma- –0.007 0.303 0.127 0.463
lactone
Itaconic acid 0.320 0.178
Alpha-ketobutyric acid 0.066 –0.208
D-malic acid 0.422 0.123 0.342 –0.283 0.147 –0.271
L-asparagine 0.807 0.139
L-phenylalanine –0.250 –0.045
L-arginine 0.394 –0.270
L-serine 0.239 0.115 0.072 0.293 0.205 –0.101
L-threonine 0.030 0.444
Glycyl-L-glutamic acid 0.202 –0.104
Phenethylamine 0.504 0.273 0.538 0.039 0.388 –0.285
Putrescine 0.173 0.220 0.238 –0.108
CONCLUSIONS
1. The application of peat can significantly improve the soil pH and reduce the available
content of Cd in soil, and lasting effect remains significant with continuous planting
3 stubbles of cabbage.
2. Applying the peat can significantly increase its biomass and reduce the shoot
Cd content after continuous planting stubbles of cabbage, even compared with the
control group. Cd content tended to increase after continuous planting 3 stubbles of
cabbage, but it is lower than the limit value of GB2762-2012.
3. Peat can increase microbial activity in soil, and peat combined with plant
increased microbial activity in soil more effective.
4. Increasing carbon utilisation ability of polymers, carbohydrates, carboxylic
acids, amino acids and other types can promote the growth of Chinese cabbage and
inhibit the absorption of Cd.
185
ACKNOWLEDGEMENTS
This work was supported by Guangdong Academy of Agricultural Sciences Dean
Fund Project (201116); Guangzhou science and technology plan project (2060404).
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in Soils by Means of Multi-indexes. Chinese J Environ Sci, 29 (9), 2606 (2008).
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M. B. KIRKHAM, K. SCHECKEL: Remediation of Heavy Metal(Loid)s Contaminated Soils – to
Mobilize or to Immobilize? J Hazard Mater, 266, 141 (2014).
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15. J. PICHTEL, D. J. BRADWAY: Conventional Crops and Organic Amendments for Pb, Cd and Zn
Treatment at a Severely Contaminated Site. Bioresour Technol, 99 (5), 1242 (2008).
Revised 17 May 2015
186
DIRECT LABELLING OF MEDICALLY INTERESTING

Tc-BENZYL PENICILLIN
99m
T. H. BOKHARIa*, M. U. AKBARa, S. HINAb,c, M. USMANa, A. HAQa,

S. ROOHId, S. SAEEDe, M. IQBALf
a
Department of Chemistry, Government College University, Faisalabad, Pakistan
E-mail: tanveer.bokhari@yahoo.com
b
Department of Bioinformatics and Biotechnology, Government College University,
c
Department of Zoology, Government College Woman University, Faisalabad,
Pakistan
d
Isotope Production Division, Pakistan Institute of Nuclear Science and
Technology, P. O. Nilore, Islamabad, Pakistan
e
Pakistan Institute of Engineering and Applied Sciences, P. O. Nilore, Islamabad,
Pakistan
f
National Centre of Excellence in Physical Chemistry, University of Peshawar,
Pakistan
ABSTRACT
More than 99% radiolabelling of benzyl penicillin with 99mTc was achieved using
SnCl2.2H2O as reducing agent. The optimised conditions for the radiolabelling of
99m
Tc- benzyl penicillin were 200 µg ligand (benzyl penicillin), 25 µg stannous chlo-
ride as a reducing agent, pH 6, room temperature and 10 min incubation time. The
radiochemical purity, stability and charge on 99mTc labelled benzyl penicillin were
determined by paper chromatography, ITLC, HPLC and electrophoresis techniques,
respectively. Biodistribution and scintigraphy studies of 99mTc-benzyl penicillin were
performed in normal mice and rabbit, respectively.
Keywords: benzyl penicillin, technetium-99m, imaging agent, HPLC, biodistribution,
scintigraphy.
AIMS AND BACKGROUND

The use of imaging technique increased day by day in both medical and research
field. Many antibiotics were labelled with different radioisotopes to fulfil this pur-
pose1–3. 99mTc is an important radionuclide among all the radionuclide because it emits
*
For correspondence.
187
gamma-radiation of 140 keV which easily penetrate in the living body and help out
the physicians to evaluate the functions of any organ4,5. 99mTc is a metastable nuclear
isomer of 99Tc having ideal half-life (6 h) and it is used approximately more than 80%
of the nuclear medicine due to this reason it is also known as medical radioisotope6–13.
Benzyl penicillin is an effective and impotent antibiotic which is generally used to treat
infections caused by susceptible bacteria. Benzyl penicillin has semi-synthetic origin
and belongs to peptidoglycan synthesis inhibitor pharmacological group on the basis
of mechanism of action. This research work deals with the radiolabelling of benzyl
penicillin with one of the most important radionuclide 99mTc. Different factors that
affecting the labelling yield of 99mTc-benzyl penicillin have been studied and bioevalu-
ation of the 99mTc-benzyl penicillin has been conducted in normal mice and rabbits.
EXPERIMENTAL
Reagents and chemicals. All chemicals which used in this research work were reactive
grade reagent and used without any further purification. The activity of technetium-99m
was obtained from 99Mo/99mTc generator produced by PINSTECH, Islamabad, Pakistan.
Labelling of 99mTc-benzyl penicillin. For the labelling of benzyl penicillin with 99mTc,
exactly 200 μg benzyl penicillin were taken in neat and clean vial, then 22–25 μg of
reducing agent (stannous chloride) were accurately added and pH 6.0 was adjusted
by using 0.5 M NaOH and 0.5 M HCl. One ml of freshly eluted 99mTcO4– (~13 mCi)
from 99Mo/99mTc generator was injected into the vial containing the above reagents
was incubated for 10 min at room temperature. All the experiments were conducted
at room temperature of 25±2°C. For each labelling experiments, labelling yields
were determined by ‘Whatman number 3’ ascending paper chromatography strips
(each 14 × 2 cm2) and ITLC-SG strips (each 7 × 1 cm2). Free 99mTcO4– in 99mTc-benzyl
penicillin was calculated by using Whatman No 3 paper as the stationary phase and
acetone as mobile phase. Reduced and hydrolysed activity in 99mTc-benzyl penicillin
was determined by using instant thin layer paper chromatography (ITLC-SG strips)
as the stationary phase and 0.05 M NaOH as a mobile phase. The radio colloids were
separated out with the help of 0.22 μm bacteria filter paper (Millipore Filter Corp).
During the passing of 99mTc-benzyl penicillin solution on filter paper, the radio col-
loids activity remaining on the filter was counted by a gamma-counter (Ludlum). The
stability of 99mTc-labelled benzyl penicillin was determined at room temperature and
different time intervals. The distribution of 99mTc-labelled benzyl penicillin, free and
hydrolysed on chromatographic strips was calculated with the help of a 2π scanner
(Berthold, Germany). In case of unavailability of 2π scanner, we can cut the strips
into 1 cm segments and counted by a gamma-counter.
Electrophoresis of 99mTc-benzyl penicillin. The charge on 99mTc-labelled benzyl peni-
cillin complex was determined by using a Deluxe electrophoresis chamber (Gelman)
system. The Whatman No 1 paper of 30 cm long and 2 cm wide strip was moistened
188
with phosphate buffer and then was introduced in electrophoresis chamber which
contained the phosphate buffer of pH 6.0. A drop of 99mTc-benzyl penicillin mix-
ture (sample) was placed at 12 cm far from the cathode edge of the paper sheet and
electrophoresis apparatus was run for 1 h at a voltage of 300 V. After completion of
electrophoresis process, the strip was dried and scanned by using a 2π scanner to
determine the charge on 99m Tc-labelled benzyl penicillin.
HPLC of 99mTc-benzyl penicillin. The 99mTc-benzyl penicillin complex was also ana-
lysed on a high performance liquid chromatography (HPLC) instrument. The HPLC
system which was used for the analysis of 99mTc-benzyl penicillin is D-200 Elite HPLC
system which contains C-18 column as a stationary phase and a mixture of water and
acetonitrile ratio 75:25 (v/v %) was used as a mobile phase. The flow rate of mobile
phase was 1 ml/min and NaI detector was used.
Stability of 99mTc-benzyl penicillin. To evaluate the stability of the 99mTc-benzyl peni-
cillin in isotonic saline after labelling, we checked its stability after different time
intervals such as 0.5, 1, 2, 3, 4, 5 and 6 h at room temperature. Any change in stability
of the 99mTc-benzyl penicillin complex was determined with the help of ITLC-SG and
paper chromatography techniques which clearly showed if any type of dissociation
of the complex occurred.
Stability in human serum. The stability of 99mTc-benzyl penicillin with human serum
was also determined. For this purpose 1.8 ml of normal human serum were mixed
with 0.2 ml of 99mTc-benzyl penicillin and incubated at 37oC up to 24 h. Then 0.2 ml
aliquots were withdrawn during the incubation at different time intervals up to 24 h
and put on to the chromatography strips for determination of 99mTc-benzyl penicillin,
reduced/hydrolysed 99mTc and free 99mTcO4–.
Biodistribution of 99mTc-benzyl penicillin in mice. Male mice were used for biodistri-
bution having weight ranges 30–50 g (4–8 weeks) and they were obtained from the
National Institute of Health (NIH), Islamabad, Pakistan. They were anesthetised with
diazepam injection. 99mTc-benzyl penicillin was prepared and then injected slowly
into the tail vein of mice. After injection of 99mTc-benzyl penicillin (2 mCi/mice),
distribution of radioactivity in various organs was calculated. The various organs
were dissected, weighed and counted for radioactivity at the various time intervals
(1, 4 and 24 h). Data were represented as the percentage of injected dose per gram
of tissue (% ID/g).
99m
Tc-benzyl penicillin scintigraphy in rabbit. The gamma-camera was used for the
scintigraphic images. The each animal was placed on a flat and hard surface with
both hind legs spread out and all legs fixed with surgical tape. Diazepam injection
(2 ml) was injected into the left thigh muscle. Saline (0.2 ml) containing 15 MBq of
99m
Tc-benzyl penicillin was then injected intravenously into the marginal ear vein of
rabbit. For the distribution study of 99mTc-benzyl penicillin in living organism (rabbit),
whole body imaging was done at 1, 2 and 24 h after post injection.
189
Benzyl penicillin is an important antibiotic which is generally used to treat infections
caused by susceptible bacteria. The results of standard chromatography techniques
show more than 99% labelling of benzyl penicillin with 99mTc. There were many fac-
tors that played their role to obtained highest labelling yield of 99mTc-benzyl penicillin
complex. As shown in Fig. 1, benzyl pencillin was poorly labelled with 99mTc at low
pH, the labelling yield was small and almost equal to 64% at pH 1 and it was noted
that by increasing the pH of the reaction mixture the labelling yield was increased
and at pH 6 the labelling yield of 99mTc-benzyl penicillin significantly increased and
reached more than 99% and then further increasing in the pH the labelling yield slowly
decrease and at pH 10 the labelling yield of 99mTc-benzyl penicillin was 81%, so furthers
experiments were conducted at pH 6. The correlation between the stannous chloride
and benzyl pencillin was examined and illustrated in Fig. 2. Maximum labelling of
benzyl penicillin with 99mTc was achieved by using the 25 µg stannous chloride as a
reducing agent. By increasing and decreasing the amount of stannous chloride from 25
µg the labelling yield of 99mTc-benzyl penicillin moves towards lower side and hence
for the labelling of benzyl penicillin (200 µg) with 99mTc (13 mCi) 25 µg reducing
agent was the optimised amount. The effect of amount of benzyl penicillin is shown
in Fig. 3. The labelling of 99mTc-benzyl penicillin complex was 56% at 50 µg of ben-
zyl penicillin and it also increased with increasing the amount of benzyl penicillin.
More than 99% labelling was observed at 200 µg of benzyl penicillin. So, it means
the optimum amount of benzyl penicillin was 200 µg. The effect of incubation time or
reaction time on the labelling of 99mTc-benzyl penicillin is described in Fig. 4. Highest
labelling yield was attained after 10 min of incubation time and it remained same up
to 30 min. It was approximately 89% after one minute of post labelling which was
low. So, for getting the highest labelling efficiency at least 10 min incubation time is
necessary. The stability of 99mTc-benzyl penicillin at room temperature is shown in
Fig. 5. The newly formed complex was highly stable in saline solution and a slight
decrease was observed with the passage of time. More than 91% was maintained in
vivo for up to 6 h (Fig. 5) and the stability of 99mTc-benzyl penicillin with normal
human serum was also excellent (Fig. 6). The newly formed 99mTc-benzyl penicillin
complex displayed no charge on paper electrophoresis because 99mTc-benzyl penicil-
lin does not moved from the origin at the applied conditions as shown in Fig. 7. The
HPLC radiochromatogram is shown in Fig. 8 and it has only one single peak at the
retention time approximately 2 min which confirmed the absence of impurities in
the formation of 99mTc-benzyl penicillin complex. The biodistribution studies of the
99m
Tc-benzyl penicillin complex were conducted in normal mice. The biodistribution
results of normal mice showed rapid accumulation in liver, lungs, spleen, kidney and
bladder (Fig. 9). The scintigraphic images of normal rabbit after administration of
99m
Tc-benzyl penicillin is shown in Fig. 10. These results strongly suggest that 99mTc-
benzyl penicillin may be used for the clinical images.
190
100
80
labelling efficiency (%)

60
40
20
0
0 1 2 3 4 5 6 7 8 9 10
pH
Fig. 1. Effect of pH on labelling of 99mTc with benzyl penicillin
110
100
90
80
70
60
50
40
30
20
10
0
0 5 10 15 20 25 30 35 40
amount of SnCl 2.2H 2O (µg)
Fig. 2. Effect of the amount of SnCl2.2H2O on labelling of 99mTc with benzyl penicillin
Fig. 3. Effect of the amount of ligand on labelling of 99mTc with benzyl penicillin
110
100
90
80
70
60
50
0 10 20 30 40
incubation time (min)
Fig. 4. Effect of the incubation time on labelling of 99mTc with benzyl penicillin
191
102

100
98
96
94
92
90
0 2 4 6 8
time (h)
Fig. 5. in vivo Stability of 99mTc-benzyl penicillin complex at room temperature
110 99m
Tc-benzyl penicillin
100
90 free pertechnetate
labeling efficiency (%)
80
70 colloid/hydrolysed
60
50
40
30
20
10
0
1 4 24
incubation time (h)
Fig. 6. Stability of 99mTc-benzyl penicillin in normal human serum
Fig. 7. Electrophoresis of 99mTc-benzyl penicillin
Fig. 8. HPLC analysis illustrating the percentage labelling of benzyl penicillin with 99mTc
192
activity accomulation in organ (%)
20 1h
4h
24 h
10
body organs
Fig. 9. After the injection of Tc-benzyl penicillin at 1, 4 and 24 h
99m
Fig. 10. Whole body gamma-camera image of rabbit injected with 99mTc-benzyl penicillin at 1.5 h post
administration (a), 4 h post administration (b) and 24 h post administration (c)
CONCLUSIONS
In conclusion, more than 99% of labelling yield of 99mTc-benzyl penicillin was suc-
cessfully achieved. The resulting complex 99mTc-benzyl penicillin has excellent sta-
bility both in vivo and in vitro and more than 91% labelling yield maintained for up
to 24 h. 99mTc-benzyl penicillin complex distributed well in the liver, kidney, bladder,
spleen and lungs. Approximately more than 16, 17 and 7% of the99mTc-benzyl peni-
cillin activity was remained in the lungs at 1, 4 and 24 h post injection, respectively.
Further studies are required to determine the application of 99mTc-benzyl penicillin
as a nuclear medicine.
193
ACKNOWLEDGEMENTS
T. H. Bokhari gratefully acknowledges the Higher Education Commission, Pakistan,
for financial support of the research project (PM-IPFP/HRD/HEC/2011/0595).
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Received 3 September 2014
Revised 20 October 2014
194
ANTIPHOSPHOLIPID SYNDROME AND ATHEROSCLEROSIS
N. STOILOVa*, VL. BOYADZHIEVAa, R. RASHKOVa, S. STEFANOVb,

B. MARINOVc, V. VLADIMIROVd
a
UMBAL ‘St. Ivan Rilski’, Clinic of Rheumatology, Medical University, Sofia,
Bulgaria
E-mail: dr_nstoilov@yahoo.com
b
SBAL ‘Detski Bolesti’ , Clinic of Rheumatology, Cardiology and Haematology,
Medical University, Sofia, Bulgaria
c
SBALAG ‘Maichin Dom’, Clinic of Pathological Pregnancy, Medical University,
Sofia, Bulgaria
d
UMBAL ‘Tzarica Joanna’, Clinic of Urology, Medical University, Sofia, Bulgaria
ABSTRACT
Antiphospholipid syndrome (APS) is an autoimmune, multisystem disease, with com-
plex pathogenesis and unknown etiology. Nearly 36% of patients with SLE exhibit
co-morbidity with APS in the European population. Antiphospholipid syndrome is
characterised by two groups of antibodies, one is directed against certain phospho-
lipids, and other, against their protein cofactors. In antiphospholipid syndrome is
used the revised classification criteria of Sydney in 2006. Atherosclerosis is the most
common pathological process affecting the vessel wall causing cerebrovascular and
cardiovascular diseases, leading to vascular events. In recent years, it is found that
role in atherogenesis plays inflammatory component of immune response.
Some autoimmune diseases such as systemic lupus erithematosus (SLE), antiphos-
pholipid syndrome (APS), rheumatoid arthritis (RA) and others are characterised by
the development of accelerated atherosclerosis.
In our group, we studied 23 patients fulfilling revised classification criteria for
antiphospholipid syndrome Sydney 2006 and established a difference in intima-media
thickness (IMT) of patients and control guppy approximately 100 μm. In statistical
processing is demonstrated strong correlation between IMT of patients and controls.
Keywords: antiphospholipid syndrome, atherosclerosis, intima-media thickness.
*
For correspondence.
195
AIMS AND BACKGROUND
Antiphospholipid syndrome (APS) is an autoimmune, multisystem disease character-
ised by thrombocytopenia, thrombosis of blood vessels and/or pathological course of
pregnancy in women (eclampsia, pre-eclampsia, recurrent spontaneous abortions, still
births) in the presence of hetrogeneous group of autoantibodies – antiphospholipid
antibodies (aPL) (Refs 1–3).
Presented alone, the disease is classified as ‘primary antiphospholipid syndrome’
(PAPS), and when in the context of another disease, ‘secondary’ (SAPS) (Ref. 4).
Most often secondary antiphospholipid syndrome associated with systemic lupus
erythematosus (SLE) (Ref. 5). Nearly 36% of patients with SLE exhibit co-morbidity
with antiphospholipid syndrome (APS) in the European population.
In other rheumatic diseases as often an APS is found in: lupus-like syndrome
(5%), Sjogren syndrome (2.2%), rheumatoid arthritis (1.8%). In other nosological
units vasculitis, myositis and others, they are about 0.5% (Refs 6–8).
The presence of antiphospholipid antibodies may be associated with other, rheu-
matic diseases: the Crohn disease, malignancies, sarcoidosis, diabetes, atheroma,
infectious diseases – leprosy, syphilis, HIV, HCV, cytomegalovirus.
Some medications may induce synthesis of aPL antibodies: hydralazine, procaina-
mide, phenytoin, kinin/quinidine, interferon, biological agents and others.
In standardised research is found that about 1% of young individuals and up to
3% of the adult population was evidence of antiphospholipid antibodies, without
leading to any clinical manifestation of APS.
Antiphospholipid syndrome is characterised by two groups of antibodies, one is
directed against certain phospholipids, and others, against their protein cofactors9,10.
Antibodies against phospholipids: cardiolipin, phosphatidylserine, phosphate acid,
phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine.
Antibodies against phospholipid binding proteins: Β2GPI, Annexin V, protein C, pro-
tein S, low and high molecular kininogens, Factor XII, •S4b binding protein, fractions
complement C4 and C5, heparan sulphate, thrombin, etc.
For determining antiphospholipid syndrome we have used the revised classifica-
tion criteria from Sydney in 2006. In clinical practice, the classification criteria are
also used as diagnostic, causing errors in diagnosis11–13. They are divided into two
groups: clinical and laboratory criteria.
CLINICAL CRITERIA
Vascular thrombosis. One or more episodes of arterial, venous and/or small vessel
thrombosis in any tissue or organ. Thrombosis must be proven by an objective vali-
dated method or histologically. In histological proof must be excluded any likelihood
of inflammation in the vessel wall.
196
Pathological course of pregnancy. One or more spontaneous abortions after 10 weeks
in morphologically healthy faetus, and this must be documented by ultrasography or
direct examination of the faetus.
– One or more births of morphologically healthy fetus before 34 weeks because
of pre-eclampsia, documented criteria adopted for placental insufficiency.
– Three or more miscarriages before 10 weeks the exclusion of all maternal
anatomical, hormonal and chromosomal pathology.
LABORATORY CRITERIA
Lupus anticoagulants registered twice in the 12 weeks examined according to gidline

of ‘International society in thrombosis and haemostasis’. Anticardiolipin antibodies,
IgG and / or IgM in high or medium high titers twice every 12 weeks were identified
by the method of ELISA.
The diagnosis is considered sure in the presence of a laboratory and a clinical
criteria or in the presence of two clinical criteria. Anti-beta 2 glicoprotein I antibod-
ies, IgG and/or IgM in high or medium high titers twice every 12 weeks out of the
plasma by the method of ELISA. Atherosclerosis is a multifocal, slowly progressive,
immune-mediated, inflammatory disease engaging arterial wall. It is characterised
by an accumulation of lipid particles in the vascular wall, leading to thickening of
intima, resulting in the reduced elasticity of the vessel wall occurs occlusion of the
lumen of the blood vessel and reduction in blood flow.
Atherosclerosis is the most common pathological process affecting the vessel
wall causing cerebrovascular and cardiovascular diseases, leading to vascular events.
In atherogenesis involved acquired factors, smoking, hypercholesterolaemia,
diabetes mellitus and others14.
In recent years, it is found that role in atherogenesis plays inflammatory component
of immune response. Some autoimmune diseases such as SLE, APS, RA and others,
are characterised by the development of accelerated atherosclerosis15. A sure sign of
this is the increased morbidity and mortality from CVD among patients with autoim-
mune rheumatic diseases compared to the general population14. This phenomenon is
conditioned by traditional risk factors for developing aterosclerosis, complications of
therapy (CS) and other autoimmune and inflammatory mechanisms that accompany
rheumatic diseases.
LDL cholesterol is a major physiological role by participating in: the construction
of cell membrane synthesis of hormones and others. In essence LDL is non-pathogenic,
non-atherogenic particle. Strong proatherogenic function has oxidised form, oxLDL.
Oxidation of LDL was proceeded in the circulation and the intima of free radicals
H2O2. Oxidised LDL is the primary component in the process of atherogenesis with
a powerful pro-inflammatory action16. After invasion of the intima, oxLDL activates
endothelial cells, which leads to the expression of adhesion molecules and chemokines
secretion by attracting leukocytes from the circulation into the subendothelial space.
Thus begins the formation of atherosclerotic plaque. Monocytes and macrophages
197
infiltrating atherosclerotic areas, as borne oxLDL and transformed into ‘foam cells’,
which in turn induce the synthesis of pro-inflammatory mediators.
B2GPI initially was considered a natural anticoagulant subsequently found in
a number of pleiotropic processes: fibrinolysis, angiogenesis, apoptosis and athero-
genesis17,18.
β2GPI recognise oxLDL forming complex – β2GPI/oxLDL, through oxidised
lipid epitopes. So b2GPI manifests antioxidant antioskdantni properties, neutralising
oxLDL. The complex β2GPI/oxLDL is with immunogenic action leading the for-
mation of autoantibodies. They interact with ‘scavenger’ receptors, resulting in the
Fc-mediated uptake of LDL by macrophages. β2GPI inhibit the uptake of oxLDL by
makrofgite, thereby playing the role of anti-atherogenic protein, which is inhibited
by anti- β2GPI. The complex oxLDL/β2GPI, can join CRP, leading to the formation
of the complex – oxLDL/β2GPI/CRP. Antibodies against complex-oxLDL/β2GPI/
CRP lead to Fcγ-mediated its takeover by macrophages.
For patients with autoimmune diseases and accelerated atherosclerosis are estab-
lished oxLDL/β2GPI/CRP complexes in the atherosclerotic plaque.
The aim of our study is to evaluate the influence of aPL in the process of athero-
genesis and the correlation between aPL and intima-media thickness (IMT).
EXPERIMENTAL
In our group, we studied 23 patients fulfilling revised classification criteria for an-
tiphospholipid syndrome Sydney 2006. Patients were tested for antiphospholipid
antibodies directed against cardiolipin (aCL), β2glikoprotein 1 (anti-β2GPI) and
prothrombin (anti-Prothrombin), by the method of ELISA. Samples were tested in
the immunological laboratory at the University Hospital ‘St. Ivan Rilski’.
The process of atherosclerosis is validated by ultrasonography of the carotid
arteries as measured thickness of the intima-media space (intima-media thickness).
Intima-media thickness was evaluated on ultrasound machine eSaote MyLab60 with
linear probe L 4–13 MHz software and hardware MEASURING intima-media (QIMT).
Measurements of IMT were made of predilection place up to 2 cm from the bifurca-
tion to A. carotis interna and A. carotis externa. Standardised scans – longitudinal
front (LA), longitudinal latral (LL) and longitudinal posterior (LP) were used, the
investigations were carried out by a single licensed investigator.
Antiphospholipid syndrome was significantly more strongly present among fe-
males, at 82.6% of the cases detected in women and 17.4% among men. The average
age at the time of the visit to the doctor is 45.4 years. Many of the patients reported
risk factors such as smoking (78.2%). In 87% of patients, the APS is in the context
of other disease and only at 13% to establish a primary APS.
198
Fig. 1. Ultrasonography image of the right carotid artery with atherosclerotic plaque
Fig. 2. Doppler ultrasonography of the right common carotis artery showing the stenosis of the blood vessel
199
Table 1. Demographic data of the APS patients
Patients with APS
Gender male – 4 (17.4%);
female – 19 (82.6%)
Age 45.4 y
Risk factors tobacco – 19 (78.2%);
alcohol – 0 (0%)
BMI 25.4
Primary APS 3 (13%)
Secondary APS 20 (87%)
For a control group selected 20 ‘healthy controls’ matched for sex, age, risk fac-
tors and BMI of patients from the antiphospholipid syndrome.
Table 2. Demographic data of the control group

Control group
Gender male – 3 (15%);
female – 17 (85%)
Age 43.6 y
Risk factors tobacco – 12 (60%);
alcohol – 0 (0%)
BMI 24.7
Primary APS NA
Secondary APS NA
In statistical processing were used the following statistical analysis: correlation

analysis Pearson and ANOVA.

Mean intima-media thickness for the group of patients are – right common carotid
artery (RCCA) – 550 μm, and for the left common carotid artery (LCCA) – 585 μm.
Mean intima-media in healthy controls were: RCCA – 496 μm, LCCA – 499 μm.
Significant differences were found in the values of IMT in patients and control
groups. There was a statistically significant difference between IMT in patients and
control group.
When carried out the Pearson correlation analysis, it was found that there was
no statistically significant correlation between the titers aPL and IMT in the group
of patients. It was found a strong correlation between IMT and age of patients, and
between aCL and b2GPI. There is no statistically significant correlation between risk
factors (smoking) and IMT in the group of patients.
200
Fig. 3. IMT in right and left carotid artery in APS patients
Fig. 4. IMT in right and left carotid artery in control group
Table 3. ANOVA results

ANOVA Sum of squares df Mean square F Sig.
VAR00007 between groups 6368.379 1 26368.379 1.369 0.255
IMTT-RIO within groups 404534.6 21 19263.551
total 430903.0 22
VAR00008 between groups 2088.039 1 2088.039 0.058 0.812
IMT-LIO within groups 753561.7 21 35883.890
total 755649.7 22
It was found a strong association of APS with dyslipidemia (Table 3). In the
group of patients it occurred in 61% of individuals and in the control group in 20%.
201
CONCLUSIONS
In our study we found a strong, statistically significant difference between the values
of IMT in patients compared to the control group.
• No statistically significant correlation between the titers of aPL and IMT;
• There is no apparent correlation between risk factors (tobacco and alcohol) and
IMT in patients;
• Dyslipidemia is considerably strongly advocated in patients with APS, as com-
pared with the control group;
• A difference was established in IMT of patients and control group approximately
100 μm;
• In statistical processing is demonstrated a strong correlation between IMT of
patients and controls.
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Received 2 December 2015
203
CLINICAL AND IMMUNOLOGICAL CORRELATIONS IN

PATIENTS WITH ANTIPHOSPHOLIPID SYNDROME
N. STOILOVa*, VL. BOYADZHIEVAa, R. RASHKOVa, S. STEFANOVb,

B. MARINOVc, V. VLADIMIROVd
a
UMBAL ‘St. Ivan Rilski’, Clinic of Rheumatology, Medical University, Sofia,
Bulgaria
E-mail: dr_nstoilov@yahoo.com
b
SBAL ‘Detski Bolesti’, Clinic of Rheumatology, Cardiology and Haematology,
Medical University, Sofia, Bulgaria
c
SBALAG ‘Maichin Dom’, Clinic of Pathological Pregnancy, Medical University,
Sofia, Bulgaria
d
UMBAL ‘Tzarica Joanna’, Clinic of Urology, Medical University, Sofia, Bulgaria
ABSTRACT
Antiphospholipid syndrome (APS) is an autoimmune, multisystem disorder charac-
terised by thrombocytopenia, venous and/or arterial thrombosis, pathological course
of pregnancy in women in the presence of a heterogeneous group of antibodies –
antiphospholipid antibodies (aPL). Our aim is to evaluate the correlation between
aPL, antinuclear antibodies (ANA) and clinical manifestations in patients with APS.
Patients were tested for the aPL directed against cardiolipin (aCL), β2glikoprotein1
(anti-β2GPI) and prothrombin (anti-prothrombin), by the method of ELISA. In our
group, secondary APS is 87.5% and primary is 12.5%. Patients with positive aCL are
85.3%, β2GPI – 74.3% and 3.7% for anti-prothrombin.
The secondary APS is in the context of: SLE – 74.2%, RA – 8.8%, vasculitis –
8.8%, others – 8.2%. The Pearson analysis establishes strong, statistically significant
positive correlation between: aCL and β2GPI, β2GPI and anti-M2, anti-Scl70, anti-
RibP, β2GPI/aCL and anti-M2. Most often positive aPL is for aCL, followed by anti-
β2GPI and most frequently anti-protrombin. In half of the patients with complicated
pregnancies is found the coexistence of aCL and anti-β2GPI.
In the conducted correlation analysis showed a statistically significant association
between miscarriage and the presence of anti-β2GPI. Severity of clinical manifesta-
tions of APS correlates directly proportional to the titer and number of aPL.
Keywords: antiphospholipid syndrome, antiphospholipid antibodies.
*
For correspondence.
204
AIMS AND BACKGROUND
Antiphospholipid syndrome (APS) is an autoimmune, multisystem disorder charac-
terised by thrombocytopenia, venous and/or arterial thrombosis, pathological course
of pregnancy in women (preeclampsia, eclampsia, miscarriages, still births) in the
presence of a heterogeneous group of antibodies – antiphospholipid antibodies (aPL)
(Ref. 1).
When the disease is presented separately is classified as ‘primary antiphospholipid
syndrome’, if it is in the context of another disease – ‘secondary’2,3. Recent studies
have shown that pathogenetically the both can not be differentiated. For this reason,
some authors have a discussion about the division.
The most common secondary antiphospholipid syndrome is associated with sys-
temic lupus erythematosus (SLE). The frequency of SLE among the female population
is 20 per 100 000 individuals4,5. Nearly 36% of SLE patients in the European population
exhibit co-morbidity with APS. In other rheumatic diseases, most oftenely an APS
is found in: lupus – like syndrome (5%), the Sjogren syndrome (2.2%), rheumatoid
arthritis (1.8%). In other nosological units vasculitis, myositis and others constitute
for about 0.5%. The formation of antiphospholipid antibodies can be associated with
other, non-rheumatic diseases: the Crohn disease, malignancy, sarcoidosis, diabetes,
atheroma, infectious diseases – leprosy, syphilis, HIV, HCV, cytomegalovirus. Some
medications may induce aPL antibodies synthesis: hydralazine, procainamide, phe-
nytoin, interferon, biological agents and the like6.
Standardised research found that within about 1% of young individuals and up to
3% of the adult population there was evidence of antiphospholipid antibodies, without
this leading to any clinical manifestation of APS.
Two main groups of antibodies are found in APS, one is directed against certain
phospholipids and the others are directed against their protein cofactors (Table 1).
Table 1. Autoantibodies in APS

1. Antibodies against phospholipids 2. Antibodies against phospholipid binding proteins
• Cardiolipin • Β2GPI
• Phosphatidylserine • Annexin V
• Phosphoric acid • Protein C
• Phosphatidyl inositol • Protein S
• Phosphatidylcholine • Low and high molecular kininogens
• Phosphatidylethanolamine • Factor XII
• S4b binding protein
• Complement C4 and C5 fractions
• Heparan sulphate
• Thrombin
• Other
The greatest clinical relevance of antibodies against phospholipids are those

directed against cardiolipin – anticardiolipin antibodies (aCL), and those against
205
phospholipid-binding proteins – β2GPI, prothrombin, annexin V, protein C and
protein S. Beta 2-glycoprotein I is an oxygen plasma co-factor that acts as a natural
anti-coagulant. Antibodies directed against Β2GPI, blocking its physiological func-
tion7,8. For positive carrier is considered when antibodies are present in two tests at
a distance of 12 weeks.
Classification criteria of Sydney (revised in 2006) are used for antiphospholipid
syndrome. In clinical practice, the classification criteria are also used as diagnostic,
causing errors in diagnosis. They are divided into two groups: clinical and laboratory
criteria7–9.
CLINICAL CRITERIA
Vascular thrombosis. One or more episodes of arterial, venous and/or small vessel
thrombosis in any tissue or organ. Thrombosis must be proven by an objective validated
method or histologically. In histological evidence should be excluded any likelihood
of inflammation in the vessel wall.
Pathological course of pregnancy. One or more spontaneous abortions after 10 weeks
in morphologically healthy fetus, and this must be documented by ultrasound or direct
examination of the fetus. One or more births of morphologically healthy fetus before
34 weeks because of preeclampsia, documented criteria adopted for placental insuf-
ficiency. Three or more miscarriages before 10 weeks the exclusion of all maternal
anatomical, hormonal and chromosomal pathology.
LABORATORY CRITERIA
• Lupus anticoagulants found twice in period of 12 weeks according to the guidelines

of ‘International Society of Thrombosis and Hemostasis’.
• Anticardiolipin antibodies, IgG and/or IgM in high or medium high titers twice
every 12 weeks identified by the method of ELISA.
• Anti-beta 2 gliprotein I antibodies, IgG and/or IgM in high or medium high
titers twice every 12 weeks out of the plasma by the method of ELISA.
The aim of this study is to investigate the relationship of antiphospholipid anti-
bodies (aPL) with clinical manifestations of antiphospholipid syndrome (APS) and
to find the correlation between aPL, antinuclear antibodies (ANA) and the clinical
manifestations in patients with APS.
EXPERIMENTAL
The patients were tested for antiphospholipid antibodies directed against cardiolipin
(aCL), β2glikoprotein 1 (anti-β2GPI) and prothrombin (anti-prothrombin) by the
method of ELISA.
Antinuclear antibodies were tested by a total titre – ANA-screen, by the method
of immunofluorescence and are typified in subtypes according to the method of immu-
206
noblot (anti-RNP/Sm, anti-Sm, anti-ss A (Ro), anti-Ro52 , anti-ss B (La), Anti-Scll70,
anti-PM-Scl, anti-Jo1, anti-Cent.B, anti-PCNA, Anti-dsDNA, anti-Nucleosomes,
anti-Histoni, anti-RibP, anti -M2).
Samples were tested in the immunological laboratory at the University Hospital
‘St. Ivan Rilski’.
The statistical processing of data was performed on the basis of the following
methods:
– The Pearson correlation analysis;
– Chi-square;
– ANOVA;
– One way ANOVA;
– Diagnostic analysis.

In our group are studied 40 patients, filling the revised classification criteria for APS
(Sydney 2006). Of these, 83% (33) were female, 17% (7) were male. The average age
of patients at the time of the review is 42. The average age of the first manifestation
of the disease was 30.5 years (Fig. 1).
Fig. 1. Distribution of APS by gender
In the group studied, APS is secondary in 87.5%. For the remaining 12.5%, the
disease is classified as primary antiphospholipid syndrome (Fig. 2).
12.50%
87.50%
primary APS secondary APS
Fig. 2. APS classification in the study group
207
Of the surveyed patients 85.3% were positive for aCL antibodies to β2GPI 74.3%
and 3.7% for anti-prothrombin (Fig. 3).
90
80
70
60
50
%
40
30
20
10
0
anti-prothrombin
Fig. 3. Positive antibodies for APS in the study group
In 47.2% of patients exhibited positive carriers of one type of aPL (aCL, anti-
β2GPI or anti-prothrombin). In 50% are found two of the subtypes antiphospholipid
antibodies, aCL/anti-β2GPI. Only in 2.8% are positive three subtypes (aCL/anti-
β2GPI/anti-prothrombin) (Fig. 4).
50
40
30
%
20
10
0
Fig. 4. Combination of aPL in the study group
The secondary APS in our group of patients is in the context of SLE in 74.2% of
patients. In other nosological units APS is distributed – RA – 8.8%, vasculitis – 8.8%,
other – 8.2% (Fig. 5).
208
8.20%
8.80%
8.80%
74.20%
vasculitis others
Fig. 5. Secondary APS in the context of other diseases
The Pearson correlation analysis between aPL and antinuclear antibodies sub-
types, establishes strong, statistically significant positive correlation between: aCL
and β2GPI; β2GPI and anti-M2; β2GPI and anti-Scl70; β2GPI/aCL and anti-M2;
β2GPI and anti-RibP.
PATHOLOGICAL PREGNANCY
Pathological course of pregnancy is one of the main manifestations of APS in 42.4%

(14). These patients have documented miscarriage, stillbirth or abortion for medical
reasons (Fig. 6).
42.40%
57.60%
Fig. 6. Pathological pregnancy
In the group of patients with pathological course of pregnancy 80% (11) are
positive for aCL, 72% (10) – for anti-β2GPI, and in 8% (1) is found the presence of
antibodies against prothrombin (Fig. 7).
209
Fig. 7. Distribution of the aPL antibodies in patients with pathological course of pregnancy
In 50% (7) was established the existence of two subtypes antiphospholipid

antibodies (aCL/anti-β2GPI). In 8% (1) there were three positive subtypes (aCL/
anti-β2GPI/anti-prothrombin), in which case 13 are registered miscarriages (Fig. 8).
Fig. 8. Distribution of the aPL antibodies in patients with pathological course of pregnancy
In 36% (5) of women with APS and pathological course of pregnancy is estab-
lished at least one thrombotic event. In 29% (4) there are two or more thrombotic
events, in this group are observed extremely high titers of aPL (three or more times
the upper limit of normal) (Fig. 9).
one thrombotic two or more

event thrombotic events
Fig. 9. Thrombotic events in women with pathological course of pregnancy
210
In 93% (13) of women with abnormal pregnancy and API was established at least
one miscarriage, while 29% (4) at least one had stillbirth and 22% had both miscarriage
and stillbirth. In 57% (8) women titer of aPL was increased threefold. In this group
the clinical course of APS was more severe than in the group with low antibody titer.
THROMBOSIS
In 46% (17) there are data for at least one thrombotic event. In 19% (7) there were
two or more thrombotic incidents. In patients with two or more thrombotic incidents,
extremely high titers of aPL (three or more times the upper value) were found (Fig. 10).
one thrombotic two or more

event thrombotic events
Fig. 10. Thrombotic events in patients with APS
Patients in the studied group have been evaluated for thrombosis of blood ves-
sels, the number of thrombosis, recurrent spontaneous abortions (number), stillbirths
(number), epilepsy, valvulopathies, myocardial infarction, vasculitis, pulmonary
embolism, pulmonitis, pulmonary fibrosis, glomerulonephritis, livedo reticularis, the
Raynaud syndrome, CNS thrombosis. Recent studies confirm our data10,11.
Clinical appearances are correlated with aPL, ANA-screen, ANA-profile. Data
were processed by the method of: ANOVA, One way ANOVA and the Pearson cor-
relation (Table 2).
Table 2. Correlation between clinical appearances and antibodies

Factor Significant meaning
Endocarditis anti-histoni, anti-Sm, anti-PM-Scl
Intracardiac thrombosis anti-M2
Epilepsy anti-histoni, anti-RNP/Sm
Pulmonary emobolism anti-M2, anti-RNP/Sm
Glomerulonephritis anti-histoni, anti-Rib P, anti-Jo1, anti-M2, anti-Ro52
Livedo reticularis anti-dsDNA, anti-ss A (Ro)
The Raynaud syndrome ANA- screen, anti-ss B (La)
Miscarriages anti – B2GPI
In this study, we examined 40 patients with antiphospholipid syndrome. Оur

aim was to investigate the relationship of aPL with clinical manifestations of the
211
disease. In our group, the incidence of secondary APS is significantly higher than the
incidence of primary.
The secondary APS is most frequently found in patients with systemic lupus
erythematosus – 74.2%. In other rheumatic diseases frequency is significantly lower
(RA 8.8%, vasculitis – 8.8% and 8.2% in other autoimmune rheumatic diseases).
In 83.0% of cases it occurs in women. Cervera et al.12 studies on clinical and im-
munologic manifestations in 1000 patients with APS from 13 European countries
(including Bulgaria) show similar results. APS is established in 82.0% of all cases in
women (820). The average age in the study group, as in our study was 42 years. In
the group studied by Cervera et al. 53.1% were patients with primary APS, 36.2% of
the cases with patients during the SLE, 5.0% with lupus like syndrome, 1.8% in RA
and 0.7% in systemic vasculitis. In 77.0% of patients with SLE a secondary APS is
established12. Comparing the international study with ours, we found many similari-
ties. Data from our study show that from the explored aPL most often positives are for
aCL following – anti-B2GPI with rarest being anti-protrombin. In 50.0% of patients
double carriage of aPL (aCL and anti-B2GPI) is more often than a positive result for
aPL (47.0%). Three positive antibodies are most rarely explored. Cervera et al. also
found that the most commonly found presence is of aCL antibodies (in 87.9% of
patients), but unlike our results, the frequency in patients positive for LA (at 53.6%)
came second4,12. Among the wide variety of clinical manifestations of APS in both
groups patients had a higher incidence of thrombotic events (46.0% in our study and
64.1% in the international group)12–15. Second in frequency is the pathological course
of pregnancy, with frequently observed miscarriages and premature births16. Our
data show that in half of the patients in our group with abnormal pregnancy there is
a combination of two types of antibodies –aCL and anti-β2GPI. Statistical analysis
showed significant correlation between miscarriage and the presence of anti-β2GPI.
Cervera et al.12 did not publish data on the type of antibodies found in patients with
complicated pregnancy.
CONCLUSIONS
The results from our study show that the severity of clinical manifestations of APS
positively correlates to the titer of antiphospholipid antibodies and the number of a
positive aPL.
REFERENCES
1. L. R. SAMMARITANO: Pediatric and Familial Antiphospholipid Syn Dromes. In: The Antiphos-
pholipid Syndrome (eds R. A. Asherson, R. Cervera, J. C. Piette, Y. Shoenfeld). CRC Press, Boca
Raton (FL), 1996, 259–266.
2. R. A. ASHERSON: The Catastrophic Antiphospholipid Syndrome. J Rheumatol, 19, 508 (1992).
3. J. T. BRANDT, D. A. TRIPLETT, B. ALVING, I. SCHARRER: Criteria for the Diagnosis of Lupus
Anticoagulants: An Update. Thromb Haemost, 74, 1185 (1995).
212
4. R. CERVERA, M. A. KHAMASHTA, J. FONT, G. D. SEBASTIANI, A. GIL, P. LAVILLA et al.:
Systemic Lupus Erythematosus: Clinical and Immunological Patterns of Disease Expression in a
Cohort of 1000 patients. Medicine (Baltimore), 72, 113 (1993).
5. E. M. TAN, A. S. COHEN, J. F. FRIES, A. T. MASI, d. j. McSHANE, N. F. ROTHFIELD et al.:
The 1982 Revised Criteria for the Classification of Systemic Lupus Erythematosus. Arthritis Rheum,
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6. E. N. HARRIS, S. S. PIERANGELI: Equivocal Antiphospholipid Syndrome. J Autoimmun, 15,
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Antiphospholipid Syndrome Classification Criteria. Ann Rheum Dis, 66, 927 (2007).
8. S. MIYAKIS, M. D. LOCKSHIN, T. ATSUMI, D. W. BRANCH, R. L. BREY, R. CERVERA,
R. H. W. M. DERKSEN, P. G. de GROOT, T. KOIKE, P. L. MERONI, G. REBER, Y. SHOEN-
FELD, A. TINCANI, P. G. VLACHOYIANNOPOULOS, S. A. KRILIS: International Consensus
Statement on an Update of the Classification Criteria for Definite Antiphospholipid Syndrome (APS).
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et al.: International Consensus Statement on Preliminary Classification Criteria for Definite An-
tiphospholipid Syndrome. Arthritis Rheum, 42, 1309 (1999).
10. R. CERVERA, M. A. KHAMASHTA, J. FONT, P. A. REYES, J. L VIANNA, A. LÓPEZ-SOTO et
al.: High Prevalence of Significant Heart Valve Lesions in Patients with the ‘Primary’ Antiphospho-
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11. E. N. HARRIS, J. K. H. CHAN, R. A. ASHERSON, V. R. ABER, A. E. GHARAVI, G. R. V. HUGHES:
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1000 Patients. Arthritis Rheum, 46, 1019 (2002)
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PINOSA et al.: Clinical Study and Follow-up of 100 Patients with the Antiphospholipid Syndrome.
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Antibody Test. Clin Exp Immunol, 68, 215 (1987).
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Features of Patients with Raised Anticardiolipin Antibodies and no Other Disorder. Ann Rheum
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213
ROLE OF GUT PROBIOTIC MICROBIOTA IN THE APOPTOSIS

OF GASTROINTESTINAL CANCER
CHAO XUa, YONGLIANG XIAa*, LULU HUANGa, HAITAO CHENa,

XIAFANG ZHANGa, QINGHUA YAOb,c*
a
First Clinical College of Zhejiang Chinese Medical University, Hangzhou, China
b
Department of Integrated Traditional Chinese and Western Medicine, Zhejiang
Cancer Hospital, Hangzhou, China
c
Key Laboratory of Integrated Traditional Chinese and Western Medicine of
Zhejiang Cancer Hospital, Hangzhou, China
E-mail: yaoqinghua2020@163.com
ABSTRACT
Some gut probiotic microbiota such as bifidobacteria and lactobacilli have been widely
used as probiotics in human and animals, and the mechanisms by which these bacteria
reduce the incidence or severity of cancers have been determined. Epidemiological
and experimental studies indicate that lactobacilli, bifidobacteria and others, as well as
their fermented dairy products and metabolites can reduce the risk of certain types of
cancer and inhibit tumor growth, and this area of research holds much clinical promise.
One potential mechanism of action is a direct influence of the gut probiotic microbiota
on gastrointestinal tumor apoptosis. Here we review the data that gut macrobiotics
and their metabolites, directly interact with cellular apoptotic pathways triggered by
p53, Bcl-2 and death receptors. These interactions may be harnessed in the future to
engineer microbiotic-based therapies for cancer prevention and treatment.
Keywords: gut microbiota, probiotics, gastrointestinal cancer, apoptosis, p53, Cas-
pase, Bcl-2.
AIMS AND BACKGROUND

25% of all neoplasms derive from the gastrointestinal (GI) tract1, accounting for 9%
of all-cause mortality globally. Gastric and colon cancers remain the leading cause
of cancer mortality throughout the world2. A flood of recent studies have attempted
to define the role of the gut microbiota in the etiology, pathology and treatment of GI
cancers. Gut microbiota, particularly probiotics, have been reported to modify several
*
For correspondence.
214
biological processes involved in the development of cancer, including apoptosis and
cellular proliferation3.
GUT MICROBIOTA: BRIEF INTRODUCTION

The GI tract is estimated to host 100 trillion microorganisms, collectively called gut
microbiota4. The gut microbiota represents an important component of the intestinal
mucosal barrier, and plays a vital function in protecting human intestines from the
invasion of bacteria, preventing the translocation of intestinal bacteria and lipopoly-
saccharide (LPS) from Gram-negative bacterium5. The gut microbiota comprises a
complex ecosystem that has been shaped by millennia of evolution that usually exists
in a symbiotic relationship with the host6,7, and could be considered to represent a
virtual organ or emergent system, with properties that need to be integrated into host
biology and physiology. Whilst the extent of the gut microbiota contribution to human
health and disease are not yet fully understood, they are known to be easily disrupted
by antibiotics, diet or surgery8.
The interdependence and interaction of intestinal probiotics and pathogenic
bacteria creates a dynamic balance of intestinal microecology, and when this balance
is disturbed, the intestine mucosal barrier is damaged. Therefore, the mechanisms of
regulation and maintenance of this intestinal microecology balance have received
increasing attention9.
Some gut microbiota (named probiotics) including bifidobacteria and lactic acid
bacteria which are associated with beneficial effects to humans and animals, can
produce active substances that not only help to maintain normal function of the gut
mucosa, but also protect mucosa from toxins, allergens and pathogens10. Meanwhile
probiotics can also contribute to digestion, prevent infection and lower the risk of
autoimmune disease. New findings suggest that some gut microbiota can also induce
apoptosis of tumor cells.
GUT MICROBIOTA AND GI CANCERS

The development of GI cancers is thought to be influenced by a combination of ge-
netics, epigenetics, immunity, environment, diet and lifestyle. These factors could all
influence the GI microflora during tumor genesis and growth11–13, and gut microbiota
have been implicated in GI diseases and disorders including GI cancer11,14. It has
been well established that Helicobacter pylori in the gut can cause gastric cancer15.
Microbiota could contribute to colorectal cancer (CRC) directly through the harmful
properties of some bacteria, or by inducing dysbiosis16, shifting in local distribution
of communities, and changing bacterial metabolic activity6. Many factors can alter
the GI ecosystem, including antibiotics, psychological and physical stress, radiation,
modified peristalsis and die17. Infection with Helicobacter pylori and Campylobacter
jejuni have been reported to be associated with the risk of developing lymphoma of the
215
small intestine, especially immunoproliferative small intestinal disease18. However, in
contrast, some gut protiotic microbiota have been reported to prevent the development
of GI cancers. Since microbiota is involved in the genesis of GI cancers, manipula-
tion of this ecosystem may have the capacity to reduce the incidence or severity of
GI cancer17. Some clinical trials have recently achieved success using probiotics for
prevention of CRC (Ref. 19).
GUT PROBIOTIC MICROBIOTA AND TUMOR CELL APOPTOSIS

Tumor cell apoptosis is energy-dependent cell death caused by activation of pro-
grammed cell suicide20. Apoptosis of gastrointestinal cancers is achieved through
three distinct pathways: the extrinsic pathway, intrinsic pathway and ER stress21. ER
stress signaling pathway is activated when cells fail to cope with prolonged ER stress,
calcium homeostasis is lost, leading to its massive release from ER, unfolded and
misfolded proteins accumulate in the ER lumen. The above pathways are associated
with the caspase family, B-Cell CLL/Lymphoma (bcl)-2 family, TRAIL family and p53.
A number of studies have demonstrated that probiotics can induce the apoptosis
of cancer cells (Table 1). Bifidobacterium longumand lactulose has the capacity to
inhibit azoxymethane-induced colonic aberrant crypt foci in rats via inducing ap-
optosis of GI cancer cells22. For instance Bifidobacterium lactis facilitate apoptosis
of carcinogen-damaged cells in rat colon14,23. Lactobacillus paracasei MPC2.1 and
Lactobacillus rhamnosus GG enhanced apoptosis of HGC-27 gastric and DLD-1 colon
cell lines24,25. And in the HGC-27 gastric cancer cell line Lactobacillus rhamnosus GG
can influence polyamine metabolism to inhibit the development of gastric cancer24.
When mice transplanted with CT-26 colon carcinoma cells were pre-inoculated with
the probiotic L. acidophilus NCFM, tumor volume growth was reduced by more than
half 28 days after tumor implants. L. acidophilus NCFM enhanced apoptosis of CT-26
cells both in dorsal-flank tumors and in segmental orthotopic CRC compared with
untreated mice. L. acidophilus NCFM pre-inoculation down regulated MHC class I
and CXCR expression in the colon and increased the rate of apoptosis in tumor tissue,
indicating that Lactobacillus acidophilus NCFM may modulate the cellular response
triggered by colon carcinogenesis. Gut microbiota can also regulate the WNT signal-
ing pathway to impact the apoptosis in colon cancer cells. Fermented Wheat Aleurone
Enriched with probiotic strains L. rhamnosus GG and Bifidobacterium Bb12 induced
down-regulation of WNT2B in colon cell lines LT97, to promote apoptosis. Milk
fermented by Propionibacterium freudenreichii also induces the apoptosis of HGT-1
human gastric cancer cells. Food fermented by probiotics may thus represent a novel
therapy to GI caners.
216
Table 1. In vitro and in vivo studies demonstrating the capacity of probiotics to influence host cell apoptosis
Bacterial strain Influence on Apoptosis References
Bifidobacterium longu- laculose suppresses azoxymethane-induced colonic 22
mand aberrant crypt foci
Bifidobacterium lactis facilitates apoptosis of carcinogen-damaged cells in 14, 23
rat colons
Lactobacillus paracasei enhances apoptosis of gastric and colon cell lines 24
MPC2.1
Lactobacillus rhamnosus enhances apoptosis of gastric and colon cell lines 24
GG, Bifidobacterium and influence polyamine metabolism to inhibit the
BB12 development of gastric cancer
Lactobacillus acidophilus reduced growth and enhanced apoptosis of trans- 25
NCFM planted CT-26 colon carcinoma cells in mouse mod-
els. L. acidophilus NCFM downregulated the CXCR
mRNA expressions in the colon and MHC class I, as
well as increase apoptosis in tumor tissue
L. rhamnosus GG down-regulates WNT2B in colon cell line LT97 to 22
promote apoptosis
Propionibacterium induce apoptosis of HGT-1 human gastric cancer cells 23
freudenreichii
Gut microbiota, particularly probiotics, have also been reported to inhibit other
cancers. The BI-TK/GCV (Bifidobacterium infantis (BI)-mediated herpes simplex
virus thymidine kinase/ganciclovir) therapy system up-regulated apoptosis in a rat
model of bladder cancer, and Fas, FasL, Cyt-C and caspase-9 were up-regulated by BI
therapy, indicating that gut microbiota played important roles in intrinsic pathways as
well as extrinsic pathways23. Probiotic Lactobacillus reuteri was reported to promote
tumor necrosis factor (TNF)-induced apoptosis in human myeloid leukemia-derived
cells by modulation of NF-kB and MAPK signaling24. Intestinal microbiota modify
activity of secondary lymphocytes following chemotherapy-induced intestinal mucosal
damage, resulting in apoptosis of tumor cells25.
However, gut microbiota can also act to inhibit apoptosis of colon cancer cells.
It was recently reported that intestinal bacteria can drive IL-17C production during
intestinal tumorigenesis and IL-17C is most likely critical for microbiota-mediated
tumor development16. Up-regulation of IL-17C restored the expression of Bcl-2 and
Bcl-xL, and antibiotic treatment significantly reduced the expression of IL-17C and
also the expression of Bcl-xL and Bcl-2 in the colon.
217
REGULATION OF APOPTOSIS BY GUT PROBIOTIC MICROBIOTA
Table 2. In vitro and in vivo studies demonstrating the capacity of gut probiotic microbacterial species
or metabolites to enhance host cell apoptosis
Bacteria or bacterial Influence on Apoptosis References
metabolite
P53 pathway
Fermented non digestible modulates p53-pathway related gene expression, 10–13
fraction of common bean induce DNA fragmentation and inhibit proliferation
of a colon adenocarcinoma cell line in vitro
Bifidobacterium lactis pluso- inhibited colon carcinogenesis caused by 1,2-di- 10–13
ligofructos/inulin methylhydrazine, and regulated intracellular p53
concentration, inducing apoptosis of colon cancer
cells in a rat model
Caspase family pathway
Ginsenoside rb1, metabo- ginsenoside compound K up-regulates caspase 3, 8 16
lised by the gut microbiota and 9 expression in colorectal cancer cell lines and
induces apoptosis of colorectal cancer cells
Probiotic compounds enhance expression of caspase 3 and 9 in the proxi- 24
mal and distal colon of AKR mice
Lactobacillus Acidophilu- sensitises colorectal cancer cells to 5-FU therapy, 25
sand L. Casei mix enhancing the expression of caspase-3, inducing
colorectal cancer cell programmed death
Bcl-2 family pathway
L. Rhamnosus GG homoge- significantly increased the Bax/Bcl-2 ratio, induc- 20
nate ing apoptosis in HGC-27 (Russo, 2007 #106) cells
Milk fermented with ameliorate the carcinogenic effects of the potent 21
L. rhamnosus GG and mycotoxin–aflatoxin B1 in a rat model of hepatocel-
L. casei strain Shirota lular carcinoma (Kumar, 2011 #82)
Cell-bound exopolysaccha- inhibited the proliferation of HT-29 colon cancer 22
rides isolated from Lacto- cells by directly affecting cell morphology rather
bacillus acidophilus 606 than cell cycle, and dramatically induced Beclin-1
and GRP78, and reduced the expression of Bcl-2
and Bak
Propionibacterium freuden- induce translocation of Bax from the cytosol to 23
reichii-produced short the mitochondria, an apoptotic event preceding
chain fatty acids Cytochrome c release
Death-inducing signaling complex pathway
Short chain fatty acids up-regulate TNFR1, and trigger Fas-mediated ap- 20
optosis in colon adenocarcinoma cells
Metabolite sodium butyrate, induced up-regulation of TRAIL receptor DR5 in 17
colon cancer cells
Lactobacillus casei enhance expression of TNFR1 and death receptor 3 18
ATCC393 in Huh7, a human hepatoma cell line
Bifidobacterium adolescen- enhanced the activity of TNF-a, inducing apoptosis 19
tis SPM0212 of colon and rectal cancer cell lines HT-29, SW 480,
and Caco-2
218
GUT PROBIOTIC MICROBIOTA AND p53
Tumor suppressor p53 regulates the expression of a variety of genes involved in cel-
lular responses to genotoxicity or cellular stress17,18. p53 stimulates a wide network
of signals that act through two major apoptotic pathways, the extrinsic pathways and
intrinsic pathways19.
Some gut probiotic microbiota have been found to directly or indirectly regulate
expression of p53 in colorectal cancer cell lines. The non-digestible fraction (NDF) of
common bean (P. vulgaris L.) (containing total dietary fibre, oligosaccharides, resistant
starch and phenolic compounds, NDF), was found to induce DNA fragmentation and
inhibit proliferation of the colon adenocarcinoma HT-29 cell line in vitro only after
fermentation by whole human gut microbiota20,21. The fermented NDF (FNDF) of
common bean cultivar Negro 8025 was also found to modulate p53-pathway related
gene expression in HT-29 cells21,22. Meanwhile symbiotic (Bifidobacterium lactis plu-
soligofructos/inulin) inhibited colon carcinogenesis caused by 1,2-dimethylhydrazine
in a rat model, and regulated intracellular p53 concentration, inducing apoptosis of
colon cancer cells23.
GUT PROBIOTIC MICROBIOTA AND CASPASE FAMILY

Caspases incise specific substrates in the implementation phase of apoptosis, then
cleave nuclear lamina proteins, inhibit DNA repair, disrupt the cytoskeleton, causing
DNA fragmentation and cell death. Expression of caspase 3 and 9 in the proximal and
distal colon of AKR mice was found to be enhanced following administration of pro-
biotic compounds24. The induction of apoptosis by commensal bacteria may represent
an important physiologic mechanism for prevention of dysplastic cell proliferation,
and thus prevention of colon cancer. Meanwhile Lactobacillus acidophilusand L.
Casei Mix was found to sensitise colorectal cancer cells to 5-FU therapy, enhancing
the expression of caspase 3, inducing colorectal cancer cell programmed death25.
Ginsenoside rb1 can be metabolised by the gut microbiota, producing Ginsenoside
compound K which up-regulates caspase 3, 8 and 9 expression in colorectal cancer
cell lines and induces apoptosis of colorectal cancer cells16.
GUT PROBIOTIC MICROBIOTA AND Bcl-2 FAMILY

Bcl-2 family proteins are involved in signaling pathways regulating the caspase 3
activities necessary for chromatin condensation and DNA fragmentation that char-
acterize apoptosis. The balance of pro- and anti-apoptotic Bcl-2 proteins is critical
for cell survival17,18.
The intrinsic apoptotic pathway is dominated by the Bcl-2 family of proteins,
which governs the release of Cytochrome c from the mitochondria19. The capacity of
milk fermented with two probiotics, lactic acid bacteria L. rhamnosus GG and L. casei
strain Shirota, to ameliorate the carcinogenic effects of the potent mycotoxin–aflatoxin
219
B1 in a rat model of hepatocellular carcinoma20. L. rhamnosus GG homogenate also
significantly increased the Bax/Bcl-2 ratio, inducing apoptosis in HGC-27 cells21.
Probiotic bacteria can also influence the bcl-2 and bak ratio in colon cancer cells.
Cell-bound exopolysaccharides (cb-EPS) isolated from Lactobacillus acidophilus
606 inhibited the proliferation of HT-29 colon cancer cells by directly affecting
cell morphology rather than cell cycle22. cb-EPS dramatically induced Beclin-1 and
GRP78, and reduced the expression of Bcl-2 and Bak22. The human probiotic Pro-
pionibacterium freudenreichii induced colorectal adenocarcinoma cell apoptosis in
vitro via its metabolites, the short chain fatty acids (SCFA), acetate and propionate,
resulting in translocation of Bax from the cytosol to the mitochondria, an apoptotic
event preceding Cytochrome c release23.
GUT PROBIOTIC MICROBIOTA AND DEATH-INDUCING SIGNALING

COMPLEX
The extrinsic apoptosis pathway involves engagement of extracellular ‘death’ receptors
that belong to the TNFR family, formation of the death-inducing signaling complex,
containing Fasl, Fas, TRAIL and TNF-α (Refs 24 and 25). The TNF-αreceptor, TNFR1,
expressed in all human tissues, forms a trimer on activation and recruits an adaptor
protein, TRADD, initiating the caspase cascade resulting in apoptosis16.
The gut microbiota fermented non-digestible fraction can contain short chain fatty
acids that have been found to up-regulate TNFR1, and trigger Fas-mediated apoptosis
in colon adenocarcinoma cells10. Sodium butyrate, also produced by gut microbiota
metabolism, induced up-regulation of TRAIL receptor DR5 in colon cancer cells17,
and Lactobacillus casei ATCC393 was found to enhance expression of TNFR1 and
death receptor 3 in Huh7, a human hepatoma cell line18. Bifidobacterium adolescentis
SPM0212 enhanced the activity of TNF-a, inducing apoptosis of colon and rectal
cancer cell lines HT-29, SW 480, and Caco-2 (Ref. 19).
CONCLUSIONS
Administration of probiotics can modify the human gut microflora by enhancing con-
centrations of beneficial bacteria species such as Lactobacillus and Bifidobacterium
and diminishing the levels of harmful microorganisms. This area of clinical research
may prove useful in developing cancer prevention and therapy. The microbiota has
also been harnessed to deliver drugs to the gut. B. longum engineered to stably ex-
press endostatin exerted a strong inhibitory effect on the growth of solid liver tumor
in a mouse model.
In summary, the gut microbiota plays important roles in the development of diges-
tive tract tumors. Probiotics can induce apoptosis of GI cancer cells both directly and
indirectly, via the action of their metabolic products, inhibiting the development of
tumors. The microbiota harness cellular apoptosis pathways involving caspases, Bcl-2,
220
p53 and death-inducing signaling complex. In the future, with increased understanding
of the human gut microbiota, engineered probiotics, may be served as chemoprotec-
tive agents for the prevention and therapy of gastrointestinal cancer. However, more
clinical trials will be required to assess their protective capacity.
ACKNOWLEDGEMENTS
This study is supported by grants from Zhejiang Provincial Natural Science Founda-
tion of China (Grant No LQ12H16006).
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16, 847 (2010).
25. A. ORLANDO, C. MESSA, M. LINSALATA, A. CAVALLINI, F. RUSSO: Effects of Lactobacillus
rhamnosus GG on Proliferation and Polyamine Metabolism in HGC-27 Human Gastric and DLD-1
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Revised 9 July 2015
222
PLASMA ANTIOXIDANT ENZYMES, LIPID PROFILES AND

PEROXIDATION PRODUCTS IN TYPE 2 DIABETIC PATIENTS
WITH HYPERTENSION
A. HAMEEDa*, T. FAROOQa*, A. HAMEEDb, M. IBRAHIMa, M. N. AHMADa,

M. A. SHEIKHc, S. A. BUKHARIa
a
Department of Applied Chemistry and Biochemistry, Government College
University, Faisalabad, Pakistan
E-mail: arrujeh@yahoo.com; tahirfarooqfsd@gmail.com
b
Nuclear Institute for Agriculture and Biology (NIAB), P.O. Box 128, Jhang Road
c
Department of Biochemistry, University of Faisalabad, Pakistan
ABSTRACT
Elevated blood glucose level in diabetic patients can induce oxidative stress. While
counteracting the oxidative stress, the antioxidant defense molecules may get exhaust
and patients could have increased risk of developing diabetic complications. The aim
of the present study was to evaluate the plasma antioxidant status, glycemic level,
protein contents, lipid profiles, lipid peroxidation and any perceivable effect of gen-
der on these biochemical parameters in type 2 diabetic patients with hypertension.
Studies on hypertensive patients established that the hypertension causes a significant
(p < 0.05) increase in plasma protein contents and levels of glucose, antioxidant en-
zymes, lipid profiles and lipid peroxidation products. However, the observed change
in plasma peroxidase (POD) level was negligible. For comparative analyses, both the
genders were excogitated and the diabetic male patients showed stupendous potential
to withstand the hypertensive conditions as compared to females. Diabetic patients
with hypertension maintained elevated glucose level, lipid profiles and peroxidation
products indicating oxidative stress damage. However, antioxidants like superoxide
dismutase (SOD) and catalase (CAT) were enhanced in response to oxidative stress
by hyperglycemia to detoxify the excessive reactive oxygen species (ROS). In con-
clusion, diabetic patients with hypertension maintained elevated glucose level, lipid
profiles and peroxidation products indicating oxidative stress damage.
Keywords: diabetes mellitus, hypertension, plasma antioxidants, superoxide dismutase,
peroxidase, catalase and lipid peroxidation.
*
For correspondence.
223
AIMS AND BACKGROUND
Diabetes and its complications are concorded with the accumulation of thiobarbituric
acid reactive substances (TBARS) which are measured as an index of the endogenous
lipid peroxidation and increased activity of free radicals1. Experimental evidences
have also suggested that oxidative damage resulting from both the cytokine-induced
production of toxic free radicals and the low antioxidant capability of the β-cell play
a substantial role in the pathogenesis of diabetes2,3.
Enhanced production of free radicals in diabetic patients exhorts the direct
contribution of hyperglycemia to oxidative stress4. The human body comprehends
a complex antioxidant defense system to control the initiation of usually uncontain-
able free radical chain reactions5. Increase in the lipid peroxidation and a perceptible
decline in the antioxidant defense may crop up at an early stage in the type 2 diabetic
patients, before the evolvement of secondary complications, execute a crucial role in
the development and further progression of diabetic dilemma1,6–10.
In this context, a course of experiments were conducted to compare the lipid
peroxidation products and plasma antioxidant enzyme activities in hypertensive pa-
tients with non-diabetic controls. Alongside, the gender effects on lipid peroxidation
products and plasma antioxidative status in the diabetic patients with hypertension
were also ascertained.
EXPERIMENTAL
Collection of samples. Blood samples of 78 diabetic patients with hypertension and 34
non-diabetics as control were collected. By centrifugation (at 3000 rpm for 15 min),
plasma was separated from blood samples and stored at –20oC for further analyses.
Protein estimation. Quantitative estimation of plasma proteins was consummated ac-
cording to modified micromethod which employed bovine serum albumin as protein
standard11.
Estimation of antioxidants. Separated plasma samples were analysed for different
antioxidants. Catalase, superoxide dismutase, and peroxidase activity levels were
measured by spectrophotometric technique. The SOD activity was assayed by meas-
uring its conceivable adequacy to restrain the photochemical reduction of nitro blue
tetrazolium12. The activities of POD and CAT were measured, exercising an established
scheme with some modification13.
Lipid peroxidation estimation. The lipid peroxidation products were quantified by fol-
lowing a well-known thiobarbituric acid (TBA) method with minor modifications1,14,15.
Malondialdehyde formed as a product of lipid peroxidation, under acidic conditions
reacted with TBA to produce a spectrochemically graspable pink-coloured product.
Further, the total cholesterol, triglycerides, HDL cholesterol and LDL cholesterol
level was estimated by using the Fluitest® Kit.
224
Statistical analysis. The descriptive statistical approaches were employed to analyse
and organize data generated as a result of above studies. For different parameters, the
significance of variance between means was measured by Student t-test (2 tailed) at
0.05 or 0.01 significance level16.
RESULTS
This study was designed to investigate different parameters like antioxidants, per-
oxidation and glycemic control effect on type 2 diabetes mellitus. A highly sex spe-
cific prevalence of type 2 diabetes was observed. In the present study, females had
53.86% type 2 diabetes mellitus as compared to males in which diabetes prevalence
was 46.15%. Hypertension is one of the major diabetic complication knows to date.
In present study, the prevalence of hypertension was 53.86% among all diabetic
complications.
Hypertension and plasma antioxidant enzymes, lipid profile and peroxidation. Indeed,
antioxidants feature an imperative role in the maintenance of cell homeostasis. They
actually quench the reactive oxygen species produced during normal and diseased
conditions17,18. The present research results indicated the increased (p < 0.05) plasma
protein, glucose (Fig. 1), catalase, superoxide dismutase (Fig. 2), malondialdehyde
(MDA) (Fig. 3) cholesterol (p < 0.001), triglycerides (TG) (p < 0.05) (Fig. 4) and high-
density lipoprotein (HDL) cholesterol (p < 0.01) (Fig. 5) level of hypertensive group
as compared to control. However, no specific change in plasma peroxidase (POD)
(Fig. 3) and low-density lipoprotein (LDL) (Fig. 4) cholesterol level in hypertensive
patients was detected.
Fig. 1. Comparison of plasma protein and glucose level in hypertensive patients and non-hypertensive
subjects
225
SOD (U/ml)
Fig. 2. Comparison of plasma catalase and SOD level in hypertensive patients and non-hypertensive
subjects
Fig. 3. Comparison of plasma peroxidase and MDA level in hypertensive patients and non-hypertensive
subjects
Fig. 4. Comparison of plasma cholesterol and triglycerides level in hypertensive patients and non-
hypertensive subjects
226
Fig. 5. Comparison of plasma LDL and HDL cholesterol level in hypertensive patients and non-
hypertensive subjects
Gender specific effects of hypertension on plasma antioxidant enzymes and lipid

peroxidation status. In hypertensive group, the effects of gender on the plasma anti-
oxidant enzymes and lipid peroxidation were also studied (Figs 6–10). Conclusively
the hypertensive males had significantly higher level of plasma CAT, SOD (Fig. 7),
POD, MDA (Fig. 8), total cholesterol, TG (Fig. 9), LDL cholesterol (Fig. 10) with
p < 0.05 than those of hypertensive females. However, no gender specific change in
protein and glucose level (Fig. 6) of hypertensive patients was calculated.
Fig. 6. Effect of gender on plasma protein and glucose level in hypertensive patients
227
Fig. 7. Effect of gender on plasma catalase and peroxidase level in hypertensive patients
Fig. 8. Effect of gender on plasma SOD and MDA level in hypertensive patients
Fig. 9. Effect of gender on plasma cholesterol and triglyceride level in hypertensive patients
228
Fig. 10. Effect of gender on plasma LDL and HDL cholesterol level in hypertensive patients
DISCUSSION
The major prevalent type of diabetes is type 2 or non-insulin dependent diabetes
(NIDDM). Our present study was also designed to investigate different parameters
like antioxidants, peroxidation and glycemic control effect on type 2 diabetes mellitus.
Females had 53.86% type 2 diabetes mellitus as compared to males in which diabetes
prevalence was 46.15%. In concord with our study, previously in the Baluchistan
rural areas it was also estimated that in females (11.1%) the type 2 diabetes was
more frequent than males (9.4%) (Ref. 19). Moreover, another report illustrated that
females were more often prone to type 2 diabetes than males in native American and
African–American youth20. Hypertension is one of the major diabetic complication
known to date21. Also in present study, the prevalence of hypertension was 53.86%
among all diabetic complications.
Antioxidants mark an imperative role in the maintenance of cell homeostasis. They
actually manage to quench the reactive oxygen species produced during normal and
diseased conditions. Previous studies comprehended to conclude that the elevated level
of glucose stimulate the oxidative stress in type 2 diabetic patients which is reflected
by the increased glucose, protein, malondialdehyde (MDA) levels and antioxidant
activities22–24. The present research results indicated the increased (p < 0.05) plasma
protein, glucose, catalase, superoxide dismutase, MDA, cholesterol (p < 0.001), triglyc-
erides (TG) (p < 0.05) and high-density lipoprotein (HDL) cholesterol (p < 0.01) level
of hypertensive group as compared to control. Conclusively the hypertensive males
had significantly higher level of plasma CAT, SOD, POD, MDA, total cholesterol,
TG, LDL cholesterol with p < 0.05 than those of hypertensive females. However, no
gender specific change in protein and glucose level of hypertensive patients was cal-
culated. Gavino et al. also reported the higher levels of antioxidants in male diabetic
229
patients as compared to female diabetics4,25. However, some contradictory results of
higher SOD and MDA levels in females than males have been reported as well24,26.
CONCLUSIONS
Altered biochemistry in hypertensive patients highlights the role of oxidative dam-
age and antioxidants in etiology and pathogenesis of hypertension in type 2 diabetic
patients.
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(2014).
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Defense System and Oxidative Stress Markers in Blood. Arab J Sci Eng, 40, 1 (2015).
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of Protein Utilizing the Principle of Protein-Dye Binding. Anal Biochem, 72, 248 (1976).
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Physiol, 59, 309 (1977).
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(1955).
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of Membrane Permeability and Lipid Peroxidation and Decreased Levels of Superoxide Dismutase
and Catalase. J Exp Bot, 32, 93 (1981).
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231
Tribochemical and corrosion interactions
EFFECT OF SiC AND Gr REINFORCEMENTS ON CORROSION

RESISTANCE OF ALUMINIUM MATRIX COMPOSITES
P. SHANMUGHASUNDARAM
Department of Automobile Engineering, Karpagam University,
641 021 Coimbatore, India
E-mail: sunramlec@rediffmail.com
ABSTRACT
The purpose of this study is to find the corrosion susceptibility of aluminium reinforced
with SiC and graphite composites in a 3.5% NaCl solution. The electrochemical behav-
iour of the composites was investigated by employing the potentiodynamic polarisation
method. The corrosion test results showed that the Al alloy–SiC composites exhibited
slightly lower corrosion resistance than Al alloy. In contrast, addition of graphite
particles increases the corrosion rate of Al alloy–SiC composite considerably since
the graphite particles act as cathodic relative to the Al alloy. The surface morphology
of the corroded surface was investigated using scanning electron microscope (SEM).
Keywords: Al alloy-SiC/Gr composite, potentiodynamic polarisation method, corro-
sion rate, surface morphology, SEM.
AIMS AND BACKGROUND

Aluminium metal matrix composites are employed as a potential material in automo-
tive and aerospace industries since they have higher stiffness and strength compared
to monolithic alloys. Corrosion characteristics of the materials must be studied before
selecting the material for the particular application where the material is exposed to
the corrosive environments. Corrosion behaviour of the particulate metal matrix com-
posites depends on the type, alloying elements and volume percentage of the matrix
and reinforcement materials, grit size, distribution of reinforcement in the matrix and
the processing method for fabricating composites. The corrosion resistance is also
greatly influenced by porosity, dislocation densities at the interface between matrix and
reinforcement, alloying segregation, intermetallic compounds and reaction products,
residual stress and the electrical conductivity of the particles1.
Jones2 reported that aluminium instantly covers its surface with an oxide film
which enhances the corrosion resistance when exposed to corroding media with pH
between 4 and 9, indicating passivity. The passive film (thickness on aluminium is
232
within 10 nm) which forms on the metal surface, acts as a barrier to further oxidation
and is easily destroyed in the solution containing active anions, such as Cl–, leading
to localised corrosion, especially pitting corrosion. Reena Kumari et al.3 investigated
the corrosion behaviour of 6061/Al – 15 vol.% SiC(p) composite and the base alloy
in NaOH solution using potentiodynamic polarisation and impedance methods. They
reported that the corrosion rate of the composite is higher than that of the base alloy.
Trowsdale et al.4 investigated the influence of silicon carbide reinforcement on the
pitting behaviour using polarisation technique in NaCl solution at 25°C. It was found
that the composites show increased susceptibility to pitting attack compared with un-
reinforced alloys and pitting arises from voids at the reinforcement/matrix interface.
Bobic et al.5 reviewed corrosion of metal matrix composites with aluminium alloy
substrate. They reported that the composites are more susceptible to corrosion attack
than the matrix alloy. Zakaria6 investigated the effect of the size and volume fraction
of SiC particulates on the microstructural and corrosion behaviour of Al/SiC metal
matrix composites. It was reported that increasing the volume fraction of the SiC
particulates increased the corrosion resistance of the Al/SiC composites. Aylor and
Moran7 analysed the effect of reinforcement on the pitting behaviour of aluminium-
graphite and aluminium-silicon carbide composites. The pitting attack on the Al/SiC
composites was distributed more uniformly across the surface, and the pits penetrated
to significantly less depths. Pardo et al.8 investigated the corrosion behaviour of cast
aluminum matrix composites in chloride media. They reported that the corrosion
process is influenced more by the concentration of alloying elements in the matrix
than by the proportion of SiC reinforcement. Candan and Bilgic9 reported that the
incorporation of SiC particles to Al–Cu alloy increased the corrosion resistance over
that of the base alloy in 3.5 wt.% NaCl solution. Kiourtsidis and Skolianos10 reported
that the intermetallic compounds surrounding the particles initiated pitting attack of
the Al alloy reinforced with SiC composites in 3.5 wt.% NaCl solution.
Graphite reinforced aluminium composites are developed for possible use in
tribological applications such as bearings and bushings owing to their good resistance
to wear and seizure. Since the graphite acts as a solid lubricant it makes the compos-
ites self-lubricating11. Akhlaghi and Zare-Bidaki12 stated that during dry sliding of Al
alloy – graphite composite forms a layer of graphite which act as a solid lubricant
between the contacting surfaces and this improves resistance to wear and machinability.
Sherif et al.13 investigated the effect of graphite on the corrosion behaviour of pure Al
in 3.5% NaCl solution by electrochemical and spectroscopic techniques. Corrosion
measurements indicated that the presence of graphite and the increase of its content
raise the corrosion rate and reduce the polarisation resistance of Al.
Although there is a considerable amount of research on the corrosion behaviour
of Al alloys and its composites, limited work has been reported in understanding
the corrosion behaviour of Al alloy reinforced with SiC/Gr hybrid composites. The
effect of SiC and graphite reinforcement on the corrosion resistance of Al alloy was
investigated using the potentiodynamic polarisation method. Scanning electron mi-
233
croscope investigation was performed on the tested specimen surface to study the
surface morphology.
EXPERIMENTAL
Microstructural analysis. In this research, Al-SiC/Gr composites were fabricated by
stir casting method14. Al alloy 7075 ingot was used as the matrix material and 5 wt.%
SiC particles with average size of 70 μm were used as the reinforcement. 2.5 wt.%
graphite particles with average size of 100 μm were used as the solid lubricant. SEM
microstructure (Fig. 1) of the Al – 5 wt.% SiC – 2.5 wt.% Gr composite specimen was
examined to explore the distribution of SiC and graphite particles in the Al alloy matrix.
Fig. 1. SEM micrograph of the Al – 5 wt.% SiC – 2.5 wt.% Gr composite
It illustrates that the clustering of SiC and graphite (black phase) particles was
not observed and particles were distributed more uniformly in the Al alloy matrix.
Electrochemical test. Corrosion is an electrochemical process where oxidation (loss
of electrons) takes place at the anode and a reduction (gain of electrons) takes place
at the cathode. The corrosion rate is computed by measuring the current flow in the
electrolyte solution with reference to potential applied. At the corrosion potential, the
rate of hydrogen is equal to the rate of metal dissolution, and this point corresponds to
the corrosion rate of the specimen expressed in terms of current density. A polarisation
technique was employed to evaluate the rate of corrosion for both unreinforced Al
alloy and composite specimens in 3.5% NaCl electrolyte solution at a pH of 7 using
a standard Gill AC series-903 potentiostat. Schematic of potentiostat corrosion test
rig is shown in Fig. 2.
Typical test rig consists of electrochemical cell which consists of NaCl electrolyte,
a calomel electrode which is a reference electrode, a platinum auxiliary or counter
electrode and a working electrode which is a specimen to be tested. A platinum
electrode provides the path for the applied current into the electrolyte solution. The
specimens were machined to 10 × 10 mm size and were ground using 600 grit emery
234
paper, rinsed in distilled water and methanol to remove surface contaminants and
finally dried. Prior to each polarisation test, the electrode was allowed to stabilise for
approximately 60 min in the test solution to attain the steady state. The specimens
were exposed to the NaCl solution for a period of 30 min at room temperature in the
corrosion test rig.
Fig. 2. Schematic of corrosion test rig
The electrochemical tests consisted of a cyclic sweep and scan was started from
400 mV at a constant sweep rate of 60 mV/min. Electrochemical polarisation reactiva-
tion curves (EPR) were obtained with the help of ACM software. Corrosion current
and corrosion rate were computed employing the following expression specified by
ASTM G1-90. Polarisation resistance can be converted to corrosion current density
Icorr according to the Stern-Geary equation:
Icorr × metal factor
corrosion rate = mm/year (1)
1000
where
ba × bc
current density, Icorr = , (2)
2.3 Rp (ba + bc)
where ba is anodic Tafel slope, V; bc – the cathodic Tafel slope, V; Rp – the polarisa-
tion resistance in Ω m2.

The effect of SiC and graphite addition on the corrosion potential of Al alloy 7075 is
presented in Fig. 3 where curves of potential (mV) versus current density (mA/cm2)
obtained through polarisation method. Corrosion resistance of the specimen would
increase if the corrosion potential shifts to more positive side and current density
235
moves to the negative side with respect to duration. The shifting of current density
values of Al-SiC-Gr composite to more positive side indicates that the addition of
Gr particulates in the Al-SiC composites increases the susceptibility to corrosion.
Fig. 3. Potentiodynamic polarisation curves for: Al alloy 7075 – 1; Al 7075 – 5 wt.% SiC – 2; Al 7075 –
5 wt.% SiC /2.5 wt.% Gr – 3
It can be seen from Fig. 3 that the current density of the Al alloy 7075 is more
negative than that of the Al–SiC composites and Al – SiC–Gr composites. It can be
concluded that Al alloy has higher corrosion resistance compared to the SiC/Gr hybrid
reinforced aluminium composites. The increased corrosion resistance of unreinforced
Al is believed to be due to the natural protective oxide layer which acts as a passive
film. Pourbaix15 reported that the pure aluminium will be rapidly covered with a dense
oxide layer of 2–3 nm thickness when it is exposed to oxygen-containing environment.
Table 1. Corrosion rate of specimens

Specimen ba (mV) bc (mV) Icorr (mA/cm²) Corrosion rate
(mm/year)
Al alloy 61.5549 63.9041 0.4359 0.00499
Al–5 wt.% SiC 59.8751 66.1261 0.5079 0.00523
Al–5 wt.% SiC/Gr 68.7339 147.639 0.7864 0.00779
The measured corrosion rate of the specimens is presented in Table 1. It can be

observed that the corrosion rate was found to be 0.00499 mm/year for the Al alloy 7075,
0.00523 for Al alloy 7075/SiC composites and 0.00779 for Al alloy/SiC/Gr composite.
The corrosion rate increased by 49% when 2.5 wt.% graphite were incorporated to
the Al alloy – SiC composite. On the other hand, corrosion rate increased by 4.8%
when 5 wt.% SiC were added to the Al alloy. It infers that the corrosion resistance of
the Al alloy was not affected considerably by the addition of SiC particles since they
are inert in the NaCl solution. The similar observation has been made by Zakaria6.
236
Fig. 4. SEM micrograph of Al – 5 wt.% SiC composite specimen
Fig. 5. SEM micrograph of Al – 5 wt.% SiC – 2.5 wt.% Gr hybrid composite specimen
It can be observed from the SEM micrographs 4 and 5 that more pits are formed
on the surface of Al-SiC/Gr hybrid composite (Fig. 5) compared to Al-SiC (Fig. 4)
composite. Moreover, pits in the Al – SiC/Gr composites were deeper than that of
the Al-SiC composites. It can be attributed to the fact that the graphite particles act
as cathodic sites since they have higher thermal conductivity. Similar observation
was noticed by Saxena et al.16 They reported that the graphite particles act as cathodic
sites compared with the Al alloy when the composite is exposed to NaCl solution.
Hence, incorporation of graphite increases cathode to anode ratio in the composites
and leads to corrosion. Hihara and Kondepudi17 have reported that the galvanic cor-
rosion is a primary corrosion mechanism for Al/Gr composites in aerated solutions.
It can be concluded that graphite is more susceptibility to corrosion in NaCl solution
than SiC particulates. It can be noted that SiC, being an insulator material, reduces
the possibility of galvanic corrosion in the composites compared to the Al alloy.
237
CONCLUSIONS
The electrochemical behaviour of Al alloy, Al – 5 wt.% SiC composite and Al –
5 wt.% SiC – 2.5 wt.% Gr hybrid composite was studied in 3.5% NaCl solution using
potentiodynamic polarisation method. The following conclusions have been drawn
from this study.
The corrosion rate increased by 4.8% when 5 wt.% SiC was incorporated to the
Al alloy. The corrosion susceptibility of the Al/SiC composites was slightly higher
than that of Al alloy. On the contrary, incorporation of graphite particles reduces the
corrosion resistance of composite drastically. The addition of 2.5 wt.% of graphite
particles to Al alloy 7075 – SiC composite increases the corrosion rate by 49%. Cor-
roded surface of Al–SiC/Gr composites reveals that pits were deeper than that of the
Al-SiC composites.
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Revised 13 June 2015
239
Technological aspects of oxidation processes of special interest to food and petrochemistry
RESEARCH ON MOISTURE CONTENT MEASURING DEVICE

OF EDIBLE OIL BASED ON CAPACITANCE DETECTION
MECHANISM
LIANG GEa,b*, QIANG ZENGa, ZHENGYIN WANGa, XIAOHUI XIEa,

JUNBI LIAOb, JUNLAN LIc
a
College of Mechanical and Electronic Engineering, Southwest Petroleum
University, 610 500 Chengdu, China
E-mail: cgroad@163.com
b
Department of Measuring and Control, Sichuan University, 610 065 Chengdu,
China
c
Southwest Branch, Engineering Design Co., Chiina National Petroleum
Corporation (CNPC), 610 413 Chengdu, China
ABSTRACT
Illegal trader adulterate water with edible oil to sail, it is important to detect the water
content of edible oil. This design uses the principle of capacitance method to measure
water content of edible oil to design detection system. The core of method is according
to permittivity difference between oil and water is large, resulting in a capacitance
value of the dielectric oil and water when the capacitive principle of the sensor is very
different. The water content of edible oil detection system has advantages of high
speed, low power consumption, low price and portable. Laboratory test shows that
the water content of edible oil detection system has the ability to accurately measure
the conductivity message of the oil, and system measurement accuracy can meet the
needs of water adulteration with edible oil detection.
Keywords: water content detection, edible oil, capacitance method, portable instru-
ment, data acquisition.
AIMS AND BACKGROUND

The watered-edible oils have a range of effect on the storage and use. As the expo-
sure of all kinds of watering edible oil incident, the quality and safety issues of the
watered-edible oil have become the focus of media attention and it is urgent to have
a fast and accurate detection to the edible oil1–6.
*
For correspondence.
240
There are many detections of moisture content of edible oil which include the
traditional distillation and electrical de-France. Although the distillation has high
measurement accuracy, its long sampling time and big sampling random restrict the
use in the laboratory measurement7–11. Because of the disability of on-line measure-
ment, it can not meet the need of automation management. The electrical de-France
operates simply but it has large measurement errors. In addition, the measurements of
moisture content of edible oil also include ray, microwave, radio frequency, infrared
spectroscopy, capacitance methods and Karl Fischer method testing edible oil trace
moisture content12–16. But the price, measurement form and using occasion of these
methods restrict the promotion of moisture content rapid measuring device and the
use of relevant administrative departments and agencies17,18. Therefore, development
of a rapidly and portable moisture content measuring device of edible oil has signifi-
cant economic and social benefits to eliminate the blind spot monitoring of edible oil
wholesale, storage and sale businesses, to eliminate abnormal loss of edible oil and
to achieve the management of edible oil monitoring information.
SYSTEM DESIGN PRINCIPLES

The edible oil permittivity is usually 2.5 and water is 80. Although the permittivity
varies from the place of production, compared with the 77 between oil and water the
variation is very little which can be omitted.
Assuming the plate length, width and spacing of the two plates were l, w and d
of the parallel plate structure capacitive sensor, respectively. Omitting the influence
of edge effects, the capacitance values C approach to:
ε0 εm l w
C= , (1)
d
where ε0 = 8.85 × 10–12 F m–1, εm is the equivalent relative permittivity of mixed

medium.
Because water molecules are polar molecules, under the action of the polaris-
ing field, polar molecules, orientation will be polarised whether the water-in-oil, or
water-in-oil. The polarised molecules interact and attract mutually, turn to the electric
field direction and connect serially as a string which can be divided to parallel to the
electric field component and perpendicular to the electric field component. Thus the
complex interface of oil-water mixed medium respected to polarisation field is parallel
and series. The formula describing the mixed polarisation state of moisture content
according to the series coefficient k can be expressed as:
2ρ
k= , (2)
5 – 3ρ
where ρ is the moisture content.
241
The relative permittivity of oil-water mixed medium is13:
εm = k (ρεw + (1 – ρ)ερ) + (1 – k)εwερ(ρεw + (1 – ρ))–1. (3)
Absolutely, in formula (3) the first is the contribution of series situation the oil
and water respected to polarisation field, and the second is the contribution of paral-
lel situation. As the ascent of moisture content these two also increase. As for the
oil-water mixed medium which has known moisture content, we can use formula (2)
and (3) to calculate the relative permittivity.
HARDWARE DESIGN
The goal of design is developing an oil product moisture content measuring system
to achieve the measurement of capacitance. Thus we can comprehend and analyse
the quality edible oil through the moisture content. The oil product moisture content
measuring system can be seemed as a portable intelligent instrument. The system will
accomplish the function of oil product moisture content parameters detection, signal
conditioning, data acquisition, data processing, data analysis and display. The design
scheme is shown in Fig. 1.
Display
Fig. 1. System block diagram
Capacitive sensor design. Since there is no useful capacitive sensor in the market,
the design adopts CAD drawing software to draw the plate of sensor probes and the
fixation. The CAD diagrams engraving machine would carve out physical. The CAD
graph is shown in Fig. 2. The silica gel is used to fix, bond and seal sensor probes.
As the measuring oil-water mixed liquid is conductive, the two capacitor plates must
be isolated to prevent short circuit of plate for the conduction of liquid and lose the
measurement results.
In the design to make the plates clean and smooth, the ethanol is used to wash
the plates firstly. Secondly, the plates copper surface would be coated with insulat-
ing materials and sealed. At last, the plate can be fixed on the fixation and seal the
electrode wire.
242
a b
Fig. 2. Computer Aided Design (CAD) graphs of plates of sensor probes and fixation AD7746 and
microcontroller interface circuit: a – sensor top and beneath plates; b – sensor fixation
Fig. 3. AD7746 and microcontroller interface circuit (VCC – volt current condenser)
The AD7746 and STC89C52 microcontroller interface circuits are shown in

Fig. 3. SDA and SCL linked with pull-up resistor are I2C two-wire serial interfaces
which receive and transmit data synchronously. For the microcontroller does not
have I2C communication bus, SCL is linked with P2.2 of microcontroller and SDA is
linked with P2.3 and through the serial analog programming, the communication will
be achieved. RDY is the sign of consummation of capacitor data conversion which
linked with P3.3 and adopts external interrupt way to transmit A/D conversion status
of AD7746 capacitor signal to microcontroller.
System power. This system is supplied by LM2596S DC-DC ultra-small adjustable
buck module which input is 3–40 VDC and output is 1.5–35 V/3ADC. The module
structure is concise and uses conveniently. When the input voltage is greater output
more than 1.5 V, its output can be sustained in regulated voltage amplitude and would
be used as stable power. In addition, the output voltage accuracy can be adjusted to
0.01 V to use as reference voltage.
Software design. The software design utilises the modular design concept constituted
with 1602 module, AD7746 capacitance digital converter module, signals processing
and computing module. Through the modular design concept, readers will understand
easily and more important is coherence during processing and repeated testing would
be avoided. The overall flow chart of the design is as shown in Fig. 4.
243
Does the extremal interrupt
I triggered?
Fig. 4. Overall flow chart of the system
SYSTEM TESTING
Testing process. Through the way of adding treated water quantitatively, we can test
the capacitance value of capacitance sensor by the system. Then compute the moisture
content as the formula (4) shown according to the volume ratio of oil and water we
had added thus establishing the doped quantitative model of capacitance value and
moisture content. At last, fitting its function formula by the software and we can ac-
complish the measurement of moisture content by testing capacitance value.
Vw
ϕ= × 100% (4)
Vw + V0
where Vw, V0 and ϕ mean the volume of treated water added, the volume of pure oil
added and the percentage of moisture content, respectively.
The system testing overall flow charter is shown in Fig. 5. When measuring the
moisture content, we need to select a standard cylinder to measure the volume of the
oil and water, and mix them sufficiently. The testing uses the JINLONG brand oil in
the market. Because the oil belongs to the mixture of alkanes, from the molecular
formula, regardless of what kind of oil, it must contain alkyl, and water contains a
hydroxyl group, the emulsified mix must use substance which contains both hydro-
carbon group and hydroxyl group. We adopt ethanol as an emulsion in the testing.
244
Fig. 5. Overall flow chart of testing
System calibration. Firstly, we measure 200 ml pure water into a container, add
quantitative treated water to the container quickly, and stir evenly. The aim is mak-
ing the treated water and oil emulsified mix fully and avoiding the measuring effects
of stratification of treated water and oil. In order to prevent increasing error in the
non-regulated measurement for adding treated water regularly, we need to add water
non-regulated every time in the testing. The measurement data are shown in Table 1.
Table 1. Moisture content data

Number of times Once water Total amount of Capacitance Moisture
added (ml) water added (ml) value (pF) content (%)
1 0 0 23.042 0
2 2 2 23.532 0.99
3 3 5 24.512 2.439
4 3 8 25.240 3.846
5 2 10 26.472 4.762
6 5 15 27.451 6.977
7 3 18 28.431 8.257
8 2 20 28.921 9.091
9 3 23 29.411 10.314
10 3 26 30.391 11.504
11 3 29 31.126 12.664
12 6 35 33.086 14.894
13 3 38 34.067 15.966
14 3 41 35.047 17.0124
15 3 44 36.519 18.033
16 3 47 37.255 19.028
17 3 50 38.236 20
245
According to the data of Table 1, the fitting curve of capacitance value and mois-
ture content is shown in Fig. 6. The fitting function formula is:
y = – 0.0328x2 + 3.3173x – 58.979.
Fig. 6. Fitting curve of capacitance value and moisture content
System test and error analysis. In order to verify the accuracy of system testing, we
conducted system testing and process the results using system calibration curve. The
testing results are shown in Table 2.
Table 2. Analysis of errors between the values of measured and actual moisture content
Actual value (%) Measured value (%) Absolute error (%)
0 –0.00757 –0.00757
0.99 0.859 –0.131
2.439 2.574 0.135
3.846 3.774 –0.072
6.977 7.256 0.279
8.257 8.708 0.451
9.091 9.431 0.340
10.314 10.102 –0.216
11.504 11.436 –0.068
12.664 12.398 –0.226
14.894 14.804 –0.090
15.966 15.916 –0.050
17.012 16.971 –0.042
18.033 18.443 0.410
19.028 19.130 0.102
20 19.993 –0.007
The maximum absolute error is 0.451%. The main source of error is due to the
influence of the parasitic capacitance, uneven distribution of oil and water, and the
246
highly volatile ethanol in the measuring process, thus resulting in fluctuations of
measuring capacitance value.
CONCLUSIONS
(1) This paper presents an edible oil moisture content measurement system design
which explained the design process in detail from hardware and software design.
(2) The moisture content information can be mastered by inspectors through
the edible oil moisture content measurement system to learn whether the edible oil
mixed with water.
(3) The system has the advantages of small size, low power consumption, speedy
test, automatic range conversion and good repeatability, thus can be used to actual
testing.
(4) Due to a wide range of edible oil in our nation currently, the edible oil of vari-
ous manufacturers has different capacitance value after watering. So, it is essential to
establish an edible oil capacitance value database and an expert management system
to achieve the accurate measurement of moisture content of edible oil.
ACKNOWLEDGEMENTS
This work is supported by scientific research starting project of SWPU (No
2014QHZ029), national natural science foundation of China (No 21204139), the
State Administration of National Security (No sichuan-009-2013AQ, No sichuang-
0021-2014AQ), and the Sichuan Educational Committee (No 15ZB0060, 14ZB0052).
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Received 9 May 2015
Revised 23 June 2015
248
EXPERIMENTAL STUDY ON REDUCING THE

INJECTION PRESSURE OF LOW INTERFACIAL TENSION
DISPLACEMENT SYSTEM TC-1 IN LOW PERMEABILITY
RESERVOIR
F. X. LIa , Z. K. WANGa*, H. L. LIUa, Y. Y. TIANb, J. N. XUa

a
Chongqing University of Science and Technology, 401 331 ChongQing, China
b
Northwest SiChuan Gas Field Southwest Oil and Gasfield Company,
621 000 Jianyou Sichuan, China
E-mail: wzk0408@126.com
ABSTRACT
As the low permeability reservoir is affected by such factors as poor physical proper-
ties, low relative permeability of water phase, strong heterogeneity, etc. The reservoir
stratum has poor water extraction efficiency, causing elevated water injection pres-
sure, increased load of water injection system and decreased rate of recovery. Based
on the optimisation of single surface active agent TPS-1 and CA-2, TC-1 system can
be formed through reciprocal preparation. Through laboratory experiments, the salt
resistance and thermal stability of system are evaluated, the impacts of low tension
system on the wettability of reservoir rock, injection pressure and water-test perme-
ability are analysed, the impacts of injection speed on the injection pressure and the
impacts of ageing time on the injection time and permeability are studied. As indicated
in the research results, the salt resistance capability of TC-1 system is high, and the
temperature resistance capability is relatively strong. The interfacial tension is low.
It also possesses ideal capability of changing the rock contact angle. The injection
pressure of subsequent water flooding decreases to some extents under different flow
velocities, the combination solution can enable changes in the wettability of reservoir
rock surface, and possess significant effects of decompression and augmented injection.
Keywords: low permeability reservoir, low tension, pressure decrease and injection
increase.
AIMS AND BACKGROUND

Low permeability reservoir has poor physical properties, lower permeability, complex
pore structure and high water flooding residual oil saturation and low water-phase rela-
*
For correspondence.
249
tive permeability as well as strong heterogeneity and low oil saturation1. In addition,
with the impacts of contamination in immediate vicinity of wellbore, it gives rise to
lowered water absorption capacity of the reservoir and increased injection pressure and
thus results in more loads on the injection system, damages in casing and decreased
rate of oil production. Pore permeability may be changed in the low permeability
reservoir by way of acid fracturing to enhance the injection capacity of water wells,
so as to increase oil recovery2; however, it costs much. It is the nature of crude oil that
determines the rate of absorption and compatibility of surfactant lipophilic group in oil
phase. Transient interfacial tension and balanced interfacial tension of the surfactant
are thus affected. When alkali is found in displacement components, the acidoid in
crude oil has a material effect on the interfacial tension. With adjustment salt content
in water and in lipophilic-hydrophilic balance of surfactant, the minimum interfacial
tension can be obtained to improve oil-water seepage characteristic, water-phase
relative permeability and reduce the injection pressure. The low tension displacement
system is developed according to the problems found in water injection development
of low permeability reservoir. A change in the wetting contact angle of rocks θ and a
decrease of oil-water interfacial tension σ can be achieved by changing the interfacial
tension among oil, water and rock and rock wettability without changes in poroperm
characteristics of low permeability reservoir so as to reduce the injection pressure of
injection wells in the low permeability reservoir and enhance water flooding effects
of such reservoir.
EXPERIMENTAL
EXPERIMENTAL INSTRUMENT
Contact Angle Instrument is produced in Japan; TX-500 Interface Tensiometer with

resolution of 0.001 mN/m; scale, with sensitive quality of 0.1 mg; multifunctional
core displacement device; oven, with room temperature of ~300°C and temperature
error of ±2°C; constant temperature bath oscillator, with room temperature of ~100°C,
temperature error of ± 2°C and oscillating frequency of 0–300 r/min.
Surfactants used was petroleum sulphonate surfactant TPS-1 (mass fraction of
effective content: 45%), Gemini surfactants CA-2 (mass fraction of effective content:
40%); crude oil: Changqing degassed crude; water: formation water used for indoor
experiment is simulated formation water of Changqing Oilfield, with composition as
shown in Table 1.
Table 1. Analysis data table of oilfield formation water

No of Ion content (mg/l) Miner- pH Water
well alisation type
K Na
+ +
Ca 2+
Mg 2+
Cl–
SO4 2–
HCO3 –
degree
D5-2 24795 1399 905 42897 411 399 70808 5.6 CaCl2
H1-7 25119 2582 1077 45120 1855 84 75840 5.3 CaCl2
250
EXPERIMENTAL METHOD
The simulated formation water from Changqing Oilfield serves as the formation water
was used for laboratory experiment. The experimental procedure is described in Fig. 1.
Fig. 1. Flow chart of experiment
(1) The drill cores that have gone through the process of extraction, washing,
drying and surface leveling are weighed in the air. Then, the formation water is satu-
rated under vacuum condition, the rock specimen with saturated formation water is
weighed and the pore volume of drill core is calculated:
Vp = (W2 – W1)/ρ (1)
where Vp is the pore volume of drill core; W1 – the weight of drill cores that have gone
through the process of extraction, washing, drying and surface levelling; W2 – the
weight of drill core saturated with the formation water; ρ – the density of formation
water.
(2) The formation water is injected into the drill core with a certain pump speed,
and the displacement pressure with the multiples of different pore volume is recorded.
After the displacement pressure becomes stable, the pressure is recorded and then the
matrix permeability (K) is determined.
(3) Oil displacement of water-test oleic permeability (K0): During the displacement
process, flow velocity is required to regulate from low to high until the state of bound
water is reached. Afterwards, it is required to flood the displacement with the same
flow rate as the last step, and the displacement pressure with the multiples of different
pore volumes is recorded. After the oil displacement keeps the displacement pressure
stable, the pressure is recorded and the oil relative permeability (Krw) is determined.
(4) Water displacement: The displacement pressure at the different time of dis-
placement is recorded. After water drive keeps the displacement pressure stable, the
pressure is recorded and the relative permeability of oilout (Kro) is determined.
(5) The low tension system is injected and kept for a period of time.
251
(6) The displacement pressure with the multiples of different pore volumes is
recorded. After water drive keeps the displacement pressure stable, the pressure is
recorded and the water relative permeability (Krw) is determined.
(7) The step 4 is repeated and the relative permeability of oil (Kro′) is determined.
LOW-TENSION FOAM SYSTEM COMPOSITION AND BASIC PERFORMANCE

EVALUATION
Low-tension foam system composition. Most of low-tension systems contain surfactant

or are dominated by surfactant and supplemented by additive. When two or more
surfactants mix with each other, its solution property is different from that of single
surfactant, mainly because antagonistic effect or synergistic effect will generate under
interreaction between surfactant molecules mixed or generated complex or electrostatic
attraction, exclution or other physical and chemical action3. Although synergistic effect
may also generate when surfactant solution is added with certain inorganic salt and
organic compounds (such as alcohol, high-molecular compound, etc.), by contrast,
mixed surfactant often has less dosage, low cost and obvious economic benefit than
that of single surfactant.
For low-tension system, based on the optimisation of single surfactant, TPS-1 and
CA-2 with good capability to reduce interfacial tension will be used for interworking
to form TC-1 system and carry out experimental analysis, with experimental result
as shown in Fig. 2.
TPS-1 CA-2 TC-1

interfacial tension (mN/m)
0.1
0.01
0.001
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
effective concentration (%)
Fig. 2. Influence of effective concentration of single surfactant and system on oil-water interfacial tension
Experimental result shows that single surfactants TPS-1 and CA-2 have good
capability to reduce interfacial tension, but under low concentration, their respective ca-
pability to reduce oil-water interfacial tension is inferior to complex of both; interfacial
tension of the complex can reach 10–2 mN/m under ultra-low concentration (effective
concentration 0.01–0.05%), which shows surfactants have good synergistic effect.
252
Basic performance evaluation of low-tension system
(1) Salt tolerance of system
With the increase in formation water salinity, metal ion in water acts with system
molecule to reduce effect of foam system; therefore, get formation water with dif-
ferent mineralisation degree to make up low-tension mixture with concentration of
10 000 and 50 000 mg/l for salt tolerance test to observe salt tolerance of low-tension
mixture, with result as shown in Fig. 3.
1
mineralisation degree 50 000 mg/l

0.1
mineralisation degree 10 000 mg/l
0.01
0.001
0.0001
1 2 3 4
time (min)
Fig. 3. Relation curve for influence of mineralisation degree on tension of TC-1 system
Under mineralisation degree of 50 000 mg/l, low-tension system can still maintain
ultralow interfacial tension, which shows that this low-tension system has good salt
tolerance more than 50 000 mg/l.
(2) Thermal stability
Since oil reservoir has higher temperature in general, form stabiliser used for
displacement foam system must have certain high-temperature resistance4. By keep-
ing low-tension mixture concentration of 500 mg/l unchanged and changing the
temperature of solution system, the influence of temperature on oil-water interfacial
tension is observed. The results are shown in Fig. 4.
1
0.1
0.01
0.001
0.0001
0 20 40 60 80 100
temperature (°C)
Fig. 4. Relation curve for interfacial tension of system varying with temperature
Interfacial tension of system is always lower than 0.01 mN/m, without obvious
change in interfacial tension and system performance and the system has strong age-
ing resistance.
253
EXPERIMENTAL ANALYSIS ON PRESSURE DECREASE AND
INJECTION INCREASE OF LOW-TENSION SYSTEM
Influence of low-tension system on rock wettability in oil reservoir. The ability of
system solution to change rock surface wettability can be evaluated through meas-
uring contact angle of surfactant with rock surface. In the experiment, core chip in
actual oil reservoir will be placed into crude oil to be soaked (simulating oil-reservoir
temperature) for 10 days. Then, injection increase system selected will be dropped
on core chip soaked and wetting contact angle under different temperature conditions
will be measured as shown in Fig. 5.
80
70 20°C
contact angle (degree)
60 50°C
50 75°C
40
30
20
10
0
0 0.05 0.1 0.15 0.2 0.25
effective concentration (%)
Fig. 5. Influence of effective concentration and temperature of TC-1 system on contact angle
Result shows that under different temperature (20, 50 and 75oC) conditions,
with the increase in system concentration, contact angle of system solution and core
chip presents a decreasing trend and when system concentration is 0–0.05% (effec-
tive concentration), contact angle will have the most reduction and may have a knee
point within this concentration, which shows injection increase system can reverse
rock surface under low concentration or ultra-low concentration, namely, from lipo-
philicity turning into hydrophily5. When concentration is more than 0.05% (effective
concentration), contact angle will decline with less range, which shows that rock
surface adsorption capacity reaches a saturation value, so angle contact will decline
with less range with increase in concentration. Under different temperature conditions,
change in contact angle will vary; under concentration of 0–0.05%, temperature of
50°C will be better than 20 and 75°C; with increase of concentration, temperature of
75°C will be better than 50°C, but the advantage is not obvious, which shows that
surfactant will be optimum under 50–75°C, namely, surfactant mixture will reserve
rock surface wettability6.
Influence of system on injection pressure and permeability to water. To further re-
flect that injection increase system has good effect of decreasing pressure, 6 groups
of cores with ultra-low permeability will be selected for core displacement test as
shown in Table 2.
254
Table 2. Statistical table of injection pressure change of TC-1 system before and after injection
Core No Permeability Permeability to water Injection pressure ris-
(10–3 μm2) rising and falling ing and falling range
range (%) (%)
1-1 6.29 6.5 –35.84
2-3 5.19 10.7 –16.13
4-1 8.48 10.3 –26.59
5-3 4.28 15.6 –35.68
8-4 6.88 19.3 –24.59
9-2 3.52 6.9 –8.57
Average 5.77 11.55 –24.57
Experimental data show that injection pressure of core with ultra-low permeabil-
ity will decline obviously and the flow conductivity will be improved after injecting
surfactant, of which B-1 core will have the largest drop in injection pressure, with
reduction degree reaching 35.84% and growing rate of permeability to water reaching
6.5%. 6 groups of cores have average depressurising range of 24.57% and average
rise of 11.55% in permeability to water, so this system has good flow conductivity
for pressure decrease and increase7.
Influence of injection speed on injection pressure. After injecting surfactant, injection
pressure for subsequent water flooding will decline to some extent under different
flow rates, which shows that retention phenomenon will occur after rock surface
contacting surfactant to result in reverse of rock surface, namely, lipophilicity of rock
surface will turn into hydrophily, finally decreasing injection pressure of the system8.
Experimental result is shown in Fig. 6.
0.5
0.45
0.4
0.35
injection pressure (MPa)
0.3
0.25
0.1
0.2 0.2
0.15 0.3
injecting surfactant
0.1 after injecting surfactant 0.1
0.05 after injecting surfactant 0.2
after injecting surfactant 0.3
0
0 2 4 6 8 10 12 14 16 18
injection rate (PV)
Fig. 6. Evaluation on speed-sensitively experiment of B-4 core
Experimental result shows that water flooding injection pressure will increase as
the injection speed increases before and after injecting surfactant mixture. However,
255
after injecting surfactant mixture, injection pressure will decrease more obviously
than before injecting, which shows that injection of surfactant mixture solution will
have a depressurised effect9.
Influence of ageing time on injection pressure and permeability. As time goes on,
the system has adsorption and retention phenomenon in the rock core and causes the
transformation of rock from lipophilicity to hydrophily, which reflects the decrease
in injection pressure of system and increase in permeability to water directly10. Ex-
perimental result is shown in Fig. 7.
0.4
0.35
injection pressure (MPa)
permeability (decimals)
0.3
0.25
0.2
0.15 injection pressure
permeability to water
0.1
0.05
0
0 5 10 15 20 25 30 35
time (days)
Fig. 7. Influence of ageing time on core permeability and injection pressure
Displacement experiment result shows that with increase in ageing time, injec-
tion pressure in core will decrease gradually, but permeability to water will rise, in
which ageing time will be 0–5 days, injection pressure will decrease significantly
and permeability to water will rise obviously; during 5–25 days, injection pressure
will became stable, but permeability to water will rise continuously, after 25 days,
increase in core permeability to water will become flat and injection pressure will also
become stable, which shows that the system has table flow conductivity for pressure
decrease and increase.
CONCLUSIONS
(1) TC-1 system formed by mixing TPS-1 and CA-2 at 1:1.2 has better capability to
reduce oil-water interfacial tension than that of single aqueous surfactant solution
and under ultralow concentration, oil-water interfacial tension can reach 10–2 mN/m.
(2) Salt tolerance of TC-1 system can reach 5000 mg/l and when experimental
temperature reaches 90°C, interfacial tension will still be less than 0.01 mN/m and
temperature resistance will be strong; under 20, 50 and 75°C, it is ideal to change the
capability of rock contact angle so that the injection pressure can be reduced.
(3) Following mixing, TC-1 system enables a mean decrease of the pressure in-
jected to cores of 24.57%, and a mean increase of permeability to water of 11.55%;
256
subsequent water flooding experiences decrease in the injection pressure with differ-
ent flow rates. Solution mixture enables changes in wettability of rock surfaces in the
reservoir, yielding evident pressure decrease and injection increase effects.
REFERENCES
1. H. Y. ZHONG, H. J. YIN: Reservoir Simulation for Improving Water Flooding Performance in
Low-permeability Reservoirs. Chem Technol Fuels Oils, 49 (3), 245 (2013).
2. Y. LI: Oil Recovery by Low Salinity Water Injection into a Reservoir: A New Study of Tertiary Oil
Recovery Mechanism. Transport in Porous Media, 90 (2), 333 (2011).
3. B. Y. JAMALOEI, R. KHARRAT: Fundamental Study of Pore Morphology Effect in Low Tension
Polymer Flooding or Polymer–Assisted Dilute Surfactant Flooding. Transport Porous Med, 76 (2),
199 (2009).
4. F. X. LI, Z. K. WANG, H. L. LIU: Study on Sensitivity of Low-permeability Reservoirs of Wuqi
Oilfield Chang 6. Oxid Commun, 38 (4), 1739 (2015).
5. M. Al-MANNAI, N. S. KHABEEV, V. S. SHAGAPOV: Towards the Problem of Droplet Injection
in a Vapor with the Aim of Reducing the Pressure. Fluid Dyn, 47 (1), 101 (2012).
6. P. P. SHEN, B. ZHU, X. B. LI, Y. S. WU: An Experimental Study of the Influence of Interfacial
Tension on Water–Oil Two-phase Relative Permeability. Transport Porous Med, 85 (2), 505 (2010).
7. J. KAI, T. JEONG, T. KIM, B. KIM: Analysis and Design for Reducing Residual Stress and Distor-
tion after Ejection of Injection Molded Part with Metal-insert. Int J Prec Eng Man, 15 (12), 2533
(2014).
8. Z. W. WU, X. A. YUE, T. CHENG, J. YU, H. YANG: Effect of Viscosity and Interfacial Tension of
Surfactant–Polymer Flooding on Oil Recovery in High-temperature and High-salinity Reservoirs.
J Petrol Explor Prod Technol, 4 (1), 9 (2014).
9. V. V. ERMOLAVEV, A. P. ANDREEV: Modernization of Standard and Rapid Pressure-reducing
and Cooling Units with Injection of High Pressure Water Power. Technol Eng, 42 (4), 222 (2008).
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ment under Ultrasonic Waves. Eur Phys J B, 60 (4), 447 (2007).
257
EFFECTS OF INCREASING SOIL CALCIUM APPLICATION

ON GROWTH AND UPTAKE OF CALCIUM, PHOSPHORUS,
ZINC AND BORON IN DURUM WHEAT (Triticum durum L.)
I. KIZILGOZ
Department of Soil Science, Faculty of Agriculture, Harran University, Sanliurfa,
Turkey
E-mail: ikizilgoz@harran.edu.tr
ABSTRACT
Calcium has an antagonistic relationship with other nutrients which influence sig-
nificantly yield and quality of crops plants. These nutrient elements are phosphorus
(P), zinc (Zn) and boron (B) which were investigated in the current study and they
have positive effects on yield and quality of crop plants. Of these nutrient elements,
phosphorus (P), zinc (Zn2+) and boron (B+) are especially important for their posi-
tive effects on plants, which was the subject of the present study. In this study, the
effects of a range of soil Ca2+ applications (0, 50, 100 and 200 mg kg–1 soil) on shoot
dry weight, shoot Ca2+, P, Zn2+ and B+ concentrations were determined in five durum
wheat cultivars (Ege-88, Kiziltan-91, C1251, Fuatbey-2000 and Zenit). The results
showed that shoot dry weight increased by soil Ca2+ application of up to 100 mg kg–1,
and 200 mg Ca2+ kg–1 soil application reduced shoot dry weight to the level of 100
mg Ca2+ kg–1 soil. Shoot Ca2+ concentrations also increased by soil Ca2+ applications,
and at all levels. Compared to nill soil Ca2+ application, there was almost a three-fold
increase in shoot Ca2+ concentration at the rate of 200 mg Ca2+ kg–1 soil. In contrast,
increasing soil Ca2+ supply reduced shoot P, Zn2+ and B+ concentrations significantly.
These reductive effects of soil Ca2+ application were the largest on shoot B+ concen-
tration. B+ was also the only nutrient for which there was a genotype × soil Ca2+ ap-
plication interaction with some cultivars having deficient levels of B+ at the highest
soil Ca2+ application (200 mg kg–1). The results demonstrate that increased soil Ca2+
content reduced P, Zn2+ and B+ nutrition of durum wheats significantly, thus resulting
in lower yields. The results also show that durum cultivars differed in their sensitivity
to increased Ca2+ application and potential nutritional disorders that result from it.
Screening of a large number of cultivars/accessions for their responses to increased
Ca2+ application, and subsequent identification of those with lower sensitivity could
be useful for breeding programs that aim to maximise crop yields in soils with high
Ca2+ content.
Keywords: phosphorus, calcium, zinc, boron, durum wheat.
258
AIMS AND BACKGROUND
Boron (B+) and calcium (Ca2+) have similar functions in plants, and are also antago-
nistic to each other1,2. B+ has a close relationship with Ca2+ in both plant and soil.
Ca2+ increases the B+ requirement of plants due to similarity in function3, and in soil
it reduces the availability of B+ perhaps due to the formation of a calcium tetraborate
complex4. Sometimes this relationship is indicated by the Ca2+/B ratio of plant and
soil. In general, Ca2+/B+ ratio is a good indicator of B+ status of the plant5. Increasing
Ca2+/B+ ratio resulted in B+ deficiency. In a separate study with barley, it has been
reported that Ca2+/B+ ratio greater than 697 resulted in B+ deficiency6.
In calcareous soils, phosphorus (P) (or phosphate) sorption to calcium carbonate
(CaCO3) may be of equal or greater importance than sorption to aluminum and iron
oxides7. In a laboratory investigation with pure calcite8, it was concluded that the reac-
tion of P with CaCO3 consisted of initial sorption reactions followed by precipitation
with increasing concentrations of P. In calcareous soils, an acidic fertiliser solution
would dissolve Ca2+, and it is anticipated that most of the added P fertiliser would
precipitate initially as dicalcium phosphate dihydrate and dicalcium phosphate9,10.
These products are only moderately stable and undergo a slow conversion into com-
pounds such as octacalcium phosphate, tricalcium phosphate, or one of the apatites.
pH and CO2 are the two most important factors affecting the solubility of calcium
carbonates. The plant Ca2+ availability depends greatly on the solubility of Ca2+ in the
soil. This low availability of Ca2+ can result in Ca2+ deficiency in crops under dry land
agriculture despite the presence of high levels of Ca2+ in the soil11–13.
In soil solutions and plants, P can bind to Zn2+ thus forming insoluble zinc-
phosphate complexes. This in turn would inhibit both the uptake of Zn2+ by the root
and the movement of Zn2+ in the plant. Field and glasshouse studies showed that
increased P fertilisation enhanced plant P uptake but reduced Zn2+ uptake thereby
causing Zn2+ deficiency14–17. It has been reported that increased P application to cal-
careous soils increased Zn2+ adsorption and calcium carbonate played an important
role in the adsorption of Zn2+ (Ref. 18). In this study, the highest Zn adsorption was
observed in a soil with the highest calcium carbonate content. Other studies reported
that increased P fertilisation increased dry matter and P concentration in the plant19.
In acid soils, calcium carbonate-P interactions of positive nature were also reported20.
The aim of this study was to determine the effects of increasing soil Ca2+ applications
on growth, and Ca2+, P, Zn2+ and B+ uptake in durum wheat.
EXPERIMENTAL
Durum wheat cultivar (cv.) Ege-88, Kiziltan-91, C-1252, Zenit and Fuatbey-2000 were
used in the present study. The soil was dried, sieved and placed into polyethylene pots
of 2 kg capacity. Different rates of Ca2+ (0, 50, 100 and 200 mg kg–1 soils were ap-
plied as Ca(OH)2 in the soil. To ensure maximum growth, N and P were also added to
259
each pot (350 mg N as NH4NO3, 150 mg P as triple superphosphate and 250 mg K as
K2SO4 (Ref. 21). The physical and chemical properties of this soil are given in Table 1.
The seeds of all cultivars were sown into pots (15 seeds per pot). Once seedlings
reached 5 cm height, they were thinned to 10. After six weeks of growth, shoots were
harvested at 5 cm above the soil surface. Shoot samples were rinsed first in tap water
then in de-ionised water. The samples were oven-dried at 70°C for determination
of shoot dry matter. Dry shoot samples were ground, ashed 550°C and dissolved in
3.3% HCl (Ref. 22). P and Zn2+ concentration in the ash was determined according to
Refs 23 and 24. Boron was determined according to the Azometin-H method25. The
experiment was set up as randomised complete block design with four replications.
The analysis of variance was conducted using GENSTAT statistical program, and
pair-wise comparisons of the means were made using the Least significance differ-
ence (LSD) test at P = 0.05.
Table 1. Physical and chemical properties of the soil used in the present study
Sand (%) 22
Silt (%) 33
Clay (%) 45
pH (soil reaction) 7.7
Electrical conductivity (EC, dS m–1) 0.8
Cation exchange capacity (CEC, cmol kg–1) 37.1
Calcareous (CaCO3, %) 27.2
Average P (mg kg–1) 5.7
Average B (mg kg–1) 0.41
Average Zn (mg kg–1) 0.39

Shoot dry weight (DW) increased upto 100 mg Ca2+ kg–1 soil application in all cultivars
and declined thereafter (Table 2). The increase at 100 mg Ca kg–1 soil application com-
pared to nill soil Ca2+ application was 24.3, 17.4, 17.4 15.2 and 5.0% in Kiziltan-91,
Ege-88, Fuatbey-2000, C1252 and Zenit, respectively. The greater increase in shoot
dry weight of Kiziltan-91 was also evident in regression analysis.
Table 2. Effects of soil Ca2+ application (mg kg–1 soil) on shoot dry weight (%)
Cultivars 0 50 100 200 Mean
shoot dry weight (%)
C1252 11.2 11.8 12.9 12.0 12.0
Ege-88 11.5 12.6 13.5 12.5 12.5
Fuatbey-2000 10.9 11.3 12.8 11.9 11.7
Kiziltan-91 10.7 11.6 13.3 12.2 12.0
Zenit 12.1 12.3 12.7 11.7 12.2
Mean 11.3 11.9 13.0 12.1
LSD 0.05 soil Ca 0.4
LSD 0.05 cultivar 0.5
260
Similar to shoot dry weight, increasing soil Ca2+ applications increased Ca2+ uptake
of all five durum wheat cultivars (p < 0.001, df = 10). At nill soil Ca2+ application, all
cultivars appeared to be adequate in Ca2+ nutrition as indicated by their shoot Ca2+
concentrations (0.4–07%) falling within the adequate range (0.4.–1.0 %) established
earlier26 (Table 3). Where maxium shoot dry weight was achieved (100 mg Ca2+ kg–1
soil), the increase in shoot Ca2+ concentration ranged from 0.7 fold in C1252 to 1.1 fold
in Kiziltan-91. The increase in shoot Ca2+ concentration observed at 50 mg Ca2+ kg–1
soil application was in the order of Kiziltan-91 > Fuatbey-2000, Zenit > Ege-88 =
C1252. This order remained the same at 100 mg Ca2+ kg–1 soil application, but changed
at 200 mg Ca2+ kg–1 soil Ca2+ application (Fuatbey-2000 > Kiziltan-91 > Ege-88 >
Zenit > C1252). This change in the order of genotypes to soil Ca2+ application may
be attributed to different genotypes showing different responses to soil Ca2+ applica-
tion16. The greatest increase in shoot Ca2+ concentration was observed at the highest
soil Ca2+ application (200 mg kg–1 soil), and shoot Ca2+ concentrations of greater than
1% indicated luxury levels. These results are in agreement with earlier studies27.
Table 3. Effects of soil Ca2+ application (mg kg–1 soil) on shoot Ca2+ (%)
Cultivars Shoot Ca2+ concentration (%)
0 50 100 200 mean
C1252 0.53 0.76 1.09 1.55 0.97
Ege-88 0.39 0.55 0.73 1.28 0.85
Fuatbey-2000 0.58 0.81 1.06 1.45 0.74
Kiziltan-91 0.49 0.63 0.88 1.38 0.98
Zenit 0.63 0.81 1.10 1.34 0.98
Mean 0.52 0.71 0.97 1.40
LSD0. 05 soil Ca 0.06
Table 4. Effects of soil Ca2+ application (mg kg–1 soil) on shoot P concentration (%)
Cultivars Shoot P concentration (%)
0 50 100 200 mean
C1252 0.20 0.18 0.16 0.15 0.17
Ege-88 0.18 0.16 0.15 0.11 0.15
Fuatbey-2000 0.23 0.19 0.17 0.14 0.18
Kiziltan-91 0.20 0.17 0.15 0.12 0.16
Zenit 0.19 0.16 0.13 0.10 0.15
Mean 0.20 0.17 0.15 0.12
LSD0. 05 soil Ca 0.02
At nill Ca2+ application, shoot P concentration in all cultivars (0.18–0.23%) ap-

peared below the adequate range established earlier at a similar growth stage to that
in the present study (0.3–0.6%) (Ref. 26).
261
Increasing soil Ca2+ application reduced shoot P concentration of all durum
wheats significantly; 10.0% (C1252) to 17.4% (Fuatbey-2000), 16.7% (Ege-88) to
31.6% (Zenit), 25.0% (C1252) to 47.4% (Zenit) at 50, 100 and 200 mg Ca2+ kg–1 soil
application, respectively. It can be sourced from Ca phosphate formation27. At the
highest soil Ca2+ application (200 mg kg–1), the observed shoot P concentrations of
0.10–0.15% pointed to Ca2+ induced P deficiency. The ranking of cultivars at nill Ca2+
application (Fuatbey-2000 > Kiziltan-91 = C1252 > Zenit > Ege-88) differed with
external soil Ca2+ supply (Fuatbey-2000 > Zenit > Kiziltan-91 > Ege-88 > C1252, Zenit
> Fuatbey-2000 > Kiziltan-91 > C1252 > Ege-88, Zenit > Kiziltan-91 > Fuatbey-2000
= Ege-88 > C1252 at 50 and 100 mg Ca2+ kg–1 soil application, respectively. These
differences in shoot Ca2+ concentration at nill Ca2+ application as well as differential
responses to soil Ca2+ applications can be attributed to genetic differences amongst
the cultivars (Table 4).
Table 5. Effects of soil Ca2+ application (mg kg–1 soil) on shoot Zn+2 concentration
Cultivars Shoot Zn2+ concentration (mg kg–1)
0 50 100 200 mean
C1252 38 33 25 22 29
Ege-88 29 27 22 14 23
Fuatbey-2000 36 29 21 17 26
Kiziltan- 91 29 23 18 14 21
Zenit 33 30 24 18 26
Mean 33 28 22 17
LSD 0. 05 soil Ca 1.8
At nill external Ca2+ supply, all cultivars were adequate in their Zn nutriton (29–33
mg kg–1 DW) (Ref. 26). However, shoot Zn concentrations of all cultivars declined as
soil Ca2+ application increased. This was also evident when shoot Ca2+ concentration
was regressed againts shoot Zn2+ concentration (p < 0.001, df = 10; Table 3). It can
be higher soil pH because of Ca fertiliser16. The highest soil Ca2+ application reduced
shoot Zn2+ concentrations near or below the adequacy (25–70 mg kg–1 DW; Ref. 26)
indicating potential deficiency in Zn2+. The reduction was in the range of 9.1% (Zenit)
to 20.7% (Kiziltan-91), 24.1% (Ege-88) to 41.7% (Fuatbey-2000), C1252 (42.1%)
to 52.8% (Fuatbey-2000) at 50, 100 and 200 mg Ca2+ kg–1 soil, respectively. The
order of cultivars at nill Ca2+ application, C1252 > Fuatbey-2000 > Zenit > Ege-88 >
Kiziltan-91, changed to C1252 > Zenit > Fuatbey-2000 > Ege-88 > Kiziltan 91 at the
highest level of soil Ca2+ application (200 mg kg–1 soil) (Table 5).
Like P and Zn2+, increasing Ca2+ application reduced shoot B+ concentrations
significantly. This could also be seen from the negative and linear relationship between
shoot Ca2+ concentration and shoot B+ concentration (p < 0.001, df = 10). Of all nutri-
ents, Ca2+ had the largest effect on B+ nutrition such that highest application of Ca2+
262
reduced B+ levels from adequate (7–11 mg kg DW) to levels that were indiciative of
B+ deficiency (4–6 mg kg–1 DW) (Table 6).
Table 6. Effects of soil Ca2+ application (mg kg–1 soil) on shoot B+ concentration
Cultivars Shoot B+ concentration (mg kg–1)
0 50 100 200 mean
C1252 11.4 9.8 7.9 5.6 8.7
Ege-88 8.1 7.4 5.3 4.1 6.2
Fuatbey-2000 8.8 8.4 6.6 4.3 7.0
Kiziltan- 91 10.8 8.3 6.1 5.8 7.8
Zenit 6.9 7.0 5.3 4.5 5.9
Mean 9.2 8.2 6.2 4.8
LSD 0.05 soil Ca 0.2
Perhaps this condition comes from calcium tetraborate formation28. The reduc-
tion in shoot B+ concentration at 200 mg Ca2+ kg–1 soil ranged from 34.7% in Zenit
to 51.1% in Fuatbey-2000. While the order of cultivars in absolute B+ concentrations
remained similar at all levels of Ca2+ applications, it varied in terms of percentage
reduction. The order of cultivars in B+ shoot concentration at 50 mg Ca2+ kg–1 soil,
C1252 > Fuatbey-2000 > Kiziltan-91 > Ege-88 > Zenit, changed to Kiziltan-91 >
C1252 > Zenit > Fuatbey-2000 > Ege-88 at 200 mg Ca2+ kg–1 soil. These findings are
in agreement with previous reports27–30.
CONCLUSIONS
Increasing soil Ca2+ application reduced P, Zn2+ and in particular B+ nutriton of durum
wheat cultivars significantly as indicated by near or below the critical deficiency con-
centrations of respective nutrients and regression analysis. However, cultivars differed
in their sensitivity to increased Ca2+ application and potential nutritional disorders
that result from it. There is a need to teste large number of cultivars/accessions for
their responses to increased Ca2+ application and those with lower sensitivity could be
utilised in breeding programs to maximize crop yields in soils with high Ca2+ content.
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29. E. SAKIN, A. SEYREK, E. D. SAKIN: Comparison of the Some Physicochemical Characteristics
and Nutrient Element Status between Cultivated and Uncultivated Soils. Oxid Commun, 38 (3),
1491 (2015).
30. I. KIZILGOZ, E. SAKIN: The Effects of Increasing Soil Application of Nitrogen and Phosphorus
on Dry Matter, Dry Weight, and Zinc and Boron Concentration in Wheat and Maize. Oxid Commun,
38 (3), 1504 (2015).
Received 29 May 2015
265
PRETREATMENT TECHNOLOGY FOR PREPARATION OF

ETHANOL VIA BIOMASS FUEL
WANG CHAO*, CHANG SHUAI

Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang
Province, College of Agriculture and Food Science, Zhejiang A and F University,
Lin’an, 311 300 Zhejiang, China
E-mail: wangchao19776@163.com
ABSTRACT
In order to better understand the ethanol production, potential sources of lignocellulosic
biomass for the process are considered. In this paper, a new method of subcritical
water pretreatment was applied to corn straw pretreatment and the pretreatment tem-
perature and time were optimised by Central Composite Design for the highest total
reducing sugar content in water-soluble materials, and the preparation of fuel ethanol
for the residuals after pretreatment under the optimum conditions was investigated.
The results show that subcritical water pretreatment can effectively remove lignin
while more than 90% cellulose was reserved. The final ethanol concentration from
residual fermented was 11.6 g/l.
Keywords: preparation, biomass, central composite design, total reducing sugar
content.
AIMS AND BACKGROUND

Biofuels based on the conversion of sustainable non-food lignocellulosic biomass are
one of the primary cornerstones in the global effort to replace fossil fuels with renew-
able sources and has the potential to generate about one quarter of the world energy
production by 2035 (Refs 1–4). The investments in second generation bioethanol and
biomethane have been increasing in response to global policies aiming to achieve
greenhouse gas (GHG) emission reduction targets and diversify energy sources. In
this context, the production of multiple types of biofuels and energy products from a
commercial biorefinery represents a compelling alternative to petroleum to maximise
the energy value of available biomass resources5–7. The development of biorefiner-
ies related to the production of biofuels utilising lignocellulosic feedstocks, such as
*
For correspondence.
266
agriculture and forestry residues, municipal solid wastes, woody and herbaceous
energy crops, is still in the early commercial stage and many technical and economic
challenges must be overcome before a renewable energy industry can become a suc-
cessful and commercially viable enterprise.
Bioethanol is by far the most widely used bio-fuel for transportation worldwide,
because it is a renewable, nontoxic, biodegradable resource and it is oxygenated, there
by provides the potential to reduce particulate emissions in compression–ignition
engines8–12. Second-generation biofuels (biomass to liquid) are made from organic
materials, such as straw, wood residues, agricultural residues, reclaimed wood, saw-
dust, and low value timber. Microorganisms are a key component of the technology
used in different fermentation regimes, including ethanol.
Currently, the fermentation of cellulosic residues forces a law major step partaker
saccharification and fermentation method (separate hydrolysis and fermentation) and
simultaneous saccharification and fermentation (simultaneous saccharification and
fermentation)13–16. Studies have shown that compared with the simultaneous saccharifi-
cation and fermentation step saccharification and fermentation method, with increased
ethanol yield, simplify the process and reduce the risk of bacterial contamination, etc.
is an ideal method of fermentation.
Crop residues are abundant lignocellulosic feedstock, according to statistics, China
produced about 710 million t of straw per year, wheat straw and corn stover accounted
for 22 and 36%, respectively. Therefore, the use of corn stover for ethanol production
has sufficient sources of raw materials, and can reduce environmental pollution, turn-
ing waste into treasure17–19. But about the use of subcritical water pretreatment of corn
stover and use the residue feedstock pretreatment and enzymatic hydrolysis were also
less ethanol production reports. In this study, corn stalks as raw materials, through the
sub-critical water pretreatment, using a central composite design optimisation straw
subcritical water pretreatment conditions, to explore changes in the structure and
physicochemical properties of straw residue after pretreatment, the resulting residue
was also investigated synchronisation enzyme – ethanol fermentation process in order
to provide a reference for the use of straw resources to produce ethanol.
EXPERIMENTAL
Before the test, the determination of straw lignin, cellulose, hemicellulose, ash and al-
cohol extract benzene content was done. Then, 15 g straw powder was placed in a high
temperature autoclave, add distilled water 150 ml, set speed of 400 r/min. According
to the results of pre-test, the effect of residence time and treatment temperature was
investigated, and treatment temperature (A) was set as 160–210°C, reaction pressure
(B) – as 2–6 MPa, processing time (C) – as 0–50 min. Central composite design is
shown in Tables 1–3. After completion of the reaction, rapid cooling was carried out
through the condensate, and filtration started when the temperature dropped to 50°C.
Water-soluble fractions were collected (filtration resulting filtrate) and the water-
267
insoluble portion (residue) was filtered. The insoluble fraction of water was dried at
110°C. Benzene alcohol extract, lignin, cellulose and hemicellulose in the residue
were determined. The water-soluble fraction was hydrolysed by sulphuric acid with
volume fraction of 4%, and the determination of total sugar content was performed
by using 3,5-dinitrosalicylic acid (DNS) method.
water – insoluble part of the quality
residue yield = × 100 (1)
raw material quality
Table 1. Factor level

Factor level –1.681793 –1 0 1 1.681793
A (°C) 170 180 190 200 210
B (MPa) 2 3 4 5 6
C (min) 10 20 30 40 50
Table 2. Central composite design and response value

No A B C Content of total re-
ducing sugar (mg)
1 1 1 –1 525
2 –1 1 –1 627
3 0 0 0 788
4 1.681793 0 0 1566
5 0 0 0 1677
6 –1 –1 –1 1788
7 0 0 0 777
8 1 –1 –1 2099
9 0 1.681793 0 2345
10 1 –1 1 677
11 0 0 0 779
12 0 0 1.681793 854
13 –1 –1 1 967
14 0 0 –1.68179 1998
15 0 0 0 766
16 –1.68179 0 0 1333
17 1 1 1 2099
18 0 0 0 744
19 0 –1.68179 0 1233
20 –1 1 1 1199
268
OPTIMISATION OF PRETREATMENT CONDITIONS FOR CORN STRAW IN

SUBCRITICAL WATER
Interaction by response surface methodology (RSM). The surface plot of objective

function is drawn by Minitab in order to directly reflect the interaction between the
various factors that affect the content of total reducing sugar and the role of strength
for interaction between the various experimental factors.
The regression equation of corn straw subcritical water pretreatment optimisation
model is: content of total reducing sugar = 933 + 89A + 58B – 148C + 114A×A +
234B×B + 105C×C + 97A×B + 50A×C + 549B×C
Figures 1–3 show the response surface plots for the variations of content of total
reducing sugar according to temperature, residence time and reaction pressure. In
each plot, two factors are varied, while the rest is kept constant.
Fig. 1. Response surface plots of the interaction between treatment temperature and reaction pressure
on content of total reducing sugar
For Fig. 1, it is shown that it is shown that treatment temperature and process-
ing time have obvious effect on content of total reducing sugar. When temperature
is close to 210°C, processing time is 10 min and the content of total reducing sugar
is about 854 mg.
Figure 2 shows that processing time and reaction pressure have obvious effect on
the content of total reducing sugar. Content of total reducing sugar decreased when
processing time increased. However, the content of total reducing sugar increased
when reaction pressure increased.
269
Fig. 2. Response surface plots of the interaction between processing time and reaction pressure on
content of total reducing sugar
Figure 3 shows that treatment temperature and processing time have obvious
effect on content of total reducing sugar. When temperature is close to 210°C and
processing time is 30 min, the content of total reducing sugar is about 1750 mg.
Fig. 3. Response surface plots of the interaction between treatment temperature and processing time on
content of total reducing sugar
OPTIMISATION OF PROCESS PARAMETERS USING RSM
Objective function of Minitab response surface provides an intuitive tool for objective
optimisation and its unique response optimiser is a powerful tool for multi-objective
problem to the solution encountered in the experimental design. And the target func-
tion is optimised by Minitab response optimiser. The result is shown in Fig. 4. It is
calculated that the optimised conditions are as follows: temperature of 174°C, reaction
pressure of 2.6 MPa and processing time of 18 min.
270
Fig. 4. Optimised result for content of total reducing sugar by response optimiser
CORN STOVER CHANGE AFTER SUB-CRITICAL WATER CONTENT OF EACH

COMPONENT OF THE PRETREATMENT
Table 3 shows that in subcritical water optimum pretreatment conditions, corn stalk
residue of sugar content of 2890 mg is slightly lower than the theoretical maximum
of 3000 mg, which may be due to that at high temperature conditions, some sugar
further undergoes degradation to methyl furfural, furfural and other products.
Table 3. Contents of the compositions of corn straw before and after pretreatment in subcritical water
Material Cellulose Hemicellu- Lignin (g/ Benzyl alco- Ash (g/kg) Content of
(g/kg) lose (g/kg) kg) hol extract total reduc-
(g/kg) ing sugar
(mg)
Raw corn 466.8 223.5 198.3 12.9 67.8 –
strew
Pretreated corn 451.9 65.1 126.4 37.2 – 2890
strew
Table 3 shows that at subcritical water optimum treatment conditions, corn stover
lignin content is reduced by 31.2%, reducing the hemicellulose content of 70.9%, while
the cellulose content is reduced by only 3.2 %. A significant reduction occurs in lignin
and hemicellulose, which helps cellulose hydrolysis by cellulase, while most of the
cellulose is retained (90%), and the use value of residue after treatment is improved.
CORN STALK RESIDUE OF SIMULTANEOUS SACCHARIFICATION AND

FERMENTATION BEFORE AND AFTER SUBCRITICAL WATER PRETREATMENT
As can be seen from Fig. 5, the fermentation of glucose and ethanol concentration
fermentation (Fig. 5b) basically remains constant. Final ethanol concentration of corn
271
stover treated for fermentation is 11.6 g/l, which increased five-fold compared with no
treatment. Thus, subcritical water pretreatment can improve the quality concentration
of ethanol for the raw material after fermentation.
Fig. 5. Simultaneous saccharification and fermentation of corn straw before (a) and after pretreatment
(b) in subcritical water
CONCLUSIONS
This paper demonstrates that the subcritical water treatment process is effective in
reducing hemicellulose and lignin in corn straw, and more than 90% cellulose are
retained. Central composite design provides sufficient data to fit the quadratic models
for content of total reducing sugar. The optimisation of the models provides the opti-
272
mum conditions: temperature of 174°C, reaction pressure of 2.6 MPa and processing
time – 18 min. The final ethanol concentration from residual fermented was 11.6 g/l.
ACKNOWLEDGEMENTS
The research was funded by the Development Fund of Zhejiang Agriculture and
Forestry University (No 2014FR059).
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Ecologically friendly oxidative processes
A REVIEW OF TECHNOLOGY FOR SMALL SEWAGE

TREATMENT: THE CHINESE PERSPECTIVE
YUFEI WANGa,b, LONG YANa,b*, JIAN LIa,b, HUIDONG SHENa,

CHAO ZHANGa, TIANTIAN QUa
a
Yulin Engineering Research Centre for Industrial Water Treatment, School of
Chemistry and Chemical Engineering, Yulin University, 719 000 Yulin, China
b
Shaanxi Key Laboratory of Low Metamorphic Coal Clean Utilisation, Yulin
University, 719 000 Yulin, China
E-mail: ylyanlong@126.com
ABSTRACT
On the basis of demographic projections, it is estimated that the quantity of sewage
which will be produced in China between 2009 and 2015 will increase from 35.52
to 56.65 billion t. Sewage treatment and its management is one of the most critical
environmental issues of today. The treatment and disposal of sewage contribute a
considerable proportion of the cost. The increasing amount of sewage and more and
more legislative regulation of its disposal have stimulated the need for efficient and
economical technologies to process sewage. Currently, the predominant method
for the disposal of this sewage is biological treatment. The small sewage treatment
facilities are cost-effective and getting benefit instantly. Here we review the state of
knowledge and technology in small sewage treatment, which makes proposals to the
construction of sewage treatment.
Keywords: sewage treatment, environmental issues, technologies.
AIMS AND BACKGROUND

The term sewage refers to the wastewater produced by a community, which may
originate from three different sources: (a) domestic wastewater, generated from
bathrooms and toilets, and activities such as cooking, washing, etc.; (b) industrial
wastewater, from industries using the same sewage system for their effluents (treated
or not), and (c) rain-water, particularly in the case of sewer systems constructed for
both wastewater and storm-water (combined systems)1–10. The treatment and disposal
of sewage is one of the most critical environmental issues of today. The waste stream
*
For correspondence.
275
of sewage is rapidly growing, generating wastes which require management in com-
pliance with the law11–16. China is inhabited by 1.37 billion people with an average
population density of 140 persons/km2, and has about a territory of 9 600 000 km2, of
which 9 326 410 km2 are occupied by land. In the year 2008, more than 33 billion t
of sewage were produced. In China, only 372 cities possessed a modern wastewater
treatment plant with enhanced nitrogen and phosphorus removal. In these plants, 5.7
million t of waste was treated per day17–24.
According to the Chinese Environmental Policy and the objectives of the National
Wastewater Treatment Plan, the quantity of sewage treated in China is systematically
increasing. A measurable effect of this is, first of all, the increasing proportion of the
population being served by wastewater treatment, but also the growth of the amount
of produced sewage. On the basis of demographic projections, it is estimated that the
quantity of sewage which will be produced in China between the years 2009 and 2015
will increase from 35.52 to 56.65 billion t (Ref. 25).
Proper treatment and disposal of sewage avoiding harm to environment is an
integral part of wastewater treatment and environmental protection. Municipal sewage
has high water content, high load of organic pollutants and high content of pathogens
as well as considerable concentration of heavy metals. Generalised processes for
sewage handling are biological treatment, chemical treatment and physical treat-
ment. Currently, the predominant method for the disposal of this sewage is biological
treatment. In order to achieve a better understanding of the prospective application
of biological technology for the small sewage treatment, here a brief review of this
promising technology is presented and application of this technology and some re-
sults reported from both laboratory and full-scale studies are reviewed. The effects of
biological technology for the small sewage treatment are discussed in detail. Finally,
some concluding remarks are given and future directions are also proposed.
TECHNOLOGY EMPLOYED IN SEWAGE PROCESSING

Five options are currently available that can be used for the processing of sewage,
which contains septic tank, stabilisation pond process, land treatment system, activated
sludge method and biological membrane.
SEPTIC TANK
People began to treat sewage by septic tanks 100 years ago. More than 70% of sus-
pended solids content and 30% of BOD5 can be reduced by septic tanks, but it is still
difficult to achieve the requirements of the integrated wastewater discharge standard.
Therefore, sewage is able to discharge after it meets the integrated wastewater dis-
charge standard by centralised sewage treatment plants. This caused repeated construc-
tion investment, and reduced the efficiency of the sewage treatment plant. Currently
many cities require new district to prohibit the use of septic tanks26–29.
276
Empowered sewage treatment apparatus is an improved septic tank, which is ap-
plied to many southern cities. The apparatus does not need power, and it is flexible30.
But its civil construction cost is high and it can not ensure the water quality in the
running process, which has been banned in Japan.
STABILISATION POND PROCESS
Stabilisation pond process was applied to treat the sewage 100 years ago, but it was
not widely used until World War II. Stabilisation pond was employed to small sew-
age treatment in the United States and Europe in particular. The United States owned
stabilisation pond more than 7000 in 1983, 90% of which were built in the small
town with a population of less than 5000. Stabilisation pond was included in large
key projects of the 7th Five-year Plan in 1986, and the stabilisation pond developed
rapidly in China31.
It can take full advantage of the terrain if sewage is treated by stabilisation pond,
and infrastructure and maintenance costs are low, and sewage is able to realise resource
recovery. However, it covers a big area and the treatment effect is vulnerable to im-
pacts of climate. Land is much in Northern China, but it is not suitable for application
because of cold climate; land resources are precious in Southern China and it is not
suitable for application, which makes stabilisation pond difficult to spread in China32–34.
LAND TREATMENT SYSTEM
Wastewater land treatment system contains many forms, which is slow rate land
treatment system, rapid infiltration land treatment system, wetland treatment system,
subsurface wastewater infiltration system, which is often used together with the oxi-
dation pond. Treatment of sewage by land treatment system is researched deeply in
China during the Seventh Five-year Plan period, and the user manual of sewage treat-
ment by land treatment system is compiled. Application of land treatment system in
China contains sewage slow rate land treatment system in Shenyang, the Changping
sewage rapid infiltration land treatment system and municipal sewage land treatment
system in Tianjin35.
Artificial wetland wastewater treatment technology has unique advantages for
small sewage treatment, and it can take full advantage of the natural conditions; it has
a simple structure, and running cost is only 10–50% of the traditional craft36. A lot of
natural wetlands distribute in China from south to north, and these wetlands are han-
dled appropriately to develop artificial wetland technology, which has a broad space.
ACTIVATED SLUDGE METHOD
Because water quality and quantity of small sewage water treatment plant changes
larger and activated sludge process is particularly sensitive to the shock loading, so
too small wastewater treatment plant is not appropriate to use activated sludge treat-
ment process. Only eight use the activated sludge process within 127 small sewage
277
treatment plants (< 1000 population equivalent) in Eastern Switzerland37. In 1987,
the Norwegian Environmental Pollution Control Agency made a survey on the exist-
ing small-scale sewage treatment plants and believed that activated sludge process
often causes sludge wastage due to shock loading and running is difficult to control.
Therefore, biological membrane method was recommended to treat small sewage. If
activated sludge process is selected, sludge storage is needed to set up38–42.
BIOLOGICAL MEMBRANE
Applications of the biofilter on small sewage treatment are not too much. Researches
on the biological aerated filter and earthworm biofilter are more43–46, but practical
engineering applications are less.
The rotating biological disc began in Germany and is widely used in western
countries. Nearly 50% small sewage treatment plants that its population scale is less
than 1000 in Swiss employ the rotating biological disc technology; 21 small-scale sew-
age treatment plants of 29 in northwestern England employ rotating biological disc47.
Rotating biological disc has been applied in China, sewage treatment and reuse project
of Jinan Nanjiao hotel utilises the parallel turntable, which obtains a well-functioning.
RESEARCH PROGRESS OF SMALL SEWAGE TREATMENT

Researches on septic tank. Fu et al.48 apply the improved 3 cell septic tank to treat
sewage. The results indicate that suspended sobids (SS), chemical oxygen demand
(COD), chromium and ammonia nitrogen removal by using three different thicknesses
fillers is from 81.6 to 92.9, 17 to 60.5, 13.5–23.2 and 7.9 to 11.9%, respectively. Vari-
ous pollutants removal increases with increasing thickness of the filler. Wang et al.49
investigate the septic tank of a residential area in the typical city of northwest region
Lanzhou, the change of sewage quality and the removal of pollutants indicators after
passing septic tanks were analysed. The results indicate that after the septic tanks
treatment, the concentration of nitrogen, total phosphorus, animal and vegetable oils
in domestic wastewater reduced. The average annual removal of them reached to
83.6, 51.1, 64.3, 68.2 and 75.6%, respectively. Wang et al.50 selected a total of 168
normally running.
Three-Grille-Model septic tank in 9 cities of Jiangsu is used as object of the
research. Analysis of the samples shows that the ‘Three-Grille-Model’ septic tank
could reduce CODCr by 48.51%, total nitrogen (TN) by 6.83%, and total phosphorus
(TP) by 23.92%. Generally, its treatment efficiency is lower than of biogas-digesterc.
The treated water flowing out of the septic tanks fails to meet the criteria of the na-
tional standard for wastewater discharge and its discharge will significantly affect
the environment.
Researches on stabilisation pond process. Li et al.51 apply constructed wetland/stabi-
lisation pond process to treat rural domestic sewage. The results show that the average
removal rates of COD, BOD5, SS, TN, NH4+–N and TP are 75.1 to 87.3, 75.2 to 94.3,
278
90.2 to 97.6, 50.2 to 67.8, 65 to 75.3 and 70.6 to 85.9%, respectively. The effluent
quality meets the first class criteria specified in the Integrated Wastewater Discharge
Standard (GB 8978-1996). This process has characteristics of low investment, good
treatment effect, simple operation and low maintenance cost. He52 studies sewage of
development area in Shihezi by hydrolysis acidification stabilisation pond process,
the findings indicate that the effluent water reaches Discharge Standards for farmland
irrigation water, this process is an economic application sewage treatment process;
the best hydraulic retention time of hydrolysis acidification segment is 3 h; COD
capacity burden in the 2.09–2.73 kg COD/(m3 d) at hydrolysis acidification segment,
biodegradability of effluent water is higher. The best hydraulic retention time of
stabilisation pond is 9 days (d); BOD surface burden in the 19.44–55.56 kg/(hm2 d)
at stabilisation pond segment, the BOD and COD of effluent can reach farmland ir-
rigation standards. (GB5084-92).
Han et al.53 apply stabilisation pond process to treat rural domestic sewage. Under
the optimum parameters, stabilisation of the pond process can be use to handle rural
domestic sewage. The treatment effect was stable. The effluent quality met the national
discharge standard. That was to meet water quality requirements.
Tang et al.54 present the application of a new type stabilisation pond process,
which is double anaerotanks-oxidation pond-oxidation ditch, treating wastewater of
town. It is indicated by the practical operation project that the process has high treat-
ment efficiency and stable water quality of effluent. All indices of effluent can meet
the secondary standard in Integrated Wastewater Discharge Standard (GB8978-1996).
Huang et al.55 apply stabilisation pond process to treat the sewage and investigates
the removal of nitrogen and phosphorus. The results show that the average removal
of TN, TP, NO3––N and NH3–N by the whole system are 25.3, 50.6, 38.4 and 35.6%,
respectively. Figure 1 is the flow chart of domestic sewage treatment process.
Fig. 1. Flow chart of domestic sewage treatment process
Researches on land treatment system. Zhang et al.56 test a pilot plant of subsurface
wastewater infiltration system filled with red clay to treat rural sewage with hydraulic
loading of 2 cm/d. The results show that average removal of COD, NH4+–N, total
phosphorus and total nitrogen being 84.7, 70.0, 98.0 and 77.7%, with average efflu-
ent concentrations of COD, NH4+–N, total phosphorus and total nitrogen being 11.7,
4.0, 0.04, and 4.7 mg/l, respectively, which met the standard for water reuse issued
by the Ministry of Construction of China Guo et al.57 bring land treatment system to
process the rural sewage, and investigate the pollutants removal at low temperature.
279
The experimental results indicate that the effect of low temperature on COD removal
is less obvious, as the COD removal is 74.66%; the COD of effluent is less than 40
mg/l, which satisfies the first level B criteria specified in the discharge standard of
pollutants for municipal wastewater treatment plant (GB 18918-2002). The low
temperature has a notable effect on the removal of TP and NH4+–N, and the average
removal of TP and NH4+–N is 37.44 and 36.25%, respectively.
Zheng58 introduces subsurface wastewater infiltration system to process the rural
sewage. The results indicate that SS, COD, ammonia nitrogen, total phosphorus and
total nitrogen have good removal effect by infiltration filter system. SS removal is
above 92%, COD removal is above 85%, TP removal is 73%, TN removal is 66.6%,
NH4+–N removal is 69%. Wang and Sun59 introduce artificial fast filtration system to
process the rural sewage and discuss the factors of effluent COD removal of waste-
water land treatment as an example of artificial rapid infiltration system. The result
implies that shortening hydraulic load cycle is able to raise reoxygenation efficiency
and stabilise water quality. Zhang et al.60 design subsurface infiltration system to treat
rural domestic wastewater in Dianchi valley. The treatment ability is 30 m3/d and the
design hydraulic loading of the infiltration system is 0.08 m3/(m2 d). The COD, total
nitrogen and total phosphorus removal of the system reach 86.7, 85.5 and 96.5%, re-
spectively. The system offers advantages including lower construction and operation
costs, easy maintenance and many economic returns.
Peng et al.61 concisely introduce the history of development, principle and ad-
vantage of artificial wetland, and study artificial wetland of Shajing town. The result
implies that its respective COD removal is 83% and total nitrogen is 45%, the effluent
quality can meet the National Sewage Discharge Standards. Cui et al.62 study the sew-
age treatment by hybrid constructed wetlands. The results show that the performance
of both hybrid constructed wetlands is much better than single type wetlands. The
average removal rates of COD, BOD5 and TP by the combination of vertical-flow and
horizontal flow constructed wetland were 70.52, 69.21 and 55.56%, respectively. The
combination of two vertical-flow wetlands system had high purification efficiency for
TP, of which the average removal rate is 72.62%, and the concentration of effluent
is between 0.10 and 0.60 mg/l. The average removal rates are 64.74% for COD and
60.63% for BOD5. It is obvious that the concentrations of COD, BOD5 and TP in the
effluent could all reach to the first rank of Urban Sewage Treatment Plant Pollutants
Drainage Standards (GB 18918-2002). Figure 2 is the flow chart of domestic sewage
treatment by land treatment system.
Fig. 2. Flow chart of sewage treatment process
280
CONCLUSIONS
(1) According to the prognosis, the stream of produced sewage in China will grow;
it follows foremost from the lifestyle changes of our society, but is also due to the
increased percentage of the population connected to the sewerage network.
(2) Construction planning is the largest with regard to investment and the most
expensive from among all the tasks resulting from the implementation of the Chinese
government directives in the field of environmental protection. Treatment ability and
level will be further improved during the 12th Five-year Plan.
(3) Undoubted advantage of A/O – Membrane Bio-reactor (MBR) combination
process, in addition to the disposal of sewage, is that it becomes possible to reduce
the waste of energy. Chinese conditions also appear to present a good opportunity to
utilise this group of waste-disposal technologies.
(4) At present, China has increased the strength of environmental legislation.
The legislated limits will determine the choices for sewage disposal; the disposal
of sewage will have to be replaced in the next few years by other methods. This is
strong incentive to develop physicochemical, biochemical, and other combination
methods of sewage.
ACKNOWLEDGEMENTS
This work was sponsored in part by the National Natural Science Foundation of
China (21203163), Science and Technology Plan Project of Shaanxi Province of
China (2014KJXX-78, 2014KW16), Science and Technology Plan Project of Yulin
Government (Sf1309, Gy1309), and Scientific Research Project of Yulin University
(12GK03, 11GK36).
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284
PHENOLIC WASTEWATER TREATMENT VIA A CLEAN

TREATMENT PROCESS
DAI DEYIa,b, RAO YINGXUEc*

a
Centre of Hubei Cooperative Innovation for Emissions Trading System
(CHCIETS), Hubei University of Economics, Wuhan, Hubei Province, China
b
School of Logistics and Engineering, Hubei University of Economics, Wuhan,
Hubei Province, China
c
School of Public Management, South-Central University for Nationalities, Wuhan,
Hubei Province, China
E-mail: raoyingxuebg@126.com
ABSTRACT
Hydrogen production from waste using supercritical water gasification (SCWG) is a
promising approach towards cleaner fuel production and a solution for hard to treat
wastes. In this paper, the catalytic gasification of phenolic wastewater was investigated
in a batch reactor at 26–34 MPa, 510–550oC, residence time of 2–5 min. The results
showed that the main gas products were H2, CO, CO2 and CH4. When residence time
was 5 min, temperature is 550oC, hydrogen gasification ratio reached up to 135%.
SCWG was proved to be an efficient technology for phenolic wastewater treatment.
Keywords: hydrogen production, phenolic wastewater, wastewater treatment, super-
critical water gasification.
AIMS AND BACKGROUND

Phenolic compounds are present in the effluents of various industries such as oil
refineries, petrochemicals, pharmaceuticals, coking operations and resin manufactur-
ing industries. Improper discharges of phenolic wastewater from such industries are
likely to lead to detrimental environmental consequences since phenol is a highly toxic
substance that poses great risk to aquatic life, micro-organisms and mammals. This
necessitated many national and international environmental regulatory bodies to set
stricter discharge limits for phenols, which renders the efficient treatment of phenolic
wastewater prior to discharge as crucial for sustainable development1. Many physical
and chemical methods, such as adsorption, steam distillation, extraction, wet-air oxi-
*
For correspondence.
285
dation electrochemical oxidation and ion exchange2 have been proposed for phenolic
wastewater treatment. Despite recent advances in the research and development of
the abovementioned methods, many challenging problems still remain, such as high
energy consumption, low efficiency and secondary pollution. For example, phenol is
commonly removed from industrial effluents by adsorption using activated carbon,
which subsequently is treated by incineration. The process of incineration generates
many new compounds such as dioxins and furans, which have very severe conse-
quences on environment and human health. In addition, the biological treatment for
the removal of phenolic compounds is very ineffective due to the toxicity of phenol to
the microbial population as well as the biorecalcitrant nature of phenolic compounds.
Supercritical water gasification is a novel biomass conversion technology and
can convert biomass into hydrogen-rich gas. For the unique properties of water
above its critical point (22.1 MPa, 374.2°C), SCWG has many advantages: (1) the
homogeneous reaction environment can be formed in SCWG because most organics
and gases can be dissolved in supercritical water. This can reduce the mass transfer
resistance and improve the reaction rate; (2) SCWG is more suitable for the treatment
of high-moisture biomass or organic waste because no drying process is needed in
this technology; (3) No hazardous emission, such as NOx, SO2 and fine particles is
formed from the gasification, even when the feedstock contains nitrogen and sulphur
elements3,4. As a result, SCWG attracted much attention around the world. Consider-
able development and progress have been made in the last decades, which can be
referred in some recent researches5–10.
In this study, SCWG was applied to treat phenolic wastewater in a 0.6-l batch
autoclave under the experimental conditions of 24–36 MPa, 510–550◦C, and residence
time of 2 to 5 min. The present study reports on the operational features of SCWG
for carbon gasification ratio (RCG), hydrogen gasification ratio (RHG).
EXPERIMENTAL
Apparatus and method. All the experiments were performed in a batch reactor. As
shown in Fig. 1, the apparatus includes feed system, preheater, reactor, condenser,
gas-liquid separator and backpressure regulator. SCWG of phenolic wastewater was
carried out in a 0.6-l batch autoclave. Firstly, water and phenolic wastewater (con-
centration was 2500 mg/l) was put into the reactor, and then the system was flowed
by nitrogen to remove the air within the system; the valves around the reactor were
closed when the air was removed entirely. Liquid samples (ca. 20 ml) were periodi-
cally withdrawn from the reactor and analysed.
Chemical analysis. The gas samples were analysed according to ASTM D1946-2011,
ASTM D2597-2010 and DIN 51872-4-1990 methods, through a Perkinelmer Clarus
680 model GC coupled with two TCD and one FID detectors.
286
Fig. 1. Schematic diagram of the experimental setup
1 – oxidant container; 2 – heater; 3 – high-pressure autoclave; 4 – high pressure pump; 5 – gas-liquid
separator; 6 – nitrogen cylinder; PID – proportion-integration-differentiation
Terms and definitions. Two parameters, carbon gasification ratio (RCG), hydrogen gasi-
fication ratio (RHG) will be discussed as the measure of organic compound destruction
and gasification efficiency in the SCWG of phenolic wastewater. The RCG and RHG are
defined as equations (1) and (2):
RCG = (CPG/COC) × 100% (1)
RHG = (HPG/HOC) × 100% (2)
where CPG is the carbon amount in the gas product; COC – carbon amount in organic
compounds; HPG – the hydrogen amount in the gas product; HOC – the hydrogen
amount in organic compounds.

Effect of pressure on phenolic wastewater gasification. The effect of pressure on
phenolic wastewater gasification was investigated under the conditions of 520oC,
3 min and 26 MPa. As it is shown in Fig. 2, changing the reaction pressure had no
significant influence on RCG and RHG, and this has been proved by many researchers.
Effect of temperature and residence time on phenolic wastewater gasification. Fig-
ures 3a–d showed the results of phenolic wastewater gasification in SCW at 30 MPa,
510–550oC, 2–5 min. At first H2 molar fraction increased and the peak value was
obtained at 550 and 530oC with residence time 2 and 3 min, respectively, however,
the peak value was obtained at 540oC with residence time from 4 to 5 min. It may be
concluded that the highest H2 molar fraction can be obtained at a lower temperature
with residence time increasing.
287
Fig. 2. Effect of reaction pressure on gas fraction, RCG and RHG at 26–34 MPa, 520oC, 3 min
Fig. 3. Effect of temperature on gas fraction at 30 MPa, 510–550oC, 2–5 min

a, b, c, d represent the gas fraction at residence time of 2, 3, 4, 5 min, respectively
Minowa and Ogi, Minowa and Inoue suggested that the CH4 was not only pro-
duced by the methanation reaction of H2 and CO, but also by H2 and CO2 in SCWG
288
of organic compounds, and both the reactions could be accelerated with the increase
of temperature and residence time. Thus, the methanation reactions become dominant
in the SCWG system with the increase of temperature and residence time, and then,
the H2, CO and CO2 are consumed. So the molar fraction of H2 was reduced and the
content of carbon dioxide did not increase significantly. Finally, H2, CO, CH4 and
CO2 come to equilibrium.
Figure 4 shows the variation of RCG and RHG to reaction temperature and residence
time, respectively. All the RCG and RHG increased with temperature and residence time
increasing. RCG reached 97.96% at 550◦C and 5 min, and it is suggested the phenolic
wastewater is almost decomposed completely and all of the decomposed organic car-
bon has been already converted to gas. RHG reached 135% at 550◦C and 5 min, which
indicated some of H2 in the product gas comes from water in the SCWG process.
100
a
80
510◦C
60
520◦C
RCG (%)
530◦C
540◦C
40
550◦C
20
0
2 3 4 5
residence time (min)
140
b
120
100
510◦C
80 520◦C
RHG (%)
530◦C
60 540◦C
550◦C
40
20
0
2 3 4 5
residence time (min)
Fig. 4. Effect of residence time on RCG, RHG at 30 MPa, 510–550◦C, 2–5 min
a, b represent the RCG and RHG, respectively
289
CONCLUSIONS
In this study, SCWG was applied to treat phenolic wastewater in a 0.6 l batch auto-
clave under the experimental conditions of 26–34 MPa, 510–550oC, residence time
of 2–5 min. The main gas products were H2, CH4, CO and CO2. The results showed
that H2 can be obtained by the gasification of phenolic wastewater in SCW. Phenolic
wastewater can be converted to H2, CO, CH4 and CO2 completely; RCG and RHG reached
97.96 and 135%, respectively, at 30 MPa, 550oC and 5 min.
ACKNOWLEDGEMENTS
This paper is supported by ‘the Fundamental Research Funds for the Central Univer-
sities (CSQ14007)’.
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Revised 6 July 2015
290
IMPORTANCE OF EXTERNAL ELECTRIC FIELD IN

DEVELOPMENT OF ELECTROCATALYSIS PROMOTION
INTERNAL MICRO-ELECTROLYSIS FOR COPPER(II)
REMOVAL
TIANGUO LIa, GANG WANGb, XIAOJUN XUa*, RUI NIEa,

VO ANH KHUEa, QIANG ZHANa, JING ZHAOa
a
Faculty of Environmental Science and Technology, Kunming University of Science
and Technology, 650 500 Kunming, China
E-mail: xuxiaojun2100@163.com
b
Urban and Rural Planning and Design Institute of Yunnan, 650 228 Kunming,
China
ABSTRACT
Electrocatalysis was utilised to promote the removal efficiency of Cu(II) from
aqueous solution by iron-carbon internal micro-electrolysis (IME). SEM, EDS and
electrochemical measurements were used to investigate the importance of external
electric field (EEF) in the reaction system of electrocatalysis promotion internal
micro-electrolysis (EPIME). Results showed that copper removal belonged to the
first-order kinetic model. Kinetic analysis indicated that electrocatalysis strengthened
the removal rate of Cu(II) significantly. The zero-valent iron (ZVI) corrosion tests and
SEM, EDS characteristics analysis displayed that EEF promoted the rate of galvanic
corrosion of ZVI and increased the quantity of reaction active sites on the surface of
activated carbon (AC) cathode in iron-carbon IME system remarkably. The reinforc-
ing effects might due to the larger corrosion current density, high overpotential and
potential for chemical reduction obtained in the iron-carbon IME system under the
presence of EEF. And then the needed activation energy of Cu(II) reductive deposi-
tion was reduced, while the rate of nucleation and Cu(II) reduction of EPIME was
promoted. Therefore, EPIME effectively improved the removal efficiency, reaction
rate and withstand capacity of Cu(II). These reinforcement mechanisms were verified
clearly via cyclic voltammetry (CV) analysis.
Keywords: Cu(II), internal micro-electrolysis, electrocatalysis, external electric field,
promotion.
*
For correspondence.
291
AIMS AND BACKGROUND
Heavy metals are toxic pollutants and usually released into the soil and ground water,
along with the emissions of industrial ‘three wastes’, due to industrial activities like
mining, chemical engineering, electroplating and other metal processing processes1.
According to the investigation of the Institute of Ecology of Chinese Academy of
Sciences, at present, the cultivated land which contaminated with cadmium, arsenic,
chromium, lead, copper and other heavy metals is about 20% (2 million ha) of China,
and cut more than 1 million t of grain a year. Now, in China, the appearance of heavy
metals pollution is from the phase of gradual accumulation entering into eruption
phase with characteristics of the sudden, chain and regional distribution2. Copper is a
widespread transition metal element with high toxicity. Though copper is an essential
element, excessive copper can lead to acute or chronic poisoning. For plants, copper
can change the synthesis of chlorophyll, and decrease the membrane integrity, the
activity of enzymes and the efficiency of the primary photochemical reaction3. For
human and animals, copper in the blood exists in two forms: 85–95% of copper are
strongly bound to ceruloplasmin, and the rest loosely bound to albumin and small
molecules4. Free copper has biotoxicity as it generates reactive oxygen species such as
superoxide, hydrogen peroxide, and hydroxyl radicals. These reactive oxygen species
damage proteins, lipids and DNA (Ref. 5). High copper levels often cause digestive
tract symptoms, and influence the kidney, liver, brain, bone, blood and other organs,
even make cytolysis and connective tissue deposition. For example, Wilson disease
is a typical representative of copper poisoning6. One of the current needs is to reduce
the exposure of copper on wastewater to a level as close to zero as possible.
Various technologies such as precipitation7, adsorption, biosorption8, ion ex-
change9, reverse osmosis10, electrodialysis, ion exchange-assisted membrane separa-
tion11,12, and electrolysis, micro-electrolysis13 have been employed to remove heavy
metals from various effluents. Among numerous techniques, electrolysis and IME have
been receiving greater attention in recent years due to the distinctive advantages of
extensive adaptability to wide variations of compositions (such as chlorinated organic,
nitrate, nitroaromatic compounds and heavy metal and arsenic ions), environment
friendly, safety and uniquely capable of recycling resources from wastewater14,15.
However, when removing heavy metal ions with single electrolysis, in order to obtain
higher removal efficiencies, voltage is often increased in the form of geometric growth,
which leads to high cost of processing16. IME operates on a principle very similar to
that of electrolysis, except that the electrons are supplied from the galvanic corrosion
of many microscope sacrificial anodes instead of external power. It has several advan-
tages such as simple process, versatility and low cost. Because of this method only
uses its own galvanic corrosion reaction to catalytic metal ions electrodeposition in
the cathode, the overpotential and catalytic ability of this system for electrodeposition
generated relatively small. Thus IME has the disadvantage in low efficiency, reaction
rate and withstand capacity. Moreover, the ion residual concentration in the emission
292
effluent is high and difficult to reach zero emissions or the discharge standard. Recently,
in order to overcome the shortcomings of IME mentioned above, large numbers of
attempts have been made by environmental researchers, such as embedded Fenton
reagent (Fe2+/H2O2) (Ref. 17), microwave18, electromagnetic, etc.19,20
Electrocatalysis is widely used in catalytic degradation or removal of many kinds
of contaminants in effluent21,22 and contaminated soil23. On the basis of electrochemical
principles, electrocatalysis was combined with iron-carbon IME and applied to remove
heavy metals from aqueous solution. Early researches suggested that it could improve
the removal efficiency of Cu(II) in heavy metal wastewater. In the present work, a
homemade EPIME electrochemical batch cell was prepared, novel experiments and
a variety of modern analytical methods were performed to investigate the optimum
conditions, reaction kinetics, the rate of ZVI galvanic corrosion, surface morphology
structures and components of filler particles. All the works focus on discussing the
reinforcing effects and the electrocatalysis promotion mechanism behind it.
EXPERIMENTAL
Materials. All the chemicals (sodium hydroxide (NaOH), potassium ferricyanide
(K3[Fe(CN)6]), copper sulphate (CuSO4), sodium sulphate (Na2SO4)) used in the
study were analytical reagent (AR) grade. Agar powders (gel strength > 1300 g/cm2)
were purchased from Kunming Biorise Science and Technology Ltd, China. Com-
mercial activated carbon (AC) (10–20 mesh) was purchased from National Medicine
Group Chemical Reagent Company, China. In order to eliminate the interference of
AC absorption of target contaminants, AC was immersed in a simulated wastewater
which updated 1 time a day at least 5 days. Cast iron filings were recycled from the
metalworking practice base of Kunming University of Science and Technology.
First, cast iron filings were activated by soaking with 20% NaOH solution for 24 h to
remove superficial greasy dirt, and then soaked with 10% H2SO4 solution for 1 h to
remove surface oxide. The stock solution of Cu(II) was prepared by dissolving copper
sulphate in deionised water.
Experimental setup. The schematic of the experimental setup is shown in Fig. 1. It is a
simplified EPIME system specifically designed to investigate the response behaviour
of internal micro-electrolysis enhanced by EEF.
293
Fig. 1. Experimental setup of: flow cell (a), and experimental setup of EPIME experimental batch reac-
tor (b): 1 – reservoirs, 2 – pump, 3 – valves, 4 – rotameter, 5 – EPIME reactor, 6 – DC power supply,
7 – flocculation basin, 8 – anode, 9 – cathode, 10 – inner compartment, 11 – outer compartment, 12 –
perforated PVC clapboard, 13 – ZVI and AC fillers
As shown in Fig. 1, the electrochemical batch reactor consists of a divided PVC

cell, in which the inner and outer PVC rectangular compartments separated by perfo-
rated PVC clapboards and the geometric dimensions are 10 × 3.5 × 12 cm and 10 ×
5 × 12 cm, respectively. The inner compartment is filled with ZVI and AC fillers. The
graphite electrodes are installed on the both sides of inner compartment, and connected
to a D.C. regulated power supply.
Experimental procedure. The corrosion rate of ZVI in solution can be estimated
through the quantity of release soluble and suspended iron hydroxide precipitates24.
In this work, a simple test had been designed to reflect the speed of galvanic corrosion
intuitively. In detail, two pieces of iron filings were suspended in two quantitative
containers (container 1#, container 2#), and then AC particles were added into the
containers, respectively. 10 g agar, 0.1 mol K3[Fe(CN)6] and 0.2 mol Na2SO4 were dis-
solved into 1000 ml deionised water. The pH of the solution (indicating solution) was
adjusted to 3.0 by H2SO4 and NaOH solution. After that, 500 ml indicating solutions
were poured into container 1# and 2#, respectively. The container 1# was installed with
294
two graphite electrodes which applied to a voltage of 10 V, and container 2# was used
as a control. The ferrous formed by the corrosion of ZVI reacted with K3[Fe(CN)6] to
generate blue precipitate Fe3[Fe(CN)6]2 (equation (1)).
3Fe2+ + 2K3[Fe(CN)6] = 6K+ + Fe3[Fe(CN)6]2 ↓ (1)
5000 ml of 100 mg/l Cu(II) aqueous solution was prepared as simulated waste-
water. For the sake of optimising the Cu(II) removal conditions, the main influence
factors, including initial pH, electrode voltage, and electrolyte were investigated. The
ZVI/carbon mass ratio was 2:1.
At optimum conditions, copper-containing wastewater was treated for 30 min
in the EPIME reactor. First of all, 50 ml effluents of EPIME reactor were sampled,
immediately filtered out the flocs, clear filtrate was acidified with nitric acid, and
then analysed the concentration of Cu(II). Meanwhile, the Cu(II) removal efficiency
and breakthrough time of EPIME, single iron-carbon IME and electrolysis treated
at each optimum conditions were compared. Among them, the breakthrough time is
defined as the time when the residual concentrations of Cu(II) more than the level
3 standard (2 mg/l) of Integrated Wastewater Discharge Standard of China, namely,
removal efficiency is less than 98%. In addition, the surface morphology structures
and components of granule AC treated with and without EEF were analysed.
Analytical methods. The pH was determined by a pH meter (pHS-3C, Shanghai, China).
The concentration of Cu(II) was measured by atomic absorption spectrophotometer
(AA240FS, Varian, USA). The surface morphology structures and components of
solid samples were characterised by scanning electron microscope (SEM) (S3400N,
Hitachi, Japan) and energy dispersive spectrometer (EDS) (E-550, EDAX, USA).
The experimental results were assessed by residual concentration and removal ef-
ficiency of Cu(II).
Electrochemical characterisation of CV was performed with an electrochemical
workstation (CHI660D, CH Instruments, Inc.). Electrochemical measurements were
performed using a three-electrode electrochemical cell. Platinum sheet was used as both
working electrode and counter-electrode. A saturated Ag/AgCl electrode (3.0 mol/l
KCl solutions) acted as the reference electrode. The electrolyte was using 0.1 mol/l
Na2SO4 solution at pH 4. The experiments were performed at room temperature.
Before each measurement, the solution was previously deoxygenated with gaseous
N2 for at least 10 min.
BATH EXPERIMENTS
Effect of pH. The effect of pH on the removal of Cu(II) by EPIME reactor is demon-
strated in Fig. 2. It can be found that the optimum pH is 4, the removal efficiency of
295
Cu(II) significantly increased when pH was increased from 2 to 4, while decreased
with the increasing pH from 4 to 6.
100
90
removal efficiency (%)

pH = 2
pH = 2.5
80 pH = 3
pH = 4
pH = 5
70 pH = 6
60
50
0 10 20 30 40 50 60 70
time (min)
Fig. 2. Effect of pH on Cu(II) removal (electrolyte concentration of Na2SO4 – 0.10 mg/l, voltage of
EEF – 10 V)
Effect of voltage. The relationship between the voltage and the removal efficiency
is illustrated in Fig. 3. It can be seen that the removal efficiency and reaction rate
of Cu(II) increase with the increasing of voltage. At the first 30 min, Cu(II) removal
efficiency was greatly impacted by voltage, however, only a little difference when
continue to extend the processing period. Previous studies reported that voltage was
a driving force of electrochemical reactions, and the presence of EEF could promote
the repolarisation of conductive particles (ZVI and AC) in electrochemical reactor.
100
90 0V
3V
6V
80 10 V
12 V
13 V
70 15 V
60
50
0 10 20 30 40 50 60 70
time (min)
Fig. 3. Effect of voltage on Cu(II) removal (pH – 4; electrolyte concentration of Na2SO4 – 0.10 mg/l)
The promoted reduction and electrodeposition of Cu(II) may attribute to change

chemical potential energy, accelerate the mass transfer rate and reduce the concentra-
tion polarisation of Cu(II) when increasing the applied electric field intensity. While
higher voltage (13 and 15 V in this work) was disadvantageous to the removal of
296
Cu(II) because more side reactions would occur (such as hydrogen evolution and
oxygen evolution reaction).
Effect of electrolyte concentration. The concentration of electrolyte significantly affects
on the removal of heavy metals by electrochemical method25. Adding an electrolyte
can increase conductivity ability and catalytic efficiency, higher concentration of
electrolyte can obtain larger electric current density under the same applied cell volt-
age26. The effects of concentration of electrolyte on the removal efficiency of Cu(II) by
EPIME are illustrated in Fig. 4. As shown in Fig. 4, the removal efficiency of Cu(II)
increases significantly and reaches 98.7% only after 30 min when increasing Na2SO4
concentration to 0.10 mol/l. And the residual concentration of Cu(II) almost keeps the
same when continues to extend the processing period. However, when adding Na2SO4
concentration to 0.15 and 0.20 mgl/l, the removal efficiency of Cu(II) decreases and
even lower than that without Na2SO4. After 60 min, the removal efficiency of Cu(II)
is without distinction at different concentration of electrolyte. It is evident that the
concentration of electrolyte only influences the reaction rate, but has no influence
on the total removal efficiency of Cu(II) when the reaction period is long enough.
The decrease in the reaction rate of Cu(II) may be due to Na2SO4 act as electrolytes.
Increasing concentration of electrolyte appropriately improves the conductivity of the
solution. Larger electric current density is conducive to the electromigration and the
reduction of Cu(II) on the surface of the cathode. But when electrolyte is excessive,
most migration ions are Na+ and SO42– in the solution, which will inhibit directional
migration of Cu(II) to the surface of the cathode, and most electrons are used for
side effects.
100
90 0 mol/l
0.05 mol/l
0.10 mol/l
80 0.15 mol/l
0.20 mol/l
70
60
50
0 10 20 30 40 50 60 70
time (min)
Fig. 4. Effect of electrolyte concentration on Cu(II) removal (pH 4; voltage of EEF – 12 V)
Reaction kinetics and enhanced effects. Comparison tests of EPIME, single iron-carbon
IME and electrolysis were performed at each optimum condition that pH were 4, 3,
5 and voltage were 12 V, none, 3 V, respectively. All the initial Cu(II) concentration
and electrolyte concentration were 100 mg/l and 0.10 mol/l. In addition, for EPIME
and single iron-carbon IME, ZVI/AC mass ratio was 2:1. As shown in Fig. 5a, after
297
nearly 60 min, the residual concentration of Cu(II) processed by EPIME, single iron-
carbon IME and electrolysis are not changing anymore, and reaches 0.022, 0.268 and
11.01 mg/l, respectively. It can be found that EPIME is most efficient, only after 10
min, Cu(II) residual concentration decreases to 0.1 mg/l, while it is only 27.16 and
62.94 mg/l of single iron-carbon IME and electrolysis, respectively.
a
70
60
EPIME
residual concentration (mg/l)
IME
50 electrolysis
40
30
20
10
0
0 10 20 30 40 50 60 70
time (min)
b
(1): Y=98%, X=40.6 h
100
90 (2): Y=98%,
X=30 h
80
70
EPIME
IME
60 electrolysis
50
40
0 10 20 30 40 50 60
time (h)
Fig. 5. Comparison of Cu(II) removal efficiency and withstand capacity under respective optimum
conditions of EPIME, single iron-carbon IME and electrolysis: comparison of the removal efficiency
of different methods (a); comparison of the breakthrough time of different methods (b) (residence time
of EPIME, single iron-carbon IME and electrolysis were 30, 60, and 90 min, respectively)
The high efficiency of EPIME can be proved clearly by the analysis of reaction
kinetics and withstand capacity. Kinetic process of Cu(II) removal by EPIME, single
iron-carbon IME and electrolysis methods can be described by the pseudo-first order
kinetic model and is expressed as follows:
γ = dc/dt = – Kobs C (2)
298
After integral can rewrite equation (2) as:
ln (C0/C) = – Kobs t, (3)
where C0 is the initial concentration of Cu(II); C – the residual concentration of Cu(II);
Kobs – the rate constants of pseudo-first order kinetic model, and t – the reaction time.
Equation (3) shows a linear relationship between ln C/C0 and t with correlation coef-
ficients larger than 0.95. The EPIME, single iron-carbon IME and electrolysis were
0.3006, 0.1026 and 0.0338 h–1, and the Cu(II) removal half life were 2.31, 6.71 and
20.51 min, respectively. The removal efficiency and reaction rate of Cu(II) by EPIME
were markedly superior to the simple summation of those by single iron-carbon IME
and electrolysis. And the relative increment of Cu(II) removal efficiency was 0.25 and
10.99%, respectively, for single iron-carbon IME and electrolysis. These indicated that
there were some synergetic effects between single iron-carbon IME and electrolysis.
From Fig. 5b, it can be seen that the breakthrough time of single iron-carbon IME
and EPIME were 30 and 40.6 h, respectively, when the same dosage of ZVI and AC
filler were used. In the presence of EEF, the total quantity of purified wastewater in-
creased from 18.0 to 48.7 l in the effective time, and the removal quantity of Cu(II)
increased from 1.76 to 4.77 g. These results suggested that EPIME promoted the
reaction rate and withstand capacity of Cu(II) removal significantly. In conclusion,
electrocatalysis is a feasible means to enhance iron-carbon IME for the removal of
Cu(II) from aqueous solution.
ENHANCED PROCESSES AND MECHANISM
Corrosion effect of ZVI. The intuitive comparison results of corrosion of ZVI in system
of iron-carbon fillers co-existing with the EEF and non-existing EEF are presented
in Fig. 6. As shown in Figs 6a, b, after 2 h, the colour of blue surrounding cast iron
filings of container 1# which iron-carbon co-existing with EEF is more depth than
that of container 2# which non-existing EEF. This suggests that the presence of EEF
can accelerate the galvanic corrosion of ZVI substrate significantly. The promotion
of the potential for chemical reduction of Cu(II) is due to generating more ferrous
ions in the system of EPIME (equations (4) and (5)):
Fe(s) Fe(aq)2+ + 2e– (4)
Fe(aq)2+ + 2H2O Fe(OH)2 + 2H+ (5)
Previous studies had reported that the corrosion current density is correlated
positively with its corrosion rate of ZVI. So, this phenomenon indicates that EEF
remarkably increases the corrosion current density in the electrolyte and exchange
current density at the interface of electrolyte and iron-carbon particles. It can be in-
ferred that EPIME would obtain higher removal efficiency and reaction rate of Cu(II)
or other heavy metals.
299
a b
Fig. 6. Comparing results of corrosion of ZVI: ZVI/AC were co-existing with EEF (a); ZVI/AC were
non-existing EEF (b)
COPPER CRYSTAL MORPHOLOGY AND COMPOSITION
The surface morphologies of AC after treating copper-containing wastewater by

single iron-carbon IME and EPIME are shown in Fig. 7. From Figs 7a–d, many cop-
per crystals can be seen clearly on the surface of AC and copper crystal growth is a
multiple nuclei. The AC surfaces of EPIME (Figs 7b, d) have more crystallisation
points and copper contents compare with single iron-carbon IME (Figs 7a, c). In the
absence of EEF (Figs 7a, c), the copper crystals mainly are of large agglomerates and
pyramidal crystals with the size approximately from dozens to hundreds of microm-
eters. Whereas in the presence of EEF (Figs 7b, d), the copper crystals mainly are of
small fluffy aggregates, granular and dendritic crystals with the size around 10 μm,
smaller particles of them even achieve the nm level. This can be further confirmed by
the elementary composition of crystals (Figs 7e, f). For a single crystal of iron-carbon
IME and EPIME, the former has more percentage of copper atoms (53.66%), while
less percentage of carbon atoms (44.61%) than the latter that the percentage of copper
and carbon are 13.16 and 80.80%, respectively. In the process of electrocrystallisa-
tion, the nucleation is parallel to crystal growth and interdependent with each other26.
Smaller crystal phase indicated that the rate of nucleation is greater than the crystal
growth. The rate of nucleation is determined by the overpotential which increasing
along with the increase of current density27–29.
300
(a)
a (b)
b
c
(c) d
(d)
(e)
e (f)
f
Element Wt% At% Element Wt% At%
C 13.48 44.61 C 50.79 80.80
Cu 85.82 53.66 Cu 43.78 13.16
Ca 00.00 00.00 Ca 00.61 00.29
O 00.70 01.73 O 04.82 05.75
Fig. 7. SEM and EDS of AC after treating Cu(II) 100 mg/l aqueous at optimum conditions
a, c, e and b, d, f were after treating by single iron-carbon IME and EPIME, respectively
As discussed above, electrocatalysis increase the galvanic corrosion rate of ZVI

to obtain larger current density, overpotential, mass transfer rate and high potential
for chemical reduction in the system of iron-carbon IME. Furthermore, the exchange
current density and concentration of adatom on the interface of electrolyte and iron-
carbon particles are increased. These changes of the iron-carbon IME system, reduce
the needed activation energy of Cu(II) reduction deposition. The surface of AC cathode
has more active sites to accelerate the rate of nucleation and Cu(II) reduction. Thus,
EPIME effectively improved the removal efficiency and reaction rate of Cu(II) from
aqueous solution.
301
0.25
2
1— 0 V 3
0.20
2— 3 V
3— 6 V 4
0.15
4— 9 V
1
j (mA cm )
–2
0.10
0.05
0.00
–0.05 4o
3o
1o 2o
–0.10
–0.4 –0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2
E (V versus Ag/AgCl)
Fig. 8. Cyclic voltammograms curves for graphite and Pt electrodes in aqueous solution under EEF of
different cell voltage, v = 10 mV s−1 (concentration of Cu(II) – 100 mg/l; electrolyte concentration of
Na2SO4 – 0.10 mol/l; pH 4)
Cu(II) REDUCTION ELECTROCHEMICAL ANALYSIS
In order to further support the reinforcement mechanisms mentioned above, the CV

method was introduced to analyse the effect of EEF on reduction of Cu(II) in iron-
carbon IME aqueous solution. As shown in Fig. 8, a positive shift of anodic and ca-
thodic peak potential along with the increase of intensity of EEF was observed. The
pair of redox peaks of higher voltage is much better defined. The degree of positive
shift of the anodic oxidation and cathodic reduction peak potential was greater and
the CV curve exhibits a larger area. These phenomena suggesting that the cathodic
reduction of Cu(II) is more easy. According to previous reports30, the degree of
cathodic reduction potential peak positive shift is equal to the quantity of cathodic
electrodeposition potential reduced, and was favourable to the occurrence of reduce
reaction. So the results of CV analysis indicate that electrocatalysis can observably
promote the removal efficiency of Cu(II), which own to the reduction of the needed
activated energy of Cu(II) cathodic reduction in the system of EPIME.
CONCLUSIONS
Electrocatalysis is a feasible means to strengthen iron-carbon IME for removal of
Cu(II) from aqueous solution. Comparing with single iron-carbon IME and elec-
trolysis, the removal efficiency of Cu(II) was enhanced significantly in the system of
EPIME. Its optimum conditions were pH 4, the voltage of EEF 12 V and electrolyte
concentration 0.10 mol/l, respectively. Copper reduction deposition belongs to the
first-order kinetic model. There were some synergistic effect between iron-carbon
IME and electrolysis. The presence of EEF promoted the galvanic corrosion rate of
ZVI and reaction rate of Cu(II) reduction, in addition, effectively extended the break-
through time. The enhanced removal efficiency of Cu(II) may due to electrocatalysis
302
and can produce larger corrosion current density, overpotential and higher potential
for chemical reduction in the system of iron-carbon IME. Moreover, for Cu(II) reduc-
tion deposition, the needed activation energy was reduced, while the active sites on
the surface of the AC cathode was increased remarkably. Ultimately, electrocatalysis
accelerate the rate of nucleation and reduction deposition of Cu(II). Therefore, these
serial ripple effects mentioned above contribute to the increasing of the removal ef-
ficiency, reaction rate and withstand capacity of Cu(II) from aqueous solution.
ACKNOWLEDGEMENTS
This work was supported by the Scientific Research Foundation of Yunnan Educa-
tion Department (2013Y327); National Academician Innovative Experiment Plan
(091067424) and the Analysis and Measurement Foundation of Kunming University
of Science and Technology (20140550).
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Revised 2 July 2015
304
EFFECTIVE PROCESS FOR TREATMENT OF COPPER

LEACHED FROM CWO OF OILY WASTEWATER
LONG YANa,b*, YUFEI WANGa,b, JIAN LIa,b, HUIDONG SHENa,

CHAO ZHANGa, TIANTIAN QUa
a
Yulin Engineering Research Centre for Industrial Water Treatment, School of
Chemistry and Chemical Engineering, Yulin University, 719 000 Yulin, China
b
Shaanxi Key Laboratory of Low Metamorphic Coal Clean Utilisation, Yulin
University, 719 000 Yulin, China
E-mail: ylyanlong@126.com
ABSTRACT
The experiments on copper leached from catalytic wet oxidation (CWO) of oily
wastewater were conducted. The effects of the initial copper concentration, pressure
and concentration polarisation on the performance of nanofiltration (NF) membranes
were investigated. The experimental results showed that cupric removal was higher
than 98% and its concentrations in permeation liquid were lower than 2.0 mg/l, meet-
ing the Discharge Standard or using as water for washing electroplated products,
which verified that nanofiltration is an effective method for the removal of copper.
By condense of cupric solution, copper enrichment reached to tenfold and realise the
hope of regenerating the copper.
Keywords: NF, CWO, oily wastewater, cupric.
AIMS AND BACKGROUND

Catalytic Wet Oxidation (CWO) using heterogeneous catalysts based on low-valence
transition metals (Fe, Cu) has emerged as a clean and effective alternative among
other processes to treat wastewaters1–7. However, the process is accompanied with
some metal leaching that would result in secondary pollution and negatively impact
on a possible subsequent biological treatment step. Metal lixiviation is caused by
the exposure of the catalyst to hot acid aqueous media, a scenario difficult to avoid
since the latter oxidation intermediates are short chain acids. This effect, which can
be neglected when iron is used, can be critical if other metals are applied, e.g. copper.
Hence, the toxicity of those partially oxidised organic intermediates as well as that
*
For correspondence.
305
resulting from metal loss by leaching must still be eliminated before the effluent is
discharged into a municipal Wastewater Treatment Plant.
In principle, many partially oxidised intermediates and metal leached could be
recovered, concentrated and recirculated to the oxidation reactor by the coupling of
a membrane separation process system to the CWO process8–11. Nanofiltration (NF),
which is in the transition between ultrafiltration (UF) and reverse osmosis (RO) pro-
cesses, has properties of high rejection of multivalent ions and organic matter with
molecular weights (MW) larger than 200 Da (Refs 12–20). Therefore, coupled CWO
and NF processes could assure the rejection of the metal leached.
This paper firstly studied treatment of copper leached from CWO of oily wastewa-
ter by NF process and investigated copper concentration, operating pressure, operating
and concentration polarisation on the retention of Cu2+ nanofiltration performance
impact on the oily wastewater treatment of copper has guiding significance.
EXPERIMENTAL
Membrane. The NF90 membrane was manufactured by DOW-Filmtec (Minneapo-
lis, USA) and was gently provided free of charge. Table 1 gives the most important
characteristics of the selected membrane.
Table 1. Characteristics of NF90 membrane

MWCO (Da) 200
Support polysulphone
Isoelectric point 4
Pure water permeance (dm3 h−1 m−2 bar) 6
Active layer polyamide
Cross-flow filtration experiments: set-up and procedure. A schematic representation

of the filtration process is presented in Fig. 1. A 1.5-l jacketed glass tank contained
the filtration solution, where the temperature was set constant to 30°C. Filtration tests
were conducted in cross-flow mode, because fouling is expected to be lower than
in dead-end mode thanks to the tangential flow of the feed stream. Tangential flow
velocities of 8.4 cm/s were achieved using a Sterlitech module hosting coupons of
44 cm2 of filtration area. The trans-membrane pressure was set to 0.8 MPa. The pH
was neither adjusted nor controlled. Filtration was monitored for 3 h. Both permeate
and retentate streams exiting the membrane were recycled back to the feed tank to
maintain the feed solution concentration constant during the filtration experiment.
Only a small fraction of permeate was not recycled to the feed tank because permeate
samples were periodically withdrawn to characterise them. The permeate flux was
measured with a balance. A membrane pump was selected for feeding the solutions
to be filtered to the membrane module. A pulse dampener was installed before the
membrane module is used in order to transform the pulsing flow to nearly continuous
306
flow reproducing a real membrane operation. The pulse dampener was actuated by
means of a backpressure valve installed after it. Before every test, the membrane was
soaked in water for 1 day and then subjected at 1 MPa of pressure for 1 h.
Fig. 1. Experimental set-up of nanofiltration

1 – temperature regulator; 2 – tank; 3 – low-pressure pump; 4 – microfilter; 5 – high-pressure pump;
6 – nano-filtration instrument; 7 – gauge
Analysis and determination. By atomic absorption spectrophotometry copper con-

centrations, atomic absorption spectrophotometer copper concentration range: 0.05–5
mg/l, was adjusted with distilled water containing Cu2+ concentration of the sample
to the measurement range.

Effect of operating pressure on Cu2+ retention. Effect of different operating pressures
on Cu2+ retention is shown in Fig. 2. It is seen that NF90 membrane is effective for
Cu2+ retention. Rate of Cu2+ retention is above 99% within the range of the operating
pressure. Level of Cu2+ concentration has little effect on retention, but it is impacted
by the concentration of copper ions in produced water. Concentration of copper ions
in produced water increased with the concentration of Cu2+ in the waste increasing.
Concentration of Cu2+ in produced water by NF90 membrane is very low within the
range of experimental concentration. Concentration of Cu2+ of the produced water is
still 1.0 mg/l or less even in the case that Cu2+ concentration in CuSO4 is 1200 mg/l,
which meets the national emission standards.
99.96 b
1.8 a
99.94
1.6 [Cu2+ ] = 160 mg/l
[Cu2+] of water production (mg/l)
[Cu2+ ] = 870 mg/l 99.92

1.4
[Cu2+ ] = 1100 mg/l
1.2 99.90
retention (%)
1.0
99.88
[Cu2+ ] = 1100 mg/l
0.8
99.86 [Cu2+ ] = 870 mg/l
0.6 [Cu2+ ] = 160 mg/l
99.84
0.4
0.2 99.82
0.4 0.6 0.8 1.0 1.2 0.4 0.6 0.8 1.0 1.2
operating pressure (MPa) operating pressure (MPa)
Fig. 2. Effect of different operating pressures on [Cu2=] of water production (a) and Cu2+ retention (b)
307
Effect of concentrate process on Cu2+ retention. Effect of concentrate process on Cu2+
retention is shown in Fig. 3. It is seen from Fig. 3 that the rate of Cu2+ retention is more
than 98% as the enrichment process proceeds and permeates constantly excludes. A
certain downward trend exists, and it is because that the membrane surface was pol-
luted after a long run. Change of the retention rate throughout the enrichment process
is relatively stable. When the volume of wastewater concentrated 10 times, the feed
concentration can be concentrated for 9 to 10 times to the original, indicating loss of
original liquid is little and copper was enriched with a recovery value. While amount
of copper ions in permeate represents a significant reduction, and permeate can be
reused and recycled to save water.
100.0
99.8
99.6
retention of Cu2+ (%)
99.4
99.2
99.0
98.8
98.6
98.4
0 500 1000 1500 2000 2500 3000
concentration of Cu2+ (mg/l)
Fig. 3. Effect of concentrate process on Cu2+ retention
Concentration polarisation. Rate of CuSO4 retention with the operating pressure by

NF90 membrane is shown in Figs 4 and 5. As can be seen, when the water flow is
1500 l/h, the rate of CuSO4 retention changes little with the operating pressure increas-
ing. When the water flow is 400 l/h, and rate of CuSO4 retention decreases with the
operating pressure increasing. This phenomenon is the performance of concentration
polarisation. When the water flow is small (400 l/h), and the operating pressure in-
creases, flux increases and the water production rate increases, resulting concentration
of mother liquor increases and circulating flow decreases. The mother liquor has not
sufficient turbulence, so concentration polarisation is obvious, showing that concen-
tration of Cu2+ in produced water, increases and the apparent retention rate decreases.
When the penetration increases, and the mass transfer coefficient k decreased. So,
concentration polarisation represents more serious. However, when the water flow is
large (1500 l/h), although the same situation is represented, but concentration polari-
sation is weak, and the apparent retention rate increases. Mass transfer coefficient k
increases with flow velocity increasing, and a corresponding decline for concentra-
tion polarisation is represented. Therefore, during the actual application, full liquor
circulation flow is ensured to reduce adverse effects of concentration polarisation.
308
100
99
98
retention (%)
97
water flow 400 l/h
96 water flow 1500 l/h
95
94
0.4 0.6 0.8 1.0 1.2
operating pressure (MPa)
Fig. 4. Effect of water flow on Cu2+ retention
99.0
98.5
98.0
retention (%)
97.5
97.0
96.5
96.0
200 400 600 800 1000 1200 1400 1600

water flow (l/h)
Fig. 5. Relationship of water flow and Cu2+ retention
CONCLUSIONS
The experiments on copper leached from catalytic wet oxidation (CWO) of oily
wastewater were conducted. The experimental results showed that cupric removal was
higher than 98% and its concentrations in permeation liquid were lower than 2.0 mg/l,
meeting the Discharge Standard. Copper concentration has little effect on the retention
rate, but produce copper concentration in water increases with the concentration of
copper rising. Therefore, during the actual application, full liquor circulation flow is
ensured to reduce adverse effects of concentration polarisation.
ACKNOWLEDGEMENTS
This work was sponsored in part by the National Natural Science Foundation of
China (21203163), Science and Technology Plan Project of Shaanxi Province of
309
China (2014KJXX-78, 2014KW16), Science and Technology Plan Project of Yulin
Government (Sf1309, Gy1309), and Scientific Research Project of Yulin University
(12GK03, 11GK36).
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Revised 23 May 2015
310
TREATING ANTIBIOTIC WASTEWATER VIA A NEW

NANO-Fe2O3 CATALYST
PENG MINGGUO*, LI HUAJIE, DU ERDENG, WANG LIPING

Changzhou, China
E-mail: pengmingguo2020@163.com
ABSTRACT
Pollution of the water environment in today world has become a global issue and due
to the shortage of water resources, more and more people pay attention to pollutants.
Occurrence of trace antibiotics in the aquatic environment has received global concerns
on account of threats to the environment and human health. In this article, nano-Fe2O3
catalyst is prepared to treat antibiotic wastewater. Catalyst dose, the initial concen-
tration of antibiotic wastewater, hydrogen peroxide dose, pH, temperature and other
factors that affect the performance degradation of antibiotic wastewater were studied.
Keywords: antibiotic wastewater, catalytic oxidation, nano-Fe2O3, chemical oxygen
demand (COD).
AIMS AND BACKGROUND

Occurrence of trace antibiotics in the aquatic environment has received global con-
cerns on account of threats to the environment and human health, such as assist in
development of antibiotic-resistant bacteria1–4, toxicity to non-target organisms5–7,
contamination of drinking water resources8–10, etc. Considering the pathways by
which antibiotics enter into the aquatic environment, effluent from municipal waste-
water treatment plants (WWTPs) has been identified as a hotspot source due to the
insufficient removal of antibiotics by the traditional treatment processes11. Control
of the discharge of antibiotics from WWTPs could be an effective approach to limit
the occurrence of antibiotics in the environment, which consequently contributes to
reducing the environmental risk.
Nano-Fe2O3 is a novel nano-material, which has some special physical and
chemical properties because of its morphology and structure of the novel features.
Whether in the field of science and technology or in the civilian industry have a high
*
For correspondence.
311
the value in use, such as in medicine sensors and catalytic materials, which has played
an excellent research value and application prospect, so far people have used different
methods to prepare nano-Fe2O3 of various morphologies and structures12–15.
In this study, the experimental results of antibiotic wastewater treated by nano-
Fe2O3 and hydrogen peroxide were reported. The feasibility of the technology as a
pre-treatment to reduce toxic organic compounds was examined. The effects of the
amount of catalyst, the initial concentration of antibiotic wastewater, the amount of
hydrogen peroxide, pH, and temperature on the removal efficiency of COD were
investigated.
EXPERIMENTAL
Materials and reagents. Antibiotic wastewater used in this study was obtained from
an antibiotic industry producing amoxicillin in Changzhou. The characteristics of
antibiotic wastewater are summarised in Table 1. Hydrogen peroxide (35% w/w) was
purchased from Changzhou, Jiangsu, China.
Table 1. Antibiotic wastewater characteristics

Parameter Value
Amoxicillin (mg/l) 150
Chemical oxygen demand (COD) (mg/l) 850
Biochemical oxygen demand (BOD) (mg/l) 85
pH 6.8
Total phosphorus (TP) (mg/l) 9
Cl− (mg/l) 8
Turbidity (NTU) 52
NO3––N (mg/l) 7
Preparation of nano-Fe2O3 nanoparticles. 1.5 g of FeCl3•6H2O were dissolved in

7.5 ml of water and configured 10 mol/l NaOH solution, 10 ml FeCl3 were added
dropwise to a magnetically stirred solution of NaOH, and stirring was continued for
5 min. The mixed solution was injected into a 35-ml autoclave, and placed in an oven
at a reaction temperature 170°C for 20 h, and then cooled to room temperature until
removed. The reaction product was collected by centrifugation and the autoclave was
washed with deionised water, several times with ethanol and then placed in a vacuum
oven, and dried at 70°C for 24 h. The black product gained is nano-Fe2O3 nanoparticle.

Effect of temperature on COD removal. In the process of organic compounds treat-
ment, the temperature is an important parameter affecting the removal of pollutants.
312
Generally, the higher operating temperature, the higher pollutant removal and the
reaction rate are obtained. Figure 1 shows COD removal within a series of reaction
temperatures (20, 30, 40 and 50°C). When the temperature increased from 20 to
30°C, COD removal significantly increased from 61.02 to 95.7% with adding H2O2
and nano-Fe2O3.
Fig. 1. Effect of temperature on COD removal
Effect of temperature on the degradation of the antibiotic wastewater is mainly

discussed for two points. Firstly, temperature rising will reduce the activation energy
of the reaction, so that the reaction can not occur previously. Temperature is raised or
the original reaction can occur now more efficiently. Secondly, the solubility of the
gas decreases as the temperature increases, which affects the dissolved oxygen in the
water; thereby the oxidation activity is reduced.
Effect of H2O2 dose on COD removal. The effect of concentration on antibiotic waste-
water degradation within the range of hydrogen peroxide 0.5–1.25 mg/l is investigated.
The result is presented in Fig. 2. As can be seen in Fig. 2, adding H2O2 can increase
COD removal. H2O2 displayed high oxidation activity in the antibiotic wastewater
treatment. 91.2% of COD were removed when H2O2 dose was 1.25 mg/l.
313
Fig. 2. Effect of H2O2 dose on COD removal
Effect of pH on COD removal. Figure 3 shows COD removal in the antibiotic wastewa-
ter degradation at temperature of 50°C and pH within the range of 8–11. Approximately
COD removal is 95.7% (Fig. 3), adding H2O2 and nano-Fe2O3 after 35 min reaction.
Fig. 3. Effect of pH on COD removal
Effect of catalyst on COD removal. The results are presented in Fig. 4. As it is ex-
pected, rising catalyst dose can increase COD removal. COD removal reached 95.7%
after 35 min at 50°C. Therefore, catalyst had a significant impact on the oxidation of
antibiotic wastewater.
314
Fig. 4. Effect of catalyst dose on COD removal
CONCLUSIONS
In this work was studied the feasibility of treating antibiotic wastewater treatment by
nano-Fe2O3 and H2O2. Knowledge of the impact of the operating conditions such as
temperature, the amount of catalyst, H2O2 dosage, pH is required. Experimental results
indicated that in the nano-Fe2O3 and H2O2 process, 95.7% COD was removed after
35 min reaction at a temperature of 50°C. Adding a catalyst significantly improved
the COD removal.
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315
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(2015).
Revised 26 May 2015
316
STUDY ON HIGH PRECISION SWILL-COOKED DIRTY

OIL DETECTION SYSTEM BASED ON DATE FUSION
TECHNOLOGY
LIANG GEa,b*, PAN HUa, XIAOHUI XIEa, ZE HUc, QIANG ZENGa,

JUNBI LIAOb
a
College of Mechanical and Electronic Engineering, Southwest Petroleum
University, 610 500 Chengdu, China
E-mail: cgroad@163.com
b
Department of Measuring and Control, Sichuan University, 610 065 Chengdu,
China
c
College of Electric and Information, Southwest Petroleum University,
610 500 Chengdu, China
ABSTRACT
At present, the detection technology of edible oil adulterated with swill-cooked dirty
oil is limited to the single indicators of waste oil from single source. In addition, the
data analysis processing is superficial, which leads to the inferior specificity of the
detection technology and lacks of wide application. The goal is designing a high
precision detection system of swill-cooked dirty oil based on date fusion technology.
Whether the oil sample is adulterated with swill-cooked dirty oil and the proportion
will be detected by the system, which use the sensor space and time redundancy and
complementary information effectively, and which intelligent computing and recog-
nition are based on adaptive weighted fusion method. The detection accuracy can be
improved effectively and the detection blind can be reduced by testing date fusion
technology. In this way, the oil samples quality can be grasped comprehensively and
at the same time, swill-cooked dirty oil can be distinguished accurately to ensure the
food safety which embracing a bright prospect.
Keywords: swill-cooked dirty oil detection, date fusion technology, food safety,
electrical conductivity.
AIMS AND BACKGROUND

With the improvement of people living standards, per capita consumption of edible
oil is also growing, which results in a large number of catering waste oil1. After being
*
For correspondence.
317
collected and simply refining, the waste oil becomes swill-cooked dirty oil and flow
back to the market2. In recent years, instead of incorporating swill-cooked dirty oil
into edible oil to make huge profit3, in the food processing industry, waste oil is also
flooded. Swill-cooked dirty oil will make threat to the health of consumers and bring
about many problems such as diarrhea, abdominal pain, dyspepsia and long-term con-
sumption will improve the risk of gastrointestinal cancer4. From small oil workshop
and street restaurants to large catering units and food processing enterprises, more
and more phenomenon of using waste oil are revealed by media. Recently, Taiwan
swill-cooked dirty oil event pushed the oil quality problem of food industry to cliff.
Thus a depth study of swill-cooked dirty oil is far-reaching to the food safety.
At present, complete recycling system of swill-cooked dirty oil and the oil qual-
ity database have been established in many developed countries5, while oil quality
detection has not reached the level of special research6. Oil sample can be detected
by the appropriate testing agency through the comparison of spectroscopy, chemical
titration. There are many domestic researches such as physical and chemical evaluation
infrared detection method7, measurement of oil viscosity8 and gas chromatography9.
But the detection technology is limited to the single indicators of waste oil from
single source, it lacks of specificity and wide application10. Studies have shown that
oil detection based on electrical conductivity has advantages of high speed and easy
operation11. But because electrical conductivity can be effect by temperature easily,
this way can not be wildly applied12. In China, there is no effective swill-cooked dirty
oil detection program13. So, it is urgent to design a high precision detection system of
swill-cooked dirty oil for the imperfect waste oil management system and the lack of
high precision detection technology has made swill-cooked dirty oil diffused in the
market. In recent years, the wide application of date fusion technology in the fields14,
such as signal detection and parameter estimate provides new thinking for detection
of swill-cooked dirty oil, so this article has designed a high precision detection system
of swill-cooked dirty oil based on date fusion technology. This system is based on oil
electrical conductivity detection, and it makes temperature compensation by intelli-
gent computing. At the same time, the computing and identification of the system are
revolving the adaptive weighted fusion method. Whether the edible oil is adulterated
with swill-cooked dirty oil and the proportion will be detected by the system and the
oil quality estimate will be guaranteed.
DATE FUSION MODEL OF THE HIGH PRECISION DETECTION

SYSTEM
Date fusion technology is information processing which takes advantages of computer
technology to analyse and integrate detecting information provided by sensors ac-
cording to some criterions automatically, and complete the needed strategy decision
and estimation task15. Fusion can be seen as comprehensive information processing
in the dynamic process of system which purpose is to solve the complement problem
318
of system function16. In contrast, the fusion of complement information would make
a qualitative leap. The fundamental of data fusion is to combine spatial, temporal
redundancy or complementary information according to some fusion ways to get the
consistent explain or describe of the measured object by making full use of sensor
information resources and reasonable control and use of observational information17.
Data fusion includes wavelet transform, weighted average, least squares,
Kalman filtering, expectation maximisation, Bayes, and batch estimate18, etc. As for
the complex composition and strong regional differences of swell-cooked dirty oil,
and similar relevant indicators with edible oil, conventional means have difficulty in
detecting it accurately. Data fusion has provided new way for the detection. Special
complementary information provided by sensors can be assembled and analysed
based on the oil indicators database parameters. For different regions and different
types of oil, the same indicators can be analysed according to different thresholds
to deduce more information, get the best result and provide protection for the final
identification of the oil quality. Based on this, this system is based on the electrical
conductivity test, and utilises adaptive weighted fusion method. The model is shown
in Fig. 1 (Ref. 19). Value xi of each sensor corresponds to a weight number ωi. In the
optimum condition of minimum mean square error of measurement value, optimum
value x^ can be obtained by finding weights in adaptive way.
Fig. 1. Adaptive weighted fusion algorithm model
Let n sensors values are x1, …, xn, and each sensor detects k times. So the i-th
sensor i-th measured value is xi(j). At the same time, set each sensor corresponding
variances are σ12, …, σn2, the total average value detected by this system is x, and
mean square error is σ2.
According to multiple measurements of single sensor, overall measurement of
multi-sensor can be deduced. Let the i-th sensor k times average measurement value
is xi. There:
1 k
xi = ∑ xi ( j ) (i = 1, 2, …, n) (1)
k i =1
319
It can be seen the i-th sensor corresponding variance is:
1 k
σi 2 = ∑ ( xi ( j ) − x i )2 (i = 1, 2, …, n) (2)
k j =1
As adaptive weighted fusion algorithm model shown in Fig. 1, the fusion result is:
n
x = ∑ ωi xi (3)
i =1
And ωi is the weighting coefficient of measurement of each sensor. And:

n
∑ ωi = 1 (4)
i=1
The total average value of system is:

n
x = ∑ ωi xi = 1 (5)
i=1
The total mean square error of system is:

1 n 2 2
σ12 = ∑ ω σi
k i =1 i
(6)

Starting from the overall, weighting coefficients which satisfy the minimum mean
square error can be inversed. The total mean square error of system is:
n
σ 2 = ∑ ωi2 σi2 (7)
i =1

According to the Multivariate function extreme value theory, in the condition
of formula (4), the minimum of formula (7) can be deduced by following equations:
 n 
∑ ωi = 1 
 i =1 
 2 2  (8)
 ∂ σ = 0
 ∂ωi 
Solving the equations above according to the multi-function derivation principle
gives:
1
ωi = (9)
n
1
σi2 ∑
σi2
i =1

Apparently, from formula (9), weighting coefficients ωi is just related to mean
square error. The transformation of fusion solution of equation (9) into equation (3) is:
320
n
xi
∑σ
i =1
2
x = n
i
(10)
∑ σ1 i
i =1

In this case, the total mean square error minimum of system measurement is:
2 1
σmin = (11)
n
1
∑ σ 2
i =1 i

Set x is the average of x1, …, xn:
1 n
x= ∑ xi
n i =1
(12)

It was found that the variance in this case is:
1 n
1 n
σ x2 =
n2
∑ ( x − x)
i
2
=
n2
∑σ 2
i
(13)
i =1 i =1

Comparing formula (11) and (13), it can be seen that adaptive weighted valua-
tion and average valuation have the same fusion effect only when the variances of
each sensor are the same. But in the actual application process, interfered by many
unpredicted factors, the variances of each sensor are different. In this case, in the ap-
plication of system measurement, adaptive weighted fusion valuation is better than
average valuation.
Comparing formulas (6), (9) and (11):
σ12 = σmin2/k (14)
Apparently, the mean squared error informed by single sensor multiple measure-
ments is less than the minimum mean squared error weighted by multi-sensor, and
with the increase of k, the value decreases. So, in the high precision detection system
of swill-cooked dirty oil, starting with formulas (2) and (9) to obtain the sensor related
weighting factor, and obtaining the measurement optimum fusion value from formula
(3). The application of data fusion technology can obtain more comprehensive infor-
mation, improve detect accuracy and make more accurate decision.
SYSTEM DESIGN
Overall structural design of the high precision detection system of swill-cooked dirty
oil based on date fusion technology is as shown in Fig. 2. This system is based on
oil electrical conductivity detection, and measurement information can be computed
intelligently and identified through the adaptive weighted fusion method. Whether
321
the edible oil is adulterated with swill-cooked dirty oil and the proportion will be
detected by the system. The overall detecting system has functions which including
oil sample electrical conductivity and temperature detection, signal conditioning, data
acquisition, information fusion analysis, data display and storage, limit alarm and
automatic temperature compensation, etc. At the same time, the system can exchange
information with computers to achieve remote terminal monitory.
Fig. 2. System diagram
Electrical conductivity detection has some application in food industry for qual-
ity valuation and detection20,21. For the swill-cooked dirty oil detection system which
based on electrical conductivity detection, when dropped proportion is low, tempera-
ture effect on the measurement accuracy can not be ignored because the electrical
conductivity can be effect by temperature greatly22. The temperature compensation
structure shown in Fig. 3, which is used to eliminate the effect of temperature on the
test results. When constant X is input into swill-cooked dirty oil sensor, output X2
will deviate from the true value X as for temperature changes Dt. This temperature
compensation structure which based on intelligent computing can eliminate deviation
caused by temperature changes and ensure output X2 is equal to X.
Fig. 3. Temperature compensation structure
322
SYSTEM HARDWARE AND SOFTWARE DESIGN
HARDWARE DESIGN
Sensor selection. Different from metal, the conductivity of electrolyte solution is

related to the moves of positive and negative ions. For such ionic conductors, electri-
cal conductivity refers to conductivity of electrolyte solution between two parallel
electrodes which areas are 1 cm2 and distance is 1 cm. In order to fully experience
electrical conductivity information of oil water phrase and eliminate the effect of
temperature on system testing, this design use stainless steel plating platinum black
electrode as an electrical conductivity sensor which electrode constant is 1.0 cm–1,
working temperature is 0–50°C, pressure range is 0–0.5 MPa.
The temperature sensor is film platinum resistance which has high accuracy
measurement and the wide temperature range can adapt to a variety of atmospheric
environments.
Signal conditioning circuit design. The system uses two sensors, this design utilises
a multi-channel analog switch in order to use a common set of signal conditioning
circuit. Through a multi-channel analog switch, signals tine-gated can be achieved
to facilitate the subsequent signal processing. Signal conditioning module mainly
includes amplifier, notch filter and level lifting circuit. Instrument amplifier chip
AD620A is used to set up amplifier to enlarge sensor signals primarily; notch filter
use double T notch filter to eliminate 50 Hz frequency interference; the gain adjust-
able chip AD620A is used to set up level lifting circuit for further electrical signal
amplitude conditioning to meet the AD converter requirements of the signal voltage.
Overall signal conditioning circuit is shown in Fig. 4.
Fig. 4. Signal conditioning circuit
Data acquisition circuit design. The system MCU use Microcontroller MSP430F169
which comes with 12-bit ADC converter and the highest conversion rate is 200 Kb/s.
Associated with the acquisition program, sensor signals acquisition can be achieved
by connecting the output of signal conditioning circuit to analog input pin of Micro-
controller MSP430F169.
323
SOFTWARE DESIGN
The software of high precision detection system of swill-cooked dirty oil based on
date fusion technology includes Microcontroller software and computer software.
Microcontroller software uses C language to achieve functions including oil con-
ductivity and temperature information collection, fusion algorithm implementation
and data analysis and transmission. Computer software is designed by LabVIEW
graphical programming language to achieve collected data analysis and processing,
real-time display, etc.

The design of high precision detection system of swill-cooked dirty oil based on
date fusion technology is finished according to the hardware and software design. In
order to verify the system reliability and accuracy, doping simulation tests of peanut
oil, sunflower oil, swill-cooked dirty oil A (taken from a hot pot shop in the vicinity
of a university in Chengdu), swill-cooked dirty oil B (taken from a restaurant in the
vicinity of a university in Chengdu) were test respectively. In test, deionised water
was used to extract oil and the oil electrical conductivity could be measured by testing
water phrase electrical conductivity.
Lysing reagent electrical conductivity test. 30 ml of deionised water and 50 ml of
petroleum ether were placed into two beakers, and their electrical conductivity was
tested 5 times respectively.
From Table 1, under the experimental test conditions, electrical conductivity
values of deionised water and petroleum ether were found to be very low.
Table 1. Lysing reagent electrical conductivity test value (μS/cm)

Testing times Deionised water Petroleum ether
1 0.45 0.02
2 0.45 0.02
3 0.48 0.02
4 0.46 0.02
5 0.45 0.02
Average value 0.458 0.020
Edible oil mixed with swill-cooked dirty oil electrical conductivity test. Peanut oil,
sunflower oil, swill-cooked dirty oil A and swill-cooked dirty oil B were mixed to
formulate blended oil which dopant volume fraction are 0, 10, 20, 40, 60, 80 and
100% respectively. 10 ml of blended oil and 30 ml of deionised water were placed
into a 50-ml beaker and the solution was stirred by a magnetic stirrer. After stirring
for 2 min, place the beaker until solution is stratified. The electrical conductivity of
324
the extracted water phase was tested at room temperature. The test results are shown
in Tables 2 and 3.
Table 2. Electrical conductivity (μS/cm) of sunflower oil with different doping percentage of swill-
cooked dirty oil
Testing 0% 10% 20% 40% 60% 80% 100%
number
1 2.0 2.0 3.0 4.8 6.7 10.0 15.0
2 2.1 2.3 3.5 4.9 7.0 10.4 15.5
3 2.1 2.1 3.2 4.8 7.1 10.2 15.6
4 2.0 2.3 3.6 5.3 7.4 10.3 15.6
5 2.1 2.1 3.5 5.3 7.0 10.5 15.8
6 2.2 2.4 3.6 5.5 7.6 10.6 15.7
7 2.2 2.4 3.5 5.5 7.5 10.5 15.7
8 2.2 2.4 3.5 5.5 7.5 10.5 15.7
Average value 2.113 2.250 3.425 5.200 7.225 10.375 15.575
Fusion value 2.109 2.348 2.549 5.243 7.289 10.439 15.624
Table 3. Electrical conductivity (μS/cm) of peanut oil with different doping percentage of swill-cooked
dirty oil
Testing 0% 10% 20% 40% 60% 80% 100%
number
1 2 2.1 2.2 2.7 3 3.4 3.4
2 2.1 2.1 2.3 2.6 3.1 3.3 3.6
3 2.2 2.2 2.1 2.8 3.2 3.2 3.5
4 2.2 2.3 2.3 2.7 3.2 3.4 3.6
5 2.1 2.3 2.2 2.8 3.1 3.4 3.5
6 2.2 2.3 2.3 2.8 3.2 3.3 3.7
7 2.2 2.2 2.3 2.8 3.2 3.3 3.7
8 2.2 2.2 2.3 2.8 3.2 3.3 3.7
Average value 2.150 2.213 2.250 2.750 3.150 3.325 3.588
Fusion value 2.169 2.205 2.300 2.772 3.169 3.325 3.645
The scatter plots (Figs 5–8) give the dependence between the electrical conduc-
tivity average and the doping percentage.
325
Fig. 5. Water phase electrical conductivity average value scatter plot of sunflower oil doped with differ-
ent doping percentage of swill-cooked dirty oil A
Fig. 6. Water phase electrical conductivity fusion value scatter plot of sunflower oil doped with different
doping percentage of swill-cooked dirty oil A
326
Fig. 7. Water phase electrical conductivity average value of peanut oil doped with different doping
percentage of swill-cooked dirty oil B
Fig. 8. Water phase electrical conductivity fusion value of peanut oil doped with different doping per-
centage of swill-cooked dirty oil B
The curve fitting for the above scatter plots lead to the obtaining of the curve
fitting equations as shown in Table 4.
Table 4. Equation fitting results

Corresponding curve Fitting equation R2
Figure 5 y = 11.1537x + 2.1130 0.9183
Figure 6 y = 11.2458x + 2.1090 0.9223
Figure 7 y = 1.4327x + 2.1750 0.9663
Figure 8 y = 1.4720x + 2.1750 0.9739
327
It is seen from Tables 2 and 3 that in the designed experiment testing conditions,
the water phrase electrical conductivity range of edible oil is 2.00–2.50 μS/cm. The
electrical conductivity of swill-cooked dirty oil is more than 3 μS/cm and the differ-
ent types of swill-cooked dirty oil have larger difference. From above scatter plots
it can be seen when the doping percentage is more than 40%, electrical conductivity
increases significantly, and has a good linear relationship. From Table 4 it is seen that
the value of R2 of every fitting equation is more than 0.9 and reaches 0.9739 which
has a high degree of acquaintance with real curve. At the same time, according to
Table 4, the correlation coefficient of average value curve fitting equation is less than
correlation coefficient of fusion value curve fitting equation, which is consistent with
the theoretical analysis. Thus the swill-cooked dirty oil content in edible oil can be
measured according to the adaptive weighted fusion algorithm accurately and quickly.
CONCLUSIONS
The design described in the present paper is based on swill-cooked dirty oil detecting
system data fusion module. The development process of this system was elaborated
from hardware design and software design and mainly achieved the following results:
(1) Employed data fusion technology to swill-cooked dirty oil detection and
designed a high precision detection system of swill-cooked dirty oil based on date
fusion technology. The design process of this system was elaborated in detail from
hardware design and software.
(2) This design has used electrical conductivity sensor to measure oil sample
electrical conductivity and used adaptive weighted fusion algorithm which based on
data fusion technology in swill-cooked dirty oil detection and the result is better than
simple average algorithm. Thus, the detection accuracy of edible oil doped with waste
oil was improved effectively.
(3) This research established a doping quantitative model for edible oil and swill-
cooked dirty oil. Through the analysis of experimental data, the quality of oil is great
when the electrical conductivity is less than 2.5 μS/cm; the quality of oil is poor when
the range of electrical conductivity is 2.5–5 μS/cm; the quality of oil is poorer when
the range of electrical conductivity is 2.5–5 μS/cm; the quality of oil is terrible when
the electrical conductivity is more than 10 µS/cm.
(4) This high precision swill-cooked dirty oil detection system which based on
date fusion technology has the ability to detect swill-cooked dirty oil doped into ed-
ible oil quickly and accurately. It has advantages including low power consumption,
small volume, automatic range switching, limit alarm and automatic temperature
compensation, etc. At the same time, the system can exchange information computers
to achieve remote terminal monitoring.
328
ACKNOWLEDGEMENTS
This work is supported by Scientific Research Starting Project of SWPU (No
2014QHZ029), National Natural Science Foundation of China (No 51504211), the
State Administration of National Security (No Sichuang-0021-2014AQ), and the
Sichuan Educational Committee (No 15ZB0060).
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Received 12 August 2015
330
STATUS OF AVAILABLE NUTRIENTS SOIL AND THEIR

RELATIONSHIP WITH THE SOIL PROPERTIES IN
SOUTHEASTERN TURKEY
E. SAKINa*, M. M. MUNISb
a
Department of Soil Science and Plant Nutrition, Agriculture Faculty of Harran
University, Osmanbey Campus, Sanliurfa, Turkey
E-mail: esakin@harran.edu.tr
b
Republic of Turkey Ministry of Food, Agriculture and Livestock, Cizre District
Agriculture Directorate, Sirnak, Turkey
ABSTRACT
The purpose of the study is to identify the micro and macro (Cu, Fe, Mn, Zn, B, K and
P) nutrient status and their relationship with the soil properties. In order to conduct this
study, soil samples were taken from the area with four different depths (0–30, 30–60,
60–90 and 90–120 cm). Soil samples were analysed for available nutrient elements
and physicochemical properties. The proportions of clay, silt and sand are respectively
between 39.00–65.00%, 11.00–26.00% and 19.00–40.00%, thus the soil is classified
as clay. Organic matter ratio and calcareous content fluctuate respectively between
1.05–3.56 and 0.38–29.2%. When, the electrical conductivity (EC) (0.29–1.88 dS m-1)
and pH (7.00–8.41) levels were observed non-saline, neutral and slightly alkaline were
discovered. In 96% of the soil P, in 100% B and Zn were at insufficient levels, while
in 96% K, in 76% Fe and in 100% Mn were found to be sufficient. Cu, Fe, Mn, Zn, B,
P and K, which are micro- and macronutrient elements, exhibited positive correlation
with OM, and negative correlation with clay, pH and CaCO3 contents. When pH and
CaCO3 contents are high, micro- and macroelements availability is reduced, transforms
into an insoluble state, thus sedimentation starts. Increase in organic material would
cause a significant increase in micro- and macroelements.
Keywords: correlation between element status, microelements, physicochemical
properties.
AIMS AND BACKGROUND

Soil is fundamental for the continuation of life and agricultural factors. The quality
and the amount of the yield would be high when the soil fertility is suitable. In order
*
For correspondence.
331
to achieve the maximum yield in agricultural areas plant growth parameters needs to
be at the optimum level. High or low amount of plant nutrient materials in the soil
would limit the benefit from these nutrients, on the other hand could cause a loss in
the yield. Therefore, it is highly important to increase the soil fertility, to protect and
to keep the soil in balance. Besides the fertility status of the soil, certain physical,
chemical and biological properties must be suitable as well.
With the initiation of agriculture mankind has begun to process the lands hastily.
Rapid cultivation and utilisation of the lands caused exploitation and desertification
of the soil. Thus, fertility of the soil decreases, organic material (OM) quantities are
reduced and accordingly physical, chemical and biological properties of soil are
impaired. In order to prevent the possible deterioration soil should be utilised con-
sciously. Therefore, it is necessary to use modern agricultural techniques instead of
traditional methods.
Plants acquire nutrients directly from the soil. As acknowledged, optimum plant
growth and the product yield is not only dependent on the total nutrients contained
in the soil, depends as well on the usefulness. Usefulness of the nutrients in the soil
depends on the physicochemical properties of the soil1. The soil subject to this study
are characterised as slightly alkaline, neutral or slightly alkaline soil reactions, low
organic matter content, high clay content, soil without salt and alkalinity problems.
This characteristic of the soil adversely affects the intake of useful micronutrient ma-
terials2. The aim of this study is to determine the certain physicochemical properties,
certain macronutrients (phosphorus, potassium) and micronutrient agents (copper,
iron, manganese, zinc and boron) and to establish the correlation between them.
EXPERIMENTAL
The study area is located at 41°49'13.725" east longitude and 37°10'25.454" north
latitude, on the southwest of Cizre district, and has 3849.6 ha area. The altitude of
the study area varies between 550–650 m. The area consists of the villages Kocapi-
nar, Yalintepe and Kurumcu. From these three regions soil samples were taken from
(0–30, 30–60, 60–90 and 90–120 cm) depths and 18 profiles. The samples are dried
in air-dried and were sieved through a < 2 mm sieve. The climate of the area presents
warm and rainy profile during winter and is hot and dry during summer. Annual mean
temperature is 19°C and the mean relative humidity fluctuates between 48–51%.
In the soil samples was done by texture3, soil reaction (1:2.5) (Ref. 4) and electri-
cal conductivity (EC) saturation extraction5, content of calcareous6. Organic matter7,
available phosphorus8, available K (Ref. 4), available Fe, Mn, Cu and Zn diethylene
triamine pentaacetic acid (DTPA) + triethanolamine (TEA) (Ref. 9) were determined
with the B Azomethin-H method10. In analysis of the data, Statistical Package for the
Social Sciences (SPSS) 10.0 statistical software was used11.
332
Minimum, maximum, mean values and standard deviation of the characteristics for
the analysed soil samples are presented in Table 1. Throughout the profile, the texture
class of the soil is clay. Secondary carbonates are low since the soil formation is on
basaltic flows, yet they increase towards the lower layers12. All samples showed neutral
or slight alkaline pH and the organic matter content is low. The reason for low OM
content of soil is that the pH is alkaline and the climate is hot, in addition oxidation
is high and the soil is extremely cultivated. The correlation coefficients between the
soil samples are given in Table 2.
Table 1. Physicochemical properties and status of element in the soils

Soil characteristics Min. Max. Mean Std. dev.
Soil characteristics
Calcareous (%) (CaCO3) 0.38 29.20 5.25 6.14
Organic matter (%) 1.05 3.56 1.84 0.52
Soil reaction (pH) (sat.ext.) 7.00 8.41 7.75 0.25
Electrical conductivity (EC) (sat. ext.) 0.29 1.88 0.96 0.24
Saturation extract 50.00 96.00 65.16 8.70
Permanent wilting point (PWP) 21.70 32.50 26.97 3.15
Field capacity (FC) 29.97 42.89 36.14 3.26
Clay (%) 39.00 65.00 53.24 5.15
Silt (%) 11.00 26.00 18.60 4.65
Sand (%) 19.00 40.00 27.46 5.60
Content of micro available element (mg kg-1)
Boron (B) 0.19 1.31 0.40 0.27
Zinc (Zn) 0.19 3.10 0.42 0.39
Manganese (Mn) 1.77 15.77 5.43 2.58
Iron (Fe) 5.38 26.53 9.61 3.59
Copper (Cu) 0.73 6.02 1.63 0.94
Content of macro available element (kg da-1)
Potassium (K) 25.90 156.60 74.64 31.67
Phosphorus (P) 0.16 48.03 2.46 6.82
Macro-(P and K) and micro-(Cu, Fe, Mn, Zn and B) nutrient status for soil sam-
ples are presented with minimum, maximum, mean values and standard deviation.
Except the four samples phosphorus content of the soil is insufficient13, K content
is excessive in all samples14, Fe content is sufficient9, Cu content is sufficient15, Mn
content is insufficient9, Zn content is sufficient9 and B content is determined to be
low16 (Table 1). The correlation between the soil properties and useful nutrient status
is presented in Table 2.
333
Table 2. Correlation between element status and soil properties
334
Soil charac- OM (%) pH CaCO3 EC Sat. ext. PWP FC Clay (%) Silt (%) Sand B Zn Mn Fe Cu K P
teristics (%) (%)
OM (%) 1.000
pH (sat.ext.) –0.280 1.000
CaCO3 (%) 0.235 0.407** 1.000
EC (sat. ext.) 0.021 –0.170 –0.200 1.000
Sat. ext. 0.235 0.030 0.084 0.164 1.000
PWP 0.148 0.090 0.072 –0.065 0.012 1.000
FC 0.031 0.065 0.017 –0.053 0.055 0.898** 1.000
Clay (%) 0.266**–0.186 –0.032 –0.022 –0.218 0.470** 0.337** 1.000
Silt (%) 0.107 0.093 –0.146 0.063 0.126 0.076 0.162 –0.337** 1.000
Sand (%) 0.169 0.077 0.173 –0.093 0.095 –0.575**–0.549**–0.683**–0.345** 1.000
B (mg kg–1) 0.593* 0.219 0.251 –0.336 –0.444 0.095 0.245 –0.692** 0.387 0.630** 1.000
Zn (mg kg–1) 0.549**–0.049 –0.077 –0.277 0.277* 0.063 0.242* –0.360** 0.108 0.237 0.924** 1.000
Mn (mg kg–1) 0.457**–0.720**–0.382** 0.047 0.222 –0.110 0.038 0.026 0.053 –0.001 0.058 0.391** 1.000
Fe (mg kg–1) 0.181 –0.283* –0.211 –0.233 –0.057 0.032 0.003 0.049 –0.053 –0.016 0.026 0.178 0.461** 1.000
Cu (mg kg–1) 0.317**–0.032 –0.145 0.004 0.487** 0.165 0.277 –0.155 0.117 –0.023 0.838** 0.590** 0.326* 0.360** 1.000
K (mg kg–1) 0.317**–0.032 –0.145 0.004 0.487** 0.165 0.037 –0.058 0.131 –0.094 –0.077 0.015 –0.040 –0.528** 0.211 1.000
P (mg kg–1) 0.45** 0.008 –0.012 –0.165 0.325** 0.083 0.239* –0.378** 0.117 0.217 0.942** 0.941** 0.272* 0.046 0.683** 0.119 1.000
**p < 0.01, *p < 0.05.
Soil in the study area is usually clayey, has well-developed structural framework,
capacity to hold high amounts of nutrient materials and humidity and has low infiltra-
tion capacity. While clay minerals affect soil properties such as surface area, cation
exchange capacity (CEC) and alteration density, these factors also affect the disin-
tegration rate of organic matter17,18. As the clay content increases silt (r = –0.337**),
sand (r = –0.683**), B (r = –0.692**), Zn (r = –0.360**), P (r = –0.378**) decrease
significantly (Table 2). Since the soil formation is on basaltic main material, lime
content is low, the lime present originates from secondary carbonates12. Organic
material content is low for all samples taken from the cultivated areas. Low organic
material content in these areas would be due to the arid and semi-arid climatic, high
oxidation rate, and poor vegetation12–18.
Micro- and macronutrient elements, Cu, Fe, Mn, Zn, B, P and K, presented a
positive correlation with OM and negative correlation with clay, pH and CaCO3, as
soil properties (Table 2). As clay, silt and CEC increases, total micronutrient elements
(Cu, Fe, Mn, Zn and B) increase and pH, sand and CaCO3 contents decrease. High
percentage of calcareous content in soil and high contents of slightly alkaline pH and
clay adversely affect microelements for intake. In case of high pH, the usefulness of
micro- and macroelements decreases and these elements turn into a insoluble form,
thus starts the sedimentation. Increase in organic matter caused the increase of micro
and macroelements significantly.
Sharma et al.19 and Kumar and Babel18 obtained similar results in the studies
they conducted at Rajasthan area of India. Cu, Fe, Mn, Zn and B exhibited positive
correlation with organic material respectively (r = 0.328**, r = 0.520**, r = 0.446**,
r = 0.445*, r = 0.389**), pH (r = –0.309**, r = –0.519**, r = –0.448**, r = –0.240*,
r = –0.317**) and negative correlation with lime respectively (r = –0.312**, r =
–0.473**, r = –0.447**, r = –0.268*, r = –0.266*) (Ref. 18).
Yadav20 argued that the useful nutrient materials (Cu, Fe, Mn, Zn and B) and
organic materials are positively correlated, and with pH and CaCO3 there is a nega-
tive correlation. Yadav and Meena2, in their study pointed out that useful Zn amount
increases significantly as clay content (r = +0.597**), organic material content (r =
+0.896**), EC (r = +0.305*) and CEC (r = +0.527**) increased. On the other hand,
useful Zn amount decreases as pH (r = –0.618**) and CaCO3 (r = –0.718**) increase.
Sidhu and Sharma15 stated in their study that the useful nutrient elements (Cu, Fe,
Mn, Zn and B) increase as the organic material contents increase and decrease as the
pH, sand and CaCO3 contents increase21,22.
CONCLUSIONS
The highest Zn adsorption was observed in a soil with the highest calcium carbonate
content. Other studies reported that increased P fertilisation increased dry matter and
P concentration in the plant. In acid soils, calcium carbonate–P interactions of positive
335
nature were also reported. The aim of this study was to determine the effects of increas-
ing soil Ca2+ applications on growth, and Ca2+, P, Zn2+ and B+ uptake in durum wheat.
REFERENCES
1. R. W. BELL, B. DEL: Micronutrients for Sustainable Food, Feed, Fibre and Bioenergy Production.
1st ed. IFA, Paris, France, 2008.
2. K. K. YADAV: Micronutrient Status in Soils of Udaipur District of Rajasthan. Hydrol J, 31 (3&4),
26 (2008).
3. H. J. BOUYOUCOS: A Hydrometer Method for the Determination of Textural Classes of Soils.
Tech. Bul. 132, Michigan State Coll. Agr. Exp. Stn., 1962, 1–38.
4. M. L. JACKSON: Soil Chemical Analysis. Prentige Hall, Inc., Englewood Cliffs, New Jersey, 1958.
498 p.
5. U. S. Salınıty Laboratory Staff: Diagnosis and Improvement of Saline and Alkali Soils. USDA
Agricultural Handbook, No 60, 1954. 358 p.
6. L. E. ALLISON, C. E. MOODIE: Carbonate. In: Methods of Soils Analysis (Ed. C. A Black). Part 2.
Agronomy 9 (1), Am. Soc. of Argon., Inc., Madison,, Wisconson U.S.A, 1965, 1379–1400.
7. D. W. NELSON, L. E. SOMMERS: Total Carbon, Organic Carbon and Organic Matter. In: Methods
of Soil Analysis (Ed. D. L. Sparks). Part 3, Chemical Methods, ASA and SSSA, Madison, WI, SSSA
Book Series No 5, 1996, 961–1010.
8. S. R. OLSEN, L. E. SOMMERS: Phosphorus. In: Methods of Soil Analysis (Eds L. A. Page, R.
H. Miller, D. R. Keeney). Part 2. Chemical and Microbiological Properties, American Society of
Agronomy, Madison, Wisconsin, 1982, 539–579.
9. W. L. LINDSAY, W. A. NORVELL: Development of a DTPA Soil Test for Zinc, Iron, Manganese
And Copper. Soil Sci Soc Am Proc, 42, 421 (1978).
10. B. WOLF: The Determination of Boron in Soil Extractes, Plant Materials, Composts, Manures,
Waters and Nutrient Solutions. Soil Sci Plant Analy, 2 (5), 363 (1939).
11. SPSS Science Inc.: SPSS for Windows Users’s Guide. Version 10.0. SPSS Science, Chicago, IL,
1989, p. 354.
12. E. SAKIN: Carbon Balance and Stocks in Soils of Southeastern Turkey. Harran University Graduate
School of Natural and Applied Sciences, Department of Soil Science, Sanliurfa, 2010. 234 p.
13. F. EYUPOGLU: Determination of Fertility Status of Turkey. T.C. Prime Ministry General Directorate
of Rural Services, Soil Fertilizer and Water Research Publishes. General Publish No 220, Technical
Publish No T-67, Ankara, 1999, p. 122.
14. N. H. PIZER: Some Advisory Aspects, Soil Potassium and Magnesium. Tech Bull, 14, 184 (1967).
15. G. S. SIDHU, B. D. SHARMA: Diethylenetriaminepentaacetic Acid-extractable Micronutrients
Status in Soil under a Rice–Wheat System and Their Relationship with Soil Properties in Different
Agro-climatic Zones of Indo–Gangetic Plains of India. Commun Soil Sci Plant Analy, 41 (1), 29
(2010).
16. H. MARSCHNER: Mineral Nutrition of Higher Plants. 2nd ed. Academic Press, Oval Road London,
1997, 24–28.
17. H. R. SCHULTEN, P. LEINWEBER: New İnsights into Organic-mineral Particles: Composition,
Properties and Models of Molecular Structure. Biol Fertil Soils, 30, 399 (2010).
18. B. KUMAR, A. L. BABEL: Available Micronutrient Status and Their Relationship with Soil Proper-
ties of Jhunjhunu Tehsil, District Jhunjhunu, Rajasthan, India. J Agric Sci, 3 (2), 97 (2011).
336
19. B. D. SHARMA, H. ARORA, R. KUMAR, V. K. NAYYAR: Relationships between Soil Charac-
teristics and Total and DTPA-extractable Micronutrients in Inceptisols of Punjab. Commun Soil Sci
Plant Analy, 35 (5–6), 799 (2004).
20. R. L. YADAV, M. C. MEENA: Available Micronutrients Status and Relationship with Soil Properties
of Degana Soil Series of Rajasthan. J Indian Soc Soil Sci, 57 (1), 90 (2009).
21. E. SAKIN, A. SEYREK, E. D. SAKIN: Comparison of the Some Physicochemical Characteristics
and Nutrient Element Status between Cultivated and Uncultivated Soils. Oxid Commun, 38 (3),
1491 (2015).
22. I. KIZILGOZ, E. SAKIN: The Effects of Increasıng Soil Application of Nitrogen and Phosphorus
on Dry Matter, Dry Weight, and Zinc and Boron Concentration in Wheat and Maize. Oxid Commun,
38 (3), 1504 (2015).
337
EFFECTS OF VOLCANIC TUFF AS A PARTIAL

REPLACEMENT FOR CEMENT ON THE COMPRESSIVE
STRENGTH OF CONCRETE
H. CEYLAN
Technical Sciences Vocational School, Suleyman Demirel University,
32 260 Isparta, Turkey
E-mail: hakanceylan@sdu.edu.tr
ABSTRACT
This study examined the use of volcanic tuff from Isparta (Turkey) Central Gelincik
Village as a mineral admixture in the production of concrete. Chemically, the volcanic
tuff is 80.5% SiO2, Al2O3, Fe2O3 and therefore matches the values needed for F-type
fly ash in the ASTM C 618 standards, and meets the ≥ 70% requirement of total major
oxides according to TS EN 450 standards. In the study, for concrete classes C20, C30
and C40, 9 mix samples were prepared at 10, 15 and 20% replacement rates and tested
at 28 and 90 days for uniaxial compressive strength. The most suitable replacement
ratio (apart from deviation in the V40/10 series) was found to be 20%. When the 20%
replacement ratio is accepted, using volcanic tuff to make 1 m3 of concrete saves 65 kg
cement in C20, 84 kg in C30 and 108 kg in C40 concrete.
Keywords: volcanic tuff, mineral admixture, compressive strength, CO2 emission.
AIMS AND BACKGROUND

Rapid economic development and world population growth have placed high demands
on natural resources, including the ingredients for concrete. Such ingredients are avail-
able around world and compose concrete for a wide range of different applications1.
Global economic growth has significantly initiated progress and innovation in the
chemical industry2. The ingredients have environmental, economic and technologi-
cal benefits3. Although concrete has all of those benefits, the technology is one of the
areas of human activity involved in increase in CO2 emission, in particular cement
production, key constituent of concrete. In addition, high demand for energy and for
water and erection and demolition buildings are the reasons why concrete should
not be considered to be particularly environmentally friendly or compatible with the
demands of sustainable development4.
*
For correspondence.
338
Consequently, the concern for the environment has led to appearance of such no-
tions as green buildings, green concrete, green cement, biocement and eco-cement4,5,
which can all be defined as industrial materials and processes which are environmen-
tally friendly as well as being economically viable. Thus, they are believed to meet
the expectations of concrete technology.
The development mode of cement industry has to change since we are facing se-
vere environmental and overcapacity problems and natural resources are being drained
fast. Conventional methods of cement production which mainly depend on investment
and resources consumption must be abandoned in favour of innovative ones, which
are greener, have lower carbon emission and more recyclability. The transformation
could be painful since it entails breaking away from old fashioned practices and will
require great adjustment in both our minds and our practices6.
Partial substitutes for cement in concrete are pozzolanic materials, which rightly
have acquired great importance7. In modern concrete technology, the addition of
mineral additives such as pozzolans has had major impacts on the economies of the
concrete industry and is of great scientific significance8.
Pozzolans are siliceous or siliceous-aluminous materials that react with calcium
hydroxide in the presence of water at room temperature. In this reaction, insoluble
calcium silicate hydrate and calcium aluminate hydrate compounds form, which pos-
sess cementitious properties9,10. The pozzolans contain a large amount of silica and
alumina. In addition, they may also contain some iron oxide, calcium oxide, alkali
and carbon. Pozzolans are divided into two groups as natural and artificial. Natural
pozzolans are volcanic ash, volcanic tuffs, volcanic glass, heat-treated clays, shales
and diatomaceous earths. The artificial pozzolans are such industrial by-products as
fly ash, silica fume and granulated blast furnace slag. Pozzolanic activity is defined as
the binding capability of pozzolans reacting with slaked lime in the presence of water.
The rate of the pozzolanic reaction is dependent on the intrinsic characteristics of the
pozzolan, such as the specific surface area, chemical composition and active phase
content11. Pozzolans are used as mineral admixtures reducing an amount of cement
in concrete. They can also be used directly in the production of cement.
In recent years, many studies have been conducted on the utilisation of various
pozzolans in concrete, mortar and cement. These are stone dust12,13, marble dust114–18,
limestone powder, basalt powder, granulated blast furnace slag, fly ash19–21, diatomite,
pumice7,22, rice husk ash23 and volcanic tuff24,25.
This study examines using volcanic tuff as a mineral additive to concrete and
partial replacement for cement. Its aim was to optimise the amount of volcanic tuff
added with respect to lower water demand owing to economic and environmental
reasons. The Portland cement generates about 7% of global CO2 emissions, which are
the main pollutant leading to environmental pollution and global climate change26.
The total amount of global CO2 emissions in 2013 was 36 billion t, of which industrial
CO2 emissions accounted for about 29%, and cement industry was one of the major
industrial sectors that generated CO2 emission26. A growing intensity of extreme
339
weather conditions brought about by climate changes has become a serious social and
economic problem of the 21st century. The aim of the present day technologists is to
produce a more and more sophisticated concrete, in terms of technical parameters,
which is absolutely environmentally friendly. The potential tools and strategies needed
to meet the environmental challenges in the construction and concrete industry could
be achieved27.
EXPERIMENTAL
In this study, ordinary commercial Portland cement (OPC) type of CEM I 42.5 R
complying with TS EN 197-1 (Ref. 28) was used. The cement used had a fineness
of 3340 cm2/g. The specific gravity of the cement used was 3.12. The volcanic tuff
(VT) came from volcanic deposits in Gelincik village (Isparta–Turkey). The chemical
analysis of OPC and volcanic tuff is given in Table 1.
VT used as mineral admixtures in concrete has been classified in ASTM C 618
(Ref. 9). The three major oxides (SiO2 + Al2O3 + Fe2O3) must total more than 50%
for class C and more than 70% for class F. For this study VT was classified as F-type
according to the ASTM criterion, because the total of major oxides in the VT was
80.5%. Also it suits TS EN 450 (total of major oxides ≥ 70%) standards29.
Table 1. Chemical composition of OPC, VT

Chemical compound (%) OPC VT
SiO2 20.52 59.97
Al2O3 4.00 17.15
Fe2O3 3.45 3.38
CaO 64.28 4.68
MgO 1.63 2.09
SO3 2.53 0.16
Loss ignition 2.72 2.47
K2O 0.73 4.54
Na2O 0.13 4.30
Specific gravity 3.12 2.57
Blain (cm2/g) 3340 3760
The mineralogical compositions of mineral admixtures were determined by X-ray

diffraction and are presented in Fig. 1.Volcanic tuff quartz, sanidine phases were
determined. In order to assess the surface characteristics of the mineral admixtures,
scanning electron microscopy (SEM) was used; typical secondary electron images
are presented in Fig. 2. As seen in Fig. 2, VT presents angular shapes with a rough
surface texture.
340
Fig. 1. X-ray diffraction of VT
Fig. 2. Scanning electron microscopy of VT
Four different types of crushed limestone aggregates – crushed limestone I

(0–4 mm), crushed limestone II (4–8 mm), crushed limestone III (8–11.2 mm),
and crushed limestone IV (11.2–22.4 mm) were used while casting each of the test
specimens according to TS 802 (Ref. 30). A water-reducing admixture was added to
the mixtures at the ratio of 1–2% binder materials by weight. Also used was a poly-
carboxylate ether-type super plasticizer (SP) with a specific gravity of 1.15 g/cm3.
In order to assess the effects of volcanic tuff as a partial replacement of cement
on the behaviour of concrete, slump tests for all mortar mixtures were first performed
to obtain a constant slump value (200 ± 20 mm). According to the slump tests, water/
binder ratios (w/b) for C20, C30 and C40 were determined to be 0.56, 0.43 and 0.34,
341
respectively. Considering these findings, 9 different mixtures were prepared in order
to determine the effects of the usage of volcanic tuff as a partial replacement for ce-
ment on the compressive strength of the concrete. Mix designs of mortar are shown
in Table 2. VT was added as a partial replacement to cement by weight at rates of
10, 15 and 20%.
For compressive strength tests, six cubes of 150 mm were cast from each con-
crete mixture. Specimens were left covered with a plastic sheet. After removal from
moulds at 24 h of age, concrete specimens were immersed in water saturated with
lime at 20°C until the age of testing (Fig. 3). The compressive strengths of specimens
were determined at the age of 28 and 90 days according to TS EN 12390-3 standard31.
Table 2. Mix proportions of mortars (kg/m3)

Mix OPC VT Water Super Crushed Crushed Crushed Crushed w/b Slump
plast. limestone limestone lime- limestone (mm)
I (0–4 II (4–8 stone III IV (11.2–
mm) mm) (8–11.2 22.4 mm)
mm)
Control 20 326 – 182 3.26 693 313 193 711 0.56 220
VT 20/10 292 32.85 182 3.26 692 312 192 709 0.56 220
VT 20/15 277 48.91 182 3.26 691 312 192 708 0.56 210
VT 20/20 261 65.22 182 3.26 690 311 192 707 0.56 200
Control 30 419 – 182 6.28 662 299 184 679 0.43 220
VT 30/10 377 41.85 182 6.28 660 298 183 676 0.43 210
VT 30/15 356 62.78 182 6.28 658 297 183 675 0.43 210
VT 30/20 335 83.70 182 6.28 657 297 183 674 0.43 190
Control 40 538 – 182 10.76 621 281 173 636 0.34 220
VT 40/10 484 53.81 182 10.76 618 279 172 633 0.34 200
VT 40/15 457 80.72 182 10.76 616 278 171 632 0.34 200
VT 40/20 430 107.62 182 10.76 615 278 171 630 0.34 190
Fig. 3. Cured concrete samples
342
The 28- and 90-day resistances strength to uniaxial compressive strength of the con-
crete samples were measured to show the effects on uniaxial compressive strength
when volcanic tuff is used as an admixture to concrete. The compressive strength
resistance of concrete samples with volcanic tuff as an admixture and the resistance
of control samples are seen in the graphs in Figs 4 and 5. Furthermore, in Figs 6, 7
and 8, the uniaxial compressive strength values of C20, C30 and C40 class concrete
samples are given in comparison to volcanic tuff admixture ratios and curing times.
Fig. 4. 28 days resistance of concrete samples
Fig. 5. 90 days resistance of concrete samples
343
1.2
28 days 90 days
1
relative compressive stress (MPa)

0.8
0.6
0.4
0.2
0
control 20 VT20/10 VT 20/15 VT 20/20
Fig. 6. Comparative uniaxial compressive strength resistance of class C20 concrete samples
1.2
28 days 90 days
1
0.8
0.6
0.4
0.2
0
control 20 VT20/10 VT 20/15 VT 20/20
1.2
28 days 90 days
1
0.8
0.6
0.4
0.2
0
control 20 VT20/10 VT 20/15 VT 20/20
Overall, apart from VT 40/10, all concrete samples increased in resistance with
an increase in admixture ratio for both the 28- and 90-day curing times. The resist-
ance of concrete samples in the C20 and C30 series increased with an increase in
volcanic tuff content. The highest resistance values for uniaxial compressive strength
344
in all C20, C30 and C40 samples, closest to the control samples, were achieved from
concrete samples with 20% volcanic tuff. Only VT 40/10 samples show a different
result. Unlike the C40 series concrete samples, 10% volcanic tuff admixture gives
similar results to the C40 control cement. The most suitable volcanic tuff admixture
ratio for the C40 series is thought to be 10%.
Furthermore, higher resistance values were achieved in all the 90 days curing
time samples. Only in the C40 series VT 40/10 group were the uniaxial compressive
strength values lower after 90 days, which means a deviation.
The reliability of the study results were questioned and analysed from a statistical
point of view. The variation coefficients of these values were calculated. The varia-
tion coefficients calculated in accordance with uniaxial compressive strength based
on concrete resistance and curing time are given in Fig. 9.
9
8
coefficient of variation values (%)
28 days 90 days
7
6
5
4
3
2
1
0
control 20
control 30
control 40
VT 40/20
VT 20/15
VT 20/20
VT 30/10
VT 30/15
VT 30/20
VT 40/10
VT 40/15
VT20/10
Fig. 9. Average coefficient of variation values of compressive strengths of the series
Generally, the variation coefficients were determined to be less than 5%. In the
uniaxial compressive strength values acquired after 28 days of curing, only the VT
40/20 concrete series had a variation coefficient of 8.51%, whilst the others were all
calculated to be below 5%. In the uniaxial compressive strength values acquired after
90 days of curing, however, VT 30/10, T 30/20, C40 and VT 40/15 concrete series
had variation coefficients of 5.47, 6.59, 5.98, and 6.35, respectively. These values are
thought to be at the boundary of toleration.
CONCLUSIONS
In this study, research was carried out on the use of volcanic tuff, obtained from quar-
ries near Central Gelincik Village, Isparta (Turkey), as a mineral admixture to the
production of concrete. With this aim, 3 different resistance groups and 3 different
admixture ratios were prepared to make 9 series of concrete samples that were tested
for uniaxial compressive strength on 28- and 90-day curing durations. The following
results were obtained from analysis of the values in the study:
345
– Chemically, the total percentage of SiO2, Al2O3 and Fe2O3 in VT is 80.5 and
therefore matches the values needed for F-type fly ash in the ASTM C 618 standards.
It also meets the ≥70% requirement of total major oxides according to TS EN 450
standards. According to these values, the VT shows the properties of a typical fly ash.
– The control concrete samples and VT-added fresh concrete samples all showed
values between 190 and 220 mm in the slump tests.
– Generally, with the VT-added concrete samples, the uniaxial compressive
strength increases with an increase in VT content. Only samples from the VT 40/10
concrete series showed different results.
– Among the 10, 15 and 20% VT replacement ratios studied, the most suitable
replacement ratio was found to be 20% (apart from the deviation in the V40/10 series).
– Among the VT-added concrete samples, only the concretes from the VT40/10
series showed values close to the C40 control sample on the 28-day uniaxial compres-
sive strength tests. The uniaxial compressive strength values of the control samples
were not achieved by any of the VT added concrete series.
– The values from the study indicate that the VT from near site Central Gelin-
cik Village, Isparta (Turkey) can be used as an alternative admixture mineral in the
production of concrete.
– According to the results, when the ideal replacement ratio of VT in concrete
is accepted as 20%, 1 m3 of concrete production would save 65 kg of cement in C20
concrete, 84 kg in C30 and 108 kg in C40. It is known that the cost of cement in
concrete production is approximately 40% in Turkey. Therefore, use of VT in cement
production is expected to provide considerable economic benefits.
– Cement is a traditional yet young building material, which is not expected to
be replaced in the near future. The Portland cement industry constitutes 7% of global
CO2 emissions and thus is known as a sector with a big large carbon footprint. Envi-
ronmental awareness, low carbon emission, integration and smart development are
the inevitable trends in the transformation from the conventional to innovative. The
success of the transformation largely depends on science and technology innova-
tion5. For these reasons, reducing the use of cement and finding alternative mineral
admixtures for it is of great importance. The use of VT and similar materials as an
alternative to cement in the production of concrete will greatly reduce cement use,
resulting in both economic and environmental benefits.
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347
Overall ecology
AN OPTIMISATION MODEL OF POLLUTION-CONTROL

REGULATION ON YINGSHANG SLUICE OF HUAI RIVER,
CHINA
DONGFENG LIa*, SHIKUI LIANGb,c, HAO CHENb

a
School of Information Engineering, North China University of Water Resources
and Electric Power, 450 045 Zhengzhou, China
b
School of Water Conservancy and Environment, Zhengzhou University,
450 001 Zhengzhou, China
c
School of Water Conservancy, North China University of Water Resources and
Electric Power, 450 045 Zhengzhou, China
E-mail: lidongfeng@ncwu.edu.cn
ABSTRACT
Due to frequent pollution incidents in the middle of Huai River, effective operation
scheme is needed for practical application. Thus, this study selects the Yingshang
sluice of Shaying River (in the Huai River watershed) as a case study to investigate
optimisation model of pollution-control regulation to obtain an optimum operate
scheme. We coupled the hydrodynamic-water quality (HWQ) model and the Multi-
objective optimisation model to a new developed model. The model is resolved by
multi-objective genetic algorithm and validated by measured field hydrology and water
quality data. The result shows that Yingshang sluice regulation obtained from optimum
model can effectively reduce the negative influences of pollution incidents caused by
Shaying River sewage, with respect to reservoir profiting and pollution-control. This
model proposed in this paper can give some insight to further study about decision
making of operation rules in heavily polluted rivers.
Keywords: Yingshang sluice, optimisation model, pollution-control regulation, heavily
polluted rivers, multi-objective genetic algorithm.
AIMS AND BACKGROUND

The Huai River is the earliest river, which performs water pollution control in China.
In 1995, the State Council issued the ‘Interim Regulations of water pollution preven-
tion on Huai River’, and specified ‘under the precondition of ensuring the flood and
*
For correspondence.
348
drought, the sluice gates of the Huai River Basin should balance both upstream and
downstream water quality, and develop pollution control regulation scheme to avoid
sewage discharge concentrated’. However, in practical application, to achieve the
purpose of ‘reservoir profiting’, an amount of polluted water is impounded by sluices
and dams of Huai River. When special hydrological phenomena happened (such as
rainstorm), large sewage discharge intercepted by the sluice easily lead to sudden water
pollution incidents in downstream protect river, such as the water pollution accidents
occurred in 1990, 1994, 1995, 2000 and 2004 in Huai River. If setting ‘pollution-
control’ as the regulation goal, the amount of polluted water intercepted by sluices
and dams must be reduced. Based on experiments of Huaidian sluice regulation in
Huai River1, the aim of this paper proposes an optimisation model of pollution-control
regulation of Yingshang sluice by considering ‘reservoir profiting’ and ‘pollution-
control’. Pollution control regulation schemes under the condition of different water
quality were proposed. The study results support reasonable environmental capacity
to reduce impoundment of water pollution, and to reduce probability of sudden water
pollution incident in Huai River.
The construction of sluices and dams is an important strategy for the develop-
ment and management of water resources, and plays important roles in flood control
and disaster reduction. However, they change the spatial and temporal distribution
of pollutants in watersheds. Especially it causes much threats to river water environ-
ment. Much research is posted to focus on these problems. The main methods used
to assess the impacts of sluices and dams on river quality can be classified into four
categories: (1) analysis of water quality, water quantity, and other aspects of the water
environment is conducted through field surveys2,3; (2) exploration of the impacts of
sluices and dams on river discharge, water quality, river structure, and other aspects
of the water environment has been conducted through examination of historical
data4–7; (3) numerical simulation models, including widely-used hydrological models
(such as the Solid and Water Assessment Tool (SWAT)) coupled with water quality
models, water quality simulation models (such as the MIKE software), and other
numerical simulation analyses have been used8–10, and (4) mechanism experiments
have been carried out. Some researchers propose model experiments to analyse the
impacts of sluice regulation on flow regimes, migration, and pollutant transforma-
tion, while others have conducted field experiments to study the transportation laws
of water contaminants11,12. Above research did not involve both ‘reservoir profiting’
and ‘pollution-control’, and did not develop scientific and reasonable sluices and
dams control scheme of heavy pollution rivers, which could not effectively support
the specific practical activities in Huai River; therefore, we did this work hoping to
develop a new model to solve those problems.
349
EXPERIMENTAL
STUDY AREA
The Shaying River is one of largest tributaries of the Huai River. It starts from the
Funiu Mountain of Henan province, flows through more than 40 counties in Henan and
Anhui Province, such as cities of Pingdingshan, Luohe, Xuchang, Zhoukou, Fuyang,
etc. The length of Huai River is 620 km, and drainage area is about 40 000 km2. The
Shaying River is also the most serious polluted river in Huai River. The total amount
of sewage and pollutants accounted for about 40% of Huai River, and ammonium
in the Huai River is mainly from Shaying River. Water pollution incidents occurring
in Huai River over the years were related to the Shaying River. As control sluices
of Shaying River working in Huai River, Yingshang sluice control scheme has a
considerable impact on water environment of Huai River. The position of Yingshang
sluice is shown in Fig. 1.
Fig. 1. Map of study area and sampling locations
METHOD
Model description. This paper employs numerical simulation and multi-objective

optimisation technique methods to propose optimisation model of pollution-control
regulation of Yingshang sluice. The model includes hydrodynamic-water quality
(HWQ) model and multi-objective optimisation model. HWQ model is used to simulate
the process of river pollutant transport and diffusion under different sluice control
scenarios. According to objectives of ‘reservoir profiting’ and ‘pollution-control’,
this paper establishes two objective functions to consider constraint conditions of
reservoir water storage, discharge flow, water quantity balance, water quality balance
and water quality objectives, and to establish the optimisation model of pollution-
350
control regulation. Then partially restrained conditions are solved by HWQ model,
non-inferior solutions are verified. Finally, optimum control schemes of Yingshang
sluice are obtained. The flow map of the optimisation model is shown in Fig. 2.
Fig. 2. Framework of optimisation model of pollution-control regulation
HWQ model. Two experiments of Huaidian sluice regulation were conducted on

March 3–6 and October 7–11 in 2010. The hydraulic parameters and water quality
parameters were measured through the experiments. Simulation model covered the
area from Fuyang sluice to Lutaizi hydrologic station. Data in the model were derived
from measured river cross-section data and terrain data. The Shayinghe River consists
of 121 sections, the Huai River have 12 sections. The average distance of sections is
about 1 to 2 km.
Combined with the actual situation of the study area, two nodes were selected,
respectively in the upper and down of Yingshang sluice. There were Yingshang sluice –
Lutaizi station to calculate river reaches. In two-dimensional hydrodynamic-water
quality model, vertical acceleration and vertical density difference of water flow was
ignored, changes of vertical water quality are reduced to the average concentration
process, and the vertical and horizontal analysis of the water quality variables is
considered.
Hydrodynamic governing equation:
∂h/∂t + h(∂u/∂x + ∂v/∂y) + u(∂h/∂x) + v(∂h/∂y) = 0 (1)
∂u/∂t + u(∂u/∂x) + v(∂u/∂y) – (1/ρ)(Exx(∂2u/∂x2) + Exy(∂2u/∂y2)) + g(∂z/∂x + ∂h/∂x) = 0 (2)
∂v/∂t + u(∂v/∂x) + v(∂v/∂y) – (1/ρ)(Eyz(∂2v/∂x2) + Eyy(∂2v/∂y2)) + g(∂z/∂y + ∂h/∂y) = 0 (3)
where x and y are coordinates of horizontal and vertical direction, respectively; u and
v – water component in x and y direction, respectively; t – time; ρ – water density;
g – gravity acceleration; z – the bottom elevation; h – water depth; Exx, Eyy, Exy and
Eyz – eddy viscosity coefficients.
351
Water quality equation:
∂(ch)/∂t + ∂(uch)/∂x + ∂(vch)/∂y = ∂(Dx(∂ch/∂x))/∂x + ∂(Dy(∂ch/∂y))/∂y – k1hc + k2hc (4)
where h is water depth; c – the pollutant concentrations for a certain point (x, y); t –
time; Dx and Dy – pollutants diffusion coefficients; k1 – pollutants degradation coef-
ficient; k2 – sediment release coefficient, which reflects the influence of sediment on
water quality under different hydrodynamic conditions.
Formula (1), (2), (3), and (4) are the basic equations of hydrodynamic-pollutant
transport model under sluice control scenarios. The equation was discretised by the
Galerkin weighted residual method, solved by Newton-Raphson nonlinear iteration,
and frontal method was used during the solving process. The unit is divided into
quadrilateral or triangular of two-dimension, space integral uses the Gauss integral,
and the time derivative is replaced by the nonlinear finite difference approximation.
Model parameters. Based on the field experiments we conducted, ammonia nitrogen
is chosen as the representative pollutant. The results are as follows:
(1) Hydraulic parameters: the Manning coefficient n is 0.028, the eddy viscosity
coefficient E is 5 m2/s.
(2) Water quality parameters: horizontal and vertical diffusion coefficient is 0.3
m2/s, ammonia nitrogen degradation coefficient is 0.076 d–1. Sediment release coef-
ficient k2 of pollutant deposition is –0.05–0.25 d–1.
Multi-objective optimisation model
Objective function:
(1) ‘reservoir profiting’ objective function. In order to avoid a greater impact of
sluice regulation on local ‘reservoir profiting’, water quantity variation between the
sluice impoundment and ‘reservoir profiting’ level is required minimum. The expres-
sion is as follows:
T
min E(v) = ∑((Vt+1 – Vnor)/Vnor)2 (5)
t=1
where T is the total time; Vt+1 – the storage water at the end of the period t for Ying-
shang sluice; Vnor – the storage water of ‘reservoir profiting’ level of Yingshang sluice.
(2) ‘pollution-control’ objective function. Water quality and water quantity of
upper reaches and water quality before the sluice needs to be considered. To ensure
no sudden water pollution appearing in Huai River, the pollutants amount discharge
to the downstream is maximum during the control period, which is designed to reduce
the total amount of pollutants before the sluice, and to pursue full utilisation of pollu-
tion carrying capacity of the protected river. The expression is as follows:
T
max F(v) = ∑(QtCt)∆t (6)
t=1
where Qt and Ct are the average flow and pollutant concentrations discharged into
downstream reach in the period of t, and ∆t – calculation time step.
352
Constraints of the application. The constraints of the multi-objective optimisation
model are given by the following:
(1) constrain of water quantity balance:
Vt+1 = Vt – (Qt – qt)∆t (7)
where Vt+1 and Vt are the water storage capacity at the beginning and end of period t,
respectively; Qt and qt – the average outflow and inflow through Yingshang sluice,
respectively;
(2) constrain of water quality balance:
Vt+1Ct+1 = VtCt – (QtCtout – qtCtin)∆t – k(Vt + Vt+1)/2 (8)
where Ct+1 and Ct are the pollutants concentration at the end and beginning of period
t, respectively; Ctout and Ctin – pollutants concentrations of average outflow and inflow
in the period of t, respectively; k – pollutants degradation rate coefficient;
(3) constrain of water quality in Huai River. Yingshang sluice carries out the pol-
lution control regulation, and thus sudden water pollution incident of Huai River will
not occur, namely water quality concentration of Lutaizi section can not be greater
than 1.5 mg/l. The expression is as follows:
CL,t ≤ 1.5 (mg/l) (9)
where CL,t is water quality concentration of Lutaizi section in the period of t;
(4) constrain of reservoir storage:
VtL ≤ Vt ≤ VtU (10)
where Vt is storage water in the period of t; VtL and VtU – the minimum and maximum
storage water in the period of t, respectively;
(5) constrain of reservoir discharge flow:
QtL ≤ Qt ≤ QtU (11)
where QtL and QtU are the lower and upper limits of discharge flow in the period of
t, respectively;
(6) all variables are not negative.
Solutions of model
(1) Treatment of the objective function. Discharge flow of every period was used
as the optimisation variable. Vt+1 in the ‘reservoir profiting’ objective function (such
as formula (5)) was transformed into flow function by water quantity balance equa-
tion (such as formula (7)). Ct+1 in the ‘pollution-control’ objective function (such as
formula (4)) was transformed into flow function by water quality balance equation
(such as formula (8)).
(2) Treatment of constraint conditions. In the water quality constraint function
of monitoring sections (such as formula (9)), HWQ model was used to calculate the
maximum discharge of each period. In the water storage constraint function (such as
formula (10)), the minimum storage VtL and maximum water storage Vt0 were taken
353
the water amount corresponding to dead water level and maximum water level, respec-
tively. In the discharge flow constraint function (such as formula (11)), the minimum
discharge flow QtL was zero (the sluice was closed), and the maximum discharge could
be obtained by hydraulic calculation.
(3) Non-dominated Sorting Genetic Algorithms-II was used in this paper. This
algorithm uses the genetic operators to evolve populations, uses a fast non-dominated
sorting method to sort individuals, and uses the crowding distance method to maintain
the population diversity. According to the multi-objective optimisation model, this
paper obtained the non-inferior solution set by multi-objective optimisation function
based on the improved NSGA-II from MATLAB. Then hydrodynamic-water qual-
ity model was used to validate the solution set, not feasible regulation scheme was
deleted. Accordingly, non-inferior control scheme set was found.

Measured average flow and water quality data of April 2004 in Shaying River and
Huai River was used. We divided April 3–30 of 2004 into four periods. The specific
data are given in Table 1. Setting water storage of each sluice as the normal storage
water, we prescribe the data in Table 1 into the objective function, programmed in
Matlab language, and calculated the pollution control optimisation model of Yingshang
sluice. Results are shown in Table 2.
Table 1. Water quality and quantity data of study area

Period Fuyang sluice Huai River Remark
flow ammonia flow ammonia
(m3/s) concentration (m3/s) concentration
(mg/l) (mg/l)
1 42.5 9.60 192.5 0.65 the normal water storage capacity
2 64.4 5.28 312.6 0.31 of the reservoir is maintained, the
3 76.6 7.56 220.8 0.98 ammonia nitrogen concentration is
4 56.2 5.50 160.8 0.55 2.95 mg/l, the time step is 7 days
Table 2. Non-inferior regulation schemes of Yingshang sluice

Scheme Change Reduction Period 1 Period 2 Period 3 Period 4
percentage amount of discharge discharge discharge discharge
of storage ammonia (m3/s) (m3/s) (m3/s) (m3/s)
capacity (%) (kg)
1 –29.3 907357 58.5 102.6 45.3 61.4
2 –21.3 879561 56.9 98.3 45.3 59.6
3 –11.3 845378 54.7 92.3 45.3 58.2
4 –2.0 814486 52.5 84.6 45.3 59.2
5 1.3 804132 56.2 75.6 45.3 61.4
354
(1) Each scheme is proposed within the regulation requirements of Huai River
protection, through the analysis and comparison of the above 5 schemes. Ammonia
concentration of Lutaizi station is in the range of 0.86–1.43 mg/s, which did not cause
water pollution phenomenon in Huai River. Scheme 1 intercepts 907 357 kg ammonia
(maximum), storage water was reduced by 29.3%. Scheme 5 intercepts 804 132 kg
ammonia (minimum), storage water was increased by 1.3%. Scheme 1 emphasis on
‘pollution-control’, and Scheme 5 emphasis on ‘reservoir profiting’, other schemes
were between the two.
(2) Pollution control optimisation model established in this paper can select the
most appropriate regulation and control scheme according to the needs of different
periods. For example, after flood season, reservoir storage is small, and scheme of
small water storage variation and small ammonia reduction can be chosen at this
time to ‘reservoir profiting’. If before flood season, scheme of large water storage
variation and large ammonia reduction can be selected to reduce pollutants amount
of Yingshang sluice and to avoid downstream water pollution events.
(3) The model established in this paper can regulate the Yingshang sluice dy-
namically according upstream different water quality and quantity in both ‘reservoir
profiting’ and ‘pollution-control’. The model can protect the Huai River water environ-
ment, prevent sudden water pollution incident in the main stream of the Huai River,
and can make best utilisation of environmental capacity of Huai River to reduce water
pollution total amount intercepted by Yingshang sluice.
CONCLUSIONS
Taking Yingshang sluice as a research object on heavy polluted Shaying River,
hydrodynamic-water quality model under sluice was constructed through field tests
and numerical analysis, then pollution control regulation optimisation model was
established. Base on the results of case study, it was found that the model established
could regulate the sluice dynamically according upstream different water quality and
quantity in both ‘reservoir profiting’ and ‘pollution-control’. The model can make full
use of environmental capacity of Huai River to reduce intercepted water pollution total
amount, and to reduce probability of sudden water pollution incident in Huai River.
The establishment method and solving method of our model can be promoted
and applied to the research of sluice operation in general heavily-polluted rivers. This
paper also can provide technical support for the operation of dams group in heavily
polluted rivers, and give some insights to further related study.
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356
Overall ecology
EFFECT OF ENVIRONMENTAL WATER RELEASE ON THE

STATE OF FLORA AND VEGETATION OF THE STEPPE PLAIN
SEGMENT OF THE IRTYSH RIVER FLOODPLAIN
V. KAMKINa*, M. BEISEMBAYEVAb, O. MAZBAYEVb, K. BAZARBEKOVc

a
Department of Agrotechnology, Agricultural Faculty, ‘S. Toraigyrov’ Pavlodar
State University, Pavlodar, Kazakhstan
E-mail: kvani@rambler.ru
b
Department of Physical and Economic Geography, Faculty of Natural Sciences,
L. N. Gumilyov Eurasian National University, Astana, Kazakhstan
E-mail: manira_ter@mail.ru; ordenbek@mail.ru
c
Department of General Biology, Faculty of Natural Sciences, Pavlodar State
Pedagogical Institute, Pavlodar, Kazakhstan
ABSTRACT
The article analyses the environmental releases for the period 2001–2014 years and the
influence of changes in the hydrological regime of the floodplain ecosystem. The cur-
rent state of the Irtysh floodplain vegetation in conditions of anthropogenic-transformed
the hydrological regime of the river. Recommendations on Environmental Manage-
ment and Protection of the Irtysh River floodplain ecosystem are given in this article.
Keywords: hydrological regime of the river Irtysh, floods, flora, vegetation ecosystems.
AIMS AND BACKGROUND

Currently, there is an increased interest in the study of the natural laws of interaction
between structural ecosystems and their reaction to change this or that environmental
factors. Vegetation has the greatest environmental information of all the natural objects.
Special scientific interest is represented in dynamic ecosystems in the valleys of large
meridian-oriented rivers where the vegetation is influenced by the imposition of zonal
and intrazonal factors and high speed fluctuation and succession processes allow us
to study the dynamics of vegetation in a relatively short period of time1.
The Irtysh River is the largest transboundary water artery of Eurasia. Flora and
vegetation of its vast valleys and flood plains particularly have a huge environmental
*
For correspondence.
357
and economic value being a major supplier of green fodder, valuable resource plants
carrying soil protection, water protection, recreational and many other functions2.
Anthropogenic changes in the hydrological regime of the river combined with
unsustainable use of nature, led to a significant salinity and turning into steppe of
the floodplain ecosystems and thus leading to the transformation of vegetation. This
has resulted in reduced productivity of grasslands, and the loss of floristic and plant
community diversity and synanthropisation of flora. In the coming years the existing
problems will be worsened by construction of the channel Black Irtysh – Karamay
in China and thus it can lead to a possible reduction in water runoff3.
For the maintenance of the dynamic equilibrium of natural ecosystems and bio-
diversity there must be data on the regularities of the spatial structure of vegetation,
ecological conditions of its formation and response to the impact of anthropogenic
factors. They are required to predict the development of negative trends in the dy-
namics of ecosystems and timely response to them and the development of measures
to optimize the distribution of surface runoff of water content in different years, as
well as rational use and effective protection of vegetation at a high scientific level4.
The aim of the research was to study the influence of the spring environmental
water releases on the state of the vegetation cover of the Irtysh River floodplain in
its steppe segment.
To achieve this goal the following tasks were advanced:
– to examine the long-term nature protection parameters of water passes;
– to conduct comprehensive floristic and geobotanical researches;
– to identify environmental and plant communities peculiarities of dominant
plant species;
– to identify factors of anthropogenic transformation of the vegetation and to
assess their role in changing the floristic and plant community diversity;
– to forecast the possible dynamics of floodplain ecosystems and to develop
recommendations for the restoration and protection of vegetation.
EXPERIMENTAL
Researches were conducted by the authors during 2001–2014 years in the valley of the
Irtysh River within the administrative boundaries of Pavlodar region (steppe plains of
the river segment). Field studies covered vegetation of the floodplain and floodplain
terraces together forming a valley of the river. The total length of the surveyed area
from north to south is over 500 km. The length of cross-sectional profiles of the right
and left bank of the valley is from 6 to 9.3 km (Ref. 5).
The methodological basis of research is the ecosystem landscape-biogeoceonotic
approach. There were used as traditional methods of botanical and geographical and
geo-botanical studies (geobotanical descriptions phytocenoses landscape and ecologi-
cal profiling, mapping, collecting herbarium, etc.) and modern methods, including
the coordinates of the points in the description of GPS devices, spatial analysis of
358
satellite images, the creation of an electronic database, and others. Special attention
was paid to the spatial arrangement (structure) of vegetation in its relationship with
other components of the landscape (topography, soil) floristic composition, condition
assessment phytocenoses identification of rare species and communities. On profiles,
oriented from the river in the direction of the original bank, all plant communities
were described in detail in a number of spatial row, their borders and GPS measured
length were identified and recorded. Except profiles, explored territory was uniformly
covered with a network geobotanic site covering all the different types of ecosystems.
630 relevés were made6.
Flora analysis was performed on the basis of personally collected materials (over
1200 herbarium sheets (Ref. 7)) and data on the study area were published. Identifica-
tion of plant species was produced by a multi-volume edition ‘Flora of Kazakhstan’
(1956–1966) (Ref. 8), ‘Flora of Siberia’ (1996) (Ref. 9), an illustrated determinant
of the plants of Kazakhstan (1981) (Ref. 10) and other determinants. Latin names of
plants were refined by reference ‘Vascular plants of the USSR’ (Ref. 11).
In the process of analysis of flora the focus was paid not only to its systematic
structure, but also to the distribution of species ecomorphs, community type and eco-
nomic groups. For each type was set its own community role and areas of ecological
optimum.
All the data obtained by the analysis of flora and relevés were recorded in a
specially designed computer database and processed using the application Microsoft
Office Excel.
When assessing the degree of anthropogenic disturbance of vegetation condition-
ally ‘background’ undisturbed community and anthropogenic modifications in each
type of ecosystem were described.

Floodplain in the flat steppe of the flow of the Irtysh River is a natural biocomplex
formed and continuing to evolve mainly under the influence of river waters and floods.
A state of floodplain meadows mainly depends on the hydrogeological regime, which
determines the dynamic and complex conditions of existence of floodplain meadows.
Upper-Irtysh cascade reservoirs produce annually releases of water needed to flood
floodplain in order to increase agricultural productivity.
But the organisers of the water releases do not use the full potential and reserves
of the cascade hydropower, all sets in the interests of energy regime3.
The Irtysh River has a mixed supply, in the upper glacier. The depth at low water
reaches reach 6–9 m, and in the shallows within 1.7–2 m. The river flow is smooth
and quiet, the flow rate in the low water is 0.2–0.7 m/s and in the flood it reaches
2 m/s. The water content of the river downstream decreases sharply. The reason for
this is decrease of the amount of water during the flood on the territory of Pavlodar
359
region and an increase in the columbine area. The mean annual water consumption
is 902 m2/s (Ref. 12).
Due to the nature of power, the natural flow of the river Irtysh (before the con-
struction of the reservoir) was characterised by two floods: maximum early spring –
from the melting of snow in plains and low hills and spring-summer – from melting
of snow and glaciers in the mountains.
A maximum spring tide was an average of 12 May earliest date – 13 April (1938)
later – June 23 (1956). The flood lasted an average of 75–80 days, sometimes inhaling
100–120 days. Often there was a strong, sometimes disastrous flooding.
After the recession of the first flood of water, soil moisture was high; heat was
enough and experienced rapid growth of vegetation. Vegetation growth slowed down
with decreasing soil moisture. Then the second flood was advancing at which lowlands,
swamps were poured and the water replenished in lakes, floodplain soils was made
up with water, which is beneficial to the state of floodplain ecosystems.
In vivo the floodplain of the Irtysh River was poured with flood almost every year
for 89–97% of its area. In some years, with a frequency of 1 every 6–8 years floodplain
was flooded only by 60–70%, and a frequency of 1 every 12–15 years, flooding up
to 10% of the area. In wet years the entire area of the floodplain was flooded for a
sufficiently long period. It occurred naturally flushing channels numerous channels,
oxbow lakes and lakes, as well as waterlogged and saline areas.
During construction of the Bukhtarma reservoir (1959–1963) the Irtysh River
floodplain inundation by flood waters was discontinued, which caused a sharp
qualitative and quantitative changes in vegetation and decrease of the productivity
of grasslands by 60–70%.
Regular flooding of the floodplain of the Irtysh began in 1964. By combining
Bukhtarma release from the reservoir to the spring floods of Yertys major tributaries –
rivers Ulba and Uba flowing below Bukhtarma and Ust-Kamenogorsk hydroelectric
power. After commissioning in 1988 the Shulba reservoir improved hydrological
regime of the Irtysh. Conditions were created for the regulation of the flow in the
spring with the flooding of the floodplain on the optimum mode of release. The range
of influence of the reservoirs on the hydrological regime of the Irtysh spreads down
the river at a distance of 1500 km (Ref. 12).
Today there is only one flood – the first spring. Reducing the duration of standing
flood waters over the flood plain has meant that the area flooded floodplain reduced13.
Analysis of environmental water releases over a number of years (2001–2014
years) shows that the value and quality of flooding affect the total volume of water
discharge of the Shulba reservoir in the main period, timing and duration of releases
in environment protection, as well as maintaining the maximum per diem (3500 m/s).
In 2001 from the Upper Irtysh cascade of reservoirs in the spring was actually
dropped 8.74 km water. Then (2002–2007) – took place the reduction to 5.96–4.58 km
further (2008–2009) – up to 3.98 and 3.88 and, respectively, in 2012 – up to 1.43 km.
360
In 2012 in accordance with the decision of the interdepartmental commission
for environmental water releases, water discharge was 1.43 km (as opposed to the
required 5.4 km). This amount is not enough even to ensure that the river at least
partially burst its banks.
In 2001 the maximum inundation of the floodplain of the river Irtysh was traced –
307.6 ha (92.5%). In the subsequent period (2002–2003) there was a decrease of
flooding inundated arrays from 254.1 thousand ha to 232.8 ha, 75–69% of the total
area. In the period from 2004 to 2007 on the lood plain were flooded 80 – 77.6% of
the total area.
2008–2009 years were dry, the minimum floodplain inundation 62.9–57.7%. In
recent seasons (2010–2011) floodplain went under water at 88.2–77.8%, indicating
a favourable period of flooding due to the climate change, water balance elements
of the columbine. 2013 spring water releases were as close to a natural spring floods
inundated flood plain arrays 89%, and in 2014 81.1% flooded floodplain meadows.
A release wave simply rolled across the floodplain, creating a momentary mir-
ror coating lands and waters. When it is allowed to reset the sharp decline of Shulba
reservoir because of what the end arrays and the floodplain areas of the Irtyshsky
and Zhelezenskiy districts Pavlodar region loses the necessary amount of water to
flood the territory, the soil does not have time to absorb enough moisture and gain
water reserves in the root system. It is clear that over time a negative aftereffect for
the ecological status of the floodplain landscapes will appear.
A kind of hydraulic structures is a complex system of dams. Numerous dams
significantly alter the hydrological regime of the Irtysh floodplain, resulting in a
reduction in the rate of flow of flood waters, changing the nature of the deposits of
alluvium, uneven sinking various floodplain and floodplain yield significant array of
the influence of the floods.
On indicators of river water quality affect water availability, the seasonal and
daily dynamics of the inside reservoir processes associated with the activities of the
physicochemical, hydrological and biological factors.
Mostly snow-supply creates small water salinity during the flood period.
The river water salinity increases from 0.5–0.8 g/l – in the spring, and summer
to 1–3 g/l, and more – in the winter period14.
The hydrological regime of the Irtysh River in the upper reaches is formed by
leaching and dissolution of rocks, the surface runoff from the columbine area and
pollutants entering the drain of the river.
After the regulation of the flow of the river a change in the composition of anions
and cations in the water has taken place. Decrease in flow and water level in the river
leads to a reduction of the processes of the dilution of pollutants and self-purification,
thereby increasing the concentration of pollutants in the water that accumulates annu-
ally on the floodplain during floods, and then accumulate in the soil and living organ-
isms. For the period of flow regulation of the Irtysh River water salinity increased
from 1.1 to 1.5 times, the concentration of total iron increased 17–45 times, 2 times
361
phosphates, nitrates 7 times compared to the natural level. Today the index value of
water pollution in the Irtysh within the Pavlodar region is 1.38 that allows charac-
terising the river as a water reservoir of third class quality – moderately polluted13.
A huge amount of hot water is formed under cooling a turbine and other industrial
heat exchangers. When resetting a hot water up to 50–40°C water is thermal pollution
of the river, significantly affecting the floodplain microclimate14.
Thus, in connection with the regulated flow of the Irtysh River the hydrologi-
cal regime of its flow has changed, leading to a change in the conditions of flooding
floodplain. The result of these processes was steppe formation and salinisation of the
floodplain.
Under the influence of the hydrological regime of the river floodplain flora and
vegetation is formed. Flora of the valley of the Irtysh River within the boundaries
of the Pavlodar region is represented by 545 species of vascular plants belonging to
268 genera and 73 families of higher spore, gymnosperms and angiosperms. Most
plants – 534 species, or 98% are angiosperms, including dicots 413 and 121 species
of monocots. 9 species (1.6%) belong to the spore-bearing plants, only 2 types (0.3%)
belong to gymnosperms.
Leading families are Asteraceae – 84 species, or 15.5% of the total number of
species, Poaceae – 57 (10.5%), Fabaceae – 34 (6.3%), Brassicaceae – 30 (5.5%),
Rosaceae – 29 (5.3%), Chenopodiaceae – 28 (5.1%), Cyperaceae – 25 (4.6%), Ra-
nunculaceae – 25 (4.6%), Lamiaceae – 18 (3.32%) and Caryophyllaceae – 17 (3.1%).
The remaining 63 families account for 198 species, accounting for only 36% of the
total amount.
Flora of the investigated area which we obtained in the process of our investiga-
tions contains the data of the following rare species: Saussurea robusta Ledeb, Ser-
ratula kirghisorum Iljin, Equisetum sylvaticum, Euphorbia microcarpa, Hemerocallis-
lilio-asphodelus, Tulipabieber steiniana, Cypripedium guttatum Swartz, Delphinium
elatum, Rosa pavlovii Chrshan of these species is the Rosa pavlovii Chrshan (Red
Book of the Kazakh SSR). The number of many of these species is very low, which
makes them particularly vulnerable.
The biomorphological spectrum is dominated by herbaceous plants (485 species,
of which 358 perennials and 93 annuals 34 biennials. There are 22 species of shrubs,
trees – 16, dwarf shrubs – 9, shrubs – 5, shrubs and vines – 4 species.
Ecological structure flora is dominated by ecomorphic mesophilic retinue (292
species, or 53.6%), which indicates the general appearance of intra-zonal vegetation.
Xerophilic suite has 157 species (28.8%), hygrophilic – 56 species (10.3%), halophilic
– 22 (4%), the hydrophilic – 19 species (3.5%).
The flora of Irtysh River valley contains a large number of economically important
plant species: fodder (170), medical (147), technical (126), honey (111), ornamental
plants (87), food (72), and others.
Zonal and subclimax steppe and meadow-steppe vegetation of terraces greatly
transformed and mainly represented by secondary communities. In the floodplain the
362
following types of intra-zonal vegetation: shrubland (floodplain forests), meadow,
marsh, water and steppe, including halophyte options. Also pioneering groups of plants
are separately considered in the shallows and river braids. The main type of vegeta-
tion in the floodplain is a major source of green fodder for livestock in the region.
Table 1 summarises the dominant meadow vegetation type, depending on its
position in the longitudinal and cross-section of the floodplain.
Table 1. Distribution of dominant vegetation type in the investigated meadows

Subzone Riverine floodplain Central floodplain Terrace floodplain
1 2 3 4
???
Arid steppes Agrostis gigantea, Bro- Agrostis gigantea, Cala- Bromopsis inermis, Eq-
mopsis inermis, Carex magrostis epigeios, Poa uisetum arvense, Sangui-
praecox, Galium bo- pratensis, Sanguisorba sorba officinalis, Galium
reale, Artemisia procera officinalis, Bromop- boreale, Carex acuta
sis inermis, Elytrigia
repens, Filipendula ul-
maria, Galium boreale,
Gratiola officinalis,
Vicia cracca, Carex
acuta
Bromopsis inermis, Alopecurus praten- Alopecurus pratensis,
Calamagrostis epigeios, sis, Elytrigia repens, Elytrigia repens, Vicia
Elytrigia repens Sanguisorba officinalis, cracca, Arctium tomento-
Bromopsis inermis, sum, Bromopsis inermis,
Glycyrrhiza uralensis, Glycyrrhiza uralensis
Poa pratensis, Vicia
cracca, Galium boreale,
Rumex confertus
Dry steppe Bromopsis inermis, Alopecurus pratensis, Elytrigia repens, Agrostis
Elytrigia repens, Alope- Elytrigia repens, Bro- gigantea, Bromopsis
curus pratensis, Agrostis mopsis inermis, Glycyr- inermis
gigantean rhiza uralensis, Galium
boreale, Lathyrus pra-
tensis, Poa pratensis
Desert Agrostis gigantea, Bro- Agrostis gigantea, Cala- Bromopsis inermis, Eq-
steppes mopsis inermis, Carex magrostis epigeios, Poa uisetum arvense, Sangui-
praecox, Galium verum, pratensis, Sanguisorba sorba officinalis, Galium
Artemisia procera officinalis, Bromopsis verum,
inermis, Elytrigia repens, Carex praecox
Filipendula ulmaria,
Galium verum, Gratiola
officinalis, Vicia cracca,
Carex acuta
to be continued
363
Continuation of Table 1
1 2 3 4
Halophytes real meadows
Arid steppes – Glycyrrhiza uralensis, Artemisia pontica, Bro-
Puccinellia gigantea, mopsis inermis, Pucci-
Artemisia pontica, nellia gigantea, Glycyr-
Jacobaea vulgaris rhiza uralensis
Dry steppe – Artemisia procera, Elytrigia repens, Glycyr-
Elytrigia repens, rhiza uralensis, Pucci-
Glycyrrhiza uralensis, nellia distans, Bromopsis
Puccinellia distans, inermis, Calamagrostis
Alopecurus pratensis epigeios, Sanguisorba
officinalis, Alopecu-
rus pratensis, Sonchus
arvensis,
Poa pratensis,
Stellaria graminea
Desert steppes Carex acuta, Elytrigia Alopecurus pratensis, Alopecurus pratensis,
repens, Glycyrrhiza Bromopsis inermis, Elytrigia repens, Glycyr-
uralensis, Poa praten- Elytrigia repens, Ley- rhiza uralensis, Agrostis
sis mus ramosus, Lycyrrhi- gigantea, Puccinellia
za uralensis, Galatella distans
punctata, Sanguisorba
officinalis, Filipendula
ulmaria, Artemisia pon-
tica, Galium verum,
Rumex confertus
Patterns of spatial structure of vegetation in the valley of the transverse profile

is shown in Figs 1–3.
Fig. 1. Profile through the valley of the Irtysh River within the subzone of dry steppes in the alignment
villages Kaymanachiha-shore (53º35’ north latitude)
0 – open areas of water surface devoid of surface vegetation, 1 – willow-poplar forest, 2 – miscellaneous
herbal cereal meadow, 3 – herbal different cereal large meadow, 4 – steppe forming campfire meadow,
5 – eco series of floodplain channels, 6 – herbal different major sedge swamp meadow, 7 – average
downed cereal different herbal meadow, 8 – big miscellaneous herbal nitrophilous meadow, 9 – worm-
wood hairline group on the side of the terrace, 10 – agrocenosis in complex with sagebrush steppe grass
cereal, 11 – agrocenosis without the participation of indigenous vegetation, 12 – birch forests
364
Fig. 2. Profile through the valley of the Irtysh River within the subzone of dry steppes in the alignment
of the village rebrovka – Shawki (52º25’ north latitude)
0 – open areas of water surface devoid of surface vegetation, 1 – pioneer group at a young sandy allu-
vium, 2 – willow-poplar forest, 3 – shrub meadow vegetation complex, 4 – miscellaneous herbal cereal
real meadow, 5 – herbal different piece of lumber cereal swampy meadow, 6 – miscellaneous herbal
steppe reformer meadow, 7 – number of eco floodplain channels, 8 – weak halophytic downed cereal
grassy meadow, 9 – average downed slightly saline wormwood cereal herbal meadow, 10 – bushland,
11 – agrocenosis without the participation of indigenous vegetation, 12 – reed swamp grass, 13 – poplar
forest, 14 – wormwood wild rye grouping on the slope terraces, 15 – miscellaneous herbal wormwood
grass steppes
Fig. 3. Profile through the valley of the Irtysh River within the subzone of desert steppes in the alignment
of the village Abay-Podpusk (51º19' north latitude)
0 – open areas of water surface devoid of surface vegetation, 1 – pioneer group at a young sandy alluvium,
2 – overgrown shrub willows, 3 – willow series Echinocystis, 4 – willow-poplar forest, 5 – honeysuckle
rose hip hawthorn scrub, 6 – herbal steppe forming different halophytic wormwood cereal meadow, 7 –
miscellaneous common salt tree (Halimodendron halodendron) herbal grass wormwood, 8 – number
of eco floodplain channels, 9 – sagebrush wild rye group, 10 – sagebrush steppe feather grass fescue,
11 – poplar forest, 12 – miscellaneous herbal cereal real halophytic meadow, 13 – miscellaneous steppe
forming herbal cereal halophytic meadow, 14 – fescue Austrian wormwood halophytic meadow steppe
with Nitraria sibirica, 15 – fescue Austrian wormwood herb weed strongly knocked steppe
365
As an important highly productive natural areas, floodplain ecosystems and es-
pecially their autotrophic component – vegetation, is under strong pressure of human
impact. Biodiversity and productivity of floodplain ecosystems are recommended the
following activities15:
– Withdrawal from phytocenosis production at a rate somewhat lower than their
annual production, in compliance with the use of pasture.
– Compliance with uniformity by creating rational use of pasture and hay turnover
and increase in the number of animal species using pasture forage.
– Holding phytomeliorative work on flood areas with disturbed vegetation and soil
cover (after fertilisation and flood-winter sowing of local fodder crops), weed control.
– Regulation of recreational loads and control of environmental and fire safety
in places of public recreation.
– Annual monitoring of the state of all components of the floodplain landscape.
– Control over the synanthropisation of the Irtysh river valley vegetation to prevent
the uncontrolled invasion of new species can displace aboriginal area.
Taking into account the specific conditions prevailing in connection with inten-
sive human activities over the past decade in the valley of the Irtysh River16, these
measures to achieve the desired effect is not enough.
Urgent need to increase the volume and duration of spring flood and bring the
level of flooding of the floodplain to 89–97% of its area, which will stop the trend
towards steppe formation and salinity of the Irtysh River floodplain17.
It is desirable to produce gaps in terms of the relevant terms of the natural flooding
of the river to the regulation of its mode of cascade hydropower plants and reservoirs.
CONCLUSIONS
An anthropogenic transformation of the hydrological regime of the river Irtysh in
the form of changing the dates, reductionof the volume and duration of floods have
a negative impact on the ecological status of floodplain landscapes. This influence is
manifested in xerophytic plant and floodplain vegetation. Highly productive grassy
meadows with the dominance of Elytrigia repens are replaced by low efficiency of
different herbal cereal and cereal different herb meadows with domination on the me-
zoxerophilic various herbs, deep root system of which allows you to better withstand
the lack of moisture and soil salinity. There is a shift of the subzonal boundaries of
the zonal and intrazonal vegetation to the northern direction, as well as convergent
approximation of intrazonal communities with quasi indigenous and subclimax com-
munities corresponding subzone. Today the flora of the surveyed area contains 545
species, 268 genera and 73 families of higher plants, which characterises it as a variety
of high enough. The vegetation coverage is composed by seven types of vegetation.
Phytoceonotic diversity due to the length of the valley in three natural-climatic zones
and subzones of steppe zone and the complex influence of zonal and intrazonal abiotic
factors, refracted through the prism of the cenotic relations.
366
For preservation of the floodplain ecosystems it is extremely important to increase
the volume and duration of flood inundation, the regulation of anthropogenic pressure,
phytomeliorative work in disturbed areas, annual monitoring of all components of
biodiversity and floodplain landscapes.
REFERENCES
1. I. S. ILINA: The Vegetation Covers of the West Siberian Plain. Science, Novosibirsk, 1985, 127–134.
2. M. DOERING, M. BLAUROCK: Landscape Transformation of an Alpine Floodplain Influenced
by Humans: Historical Analyses from Aerial Images. EAWAG, Swiss Federal Institute of Aquatic
Science and Technology, 2012.
3. F. T. KUKEYEVA, L. F. DELOVAROVA, T. A. ORMYSHEVA, K. N. SHAKIROV: Sustainable
Development and Water Management Issues: Transboundary Rivers Management Gaps between
Kazakhstan and China (Case of Ili and Irtysh). Oxid Commun, 38 (3), 1480 (2015).
4. P. CASSANDRA: Rivers Recover Rapidly Once Dams Are Gone. Study Finds, 2015.
5. E. LIOUBIMTSEVA, G. M. HENEBRY: Potential Impact of Climate Change on the Grain Produc-
tivity in Central Eurasia: Human Vulnerability and Adaptations. In: Global Changes: Vulnerability,
Mitigation, and Adaptation. Sofia University ‘St. Kliment Ohridski’ University Press, Sofia, 2009,
22–27.
6. V. P. GOLOSKOKOVA: A Feasibility Study on the Establishment of the State Natural Reserve
‘Floodplain Irtysh River’. Pavlodar, 2000. 72 p.
7. N. P. OGAR: The Vegetation of River Valleys and Arid Semi-arid Regions of Continental Asia.
Thesis for the Degree of Doctor of Biological Sciences, Almaty, 1999. 273 p.
8. Flora of Kazakhstan. Alma-Ata, 1956–1966. Vol. 1–9. 4248 p.
9. G. A. PESHKOVOY (Ed.): Flora of Siberia. 14 Vols, Science, Russian Academy of Sciences Siberian
Publ. House, Novosibirsk, 1996. 5127 p.
10. V. P. GOLOSKOKOVA (Ed.): Illustrated Determinants of the Plants of Kazakhstan. Nauka, Alma-
Ata,. Vols 1–2, 1969. 1216 p.
11. S. K. CHEREPANOV: Vascular Plants of the USSR. Science, Leningrad, 1981. 292 p.; B. A. BULLS:
Geobotany. Science, Alma-Ata, 1978. 268 p.
12. A. G. TSAREGORODTSEVA: Anthropogenic Transformation of the Hydrological Regime of
Floodplain Landscapes of the Irtysh River. Ph. D. Thesis, Almaty, 2003. 146 p.
13. V. A. KAMKIN: Laws of the Spatial Structure of the Vegetation of the Valley of the Irtysh River
(within the Boundaries of the Pavlodar Region). Ph. D. Thesis, Almaty, 2009. 118 p.
14. S. E. SARSEMBEKOVA: Self-cleaning Ability of River Irtysh. Master Thesis, Pavlodar, 2003. 98 p.
15. S. V. MOGILYUK: Ecological Condition of the Irtysh River Basin within the Pavlodar Region. In:
Proc. of the Conference ‘The Influence of the Environmental Situation in the Region on Health and
Socio-economic Status of Women’, Pavlodar, 2004, 6–8.
16. A. G. TSAREGORODTSEVA: Floodplain Landscapes of Pavlodar Region. Manual. Pavlodar State
University, Pavlodar, 2003. 114 p.
17. B. A. BYKOVA (Ed.): The Red Book of the Kazakh SSR. Rare and Endangered Species of Animals
and Plants. Part 2: Plants. Science, Alma-Ata, 1981. 260 p.
Received 22 September 2015
Revised 15 November 2015
367
Overall ecology
OBTAINING THE SCIENTIFICALLY PLANNED CROP

YIELDS THROUGH APPLYING AGROPHYSICAL AND
AGROCHEMICAL SCIENTIFIC BASE
B. TASa, I. AYDINb, N. GOKCEc*, I. CHRISTOVd

a
Vocational School of Technical Science, Uludag University, Bursa, Turkey
E-mail: melik@uludag.edu.tr
b
Department of Social Science, Faculty of Necatibey Education, University of
Balikesir, Balikesir, Turkey
E-mail: ibrahimaydin10@hotmail.com
c
Department of Social Studies, Faculty of Education, Anadolu University, Eskisehir,
Turkey
E-mail: nazliu@anadolu.edu.tr
d
Poushkarov Institute for Soil Science, Agrotechnology and Plant Protection, Sofia,
Bulgaria
E-mail: ichristow@gmail.com
ABSTRACT
The paper deals with the application of recent top scientific attainments, which allow
purposeful obtaining both the maximum and the reasonable crop yields. Maximum
possible yield is necessary in plant genetic research to determine the yield genetically
set in the new plant variety or hybrid. Practically reasonable crop yield is economi-
cally acceptable to be obtained in agricultural practices.
For the first time, both new ecotechnology for monitoring, estimating and manag-
ing the water status (EMEMWS) and new scientific bridge between water and nutrient
statuses (SBWNS) enable us to estimate and create appropriate conditions for different
crops in each field. The EMEMWS is a Decision Support System (DSS) for precise
management in agriculture. Its version for scientific research was checked. The version
for friendly and easy application by farmers can be created as market tool product.
Both scientific (EMEMWS and SBWNS) attainments are verified under field
conditions. These are managing tools for obtaining planned amount and quality of
crop yield in each agricultural field. Using it, the farmer saves energy (electrical,
from fuel, etc.), irrigation water, nutrients for plant, and human labour in agricultural
practices. This way, the farmer minimises or completely removes the losses of soluble
*
For correspondence.
368
substances, which pollute the surface and underground water and soil. The EMEMWS
substitutes the periodical sampling for soil moisture determination and the use of other
methods for local (point) measuring.
Keywords: crop, maximum yield, planned yield, water status, energy level of soil
moisture, nutrient needed for fixed level of water status.
AIMS AND BACKGROUND

Increase in the efficacy of investments can be attained through practical applica-
tion of the examined (during a period of 30 years) computerised ecotechnology for
monitoring, estimating and managing the water status (EMEMWS). Creating the new
varieties and hybrids requires exact determination of their maximum amount of yield
and quality indices. For reaching this purpose, we need:
(a) the exact estimate of biologically optimum soil water status and its establish-
ment in each field for the whole growing season of each year1–3;
(b) the nutrient (nitrogen, phosphorus, potassium, microelements) supplies in soil,
which are necessary to correspond and keep the growth at this biological optimum4–6;
(c) the ecotechnology for current monitoring, estimating and managing the water
status (EMEMWS), taking into account the various plant stage susceptibilities7,8. Us-
ing EMEMWS and appropriate irrigation equipment in the field, we can create the
specific water status needed (applying minimum irrigation water) during the growing
season9–13;
(d) the appropriate irrigation equipment, water resources and executive human
team14.
The paper is aimed at revealing some great economic and ecological possibilities
of the modern ecotechnology for monitoring, estimating and managing the agroeco-
system water-nutrient status, including the creation of exact conditions needed for
obtaining the genetically-possible and practically-reasonable crop yields.

Genetically-possible crop yield. The both top scientific achievements (EMEMWS
and SBWNS) enable us to estimate and create appropriate agroecosystem statuses for
different crops in each field. Moreover, using these scientific tools, we can create the
biologically optimum water and nutrient statuses under the changing meteorological
conditions in different fields and regions. This way, we are able to determine and
obtain the genetically-possible crop yield (GPCY) in field.
This complex ecotechnology is necessary in plant genetic research to determine
the maximum yield genetically set in the new plant variety or hybrid. GPCY can be
obtained under biologically-optimum conditions in field. Moreover, the ecotechnol-
ogy is necessary to be applied in the seed production.
369
For the first time, the ecotechnology gives scientifically based decision how to
create the recommended energy level L = 5 J1/2/kg1/2 from the Class of Biological
Optimum2 in field during the growing season of crops each year, and to determine
this genetic feature of the existing and new crop varieties or hybrids and produce their
seeds under this water status.
Experimental data of 30-year period show that the maximum genetically-possible
grain yield of H 708 maize grown on Calcareous Chernozem soil in north-western
Bulgaria is on average 16.21 t/ha (Table 1).
Table 1. Soil-moisture energy levels (L, J1/2/kg1/2) recommended to be created in fields for the maize
H 708, and the corresponding minimum allowed values of soil moisture potential (ymin, J/kg) at each
stage of crop growing
Class Class name L ymin Maize grain yield
number (J1/2/kg1/2) (J/kg) (t/ha)
І Class of biological optimum 5 –50 16.21 (e)
16.69 (c)
ІІІ Class of slightly lowered levels 15 –225 11.91 (e)
11.18 (c)
The values of grain yield (both maximum genetically-possible amount at L = 5 J1/2/kg1/2 and reasonable
one at L = 15 J1/2/kg1/2) are experimentally (e) obtained in field and calculated (c) using the regression
equations based on numerous experimental data2.
Table 2 shows some detailed data on the H 708 maize yield, soil-moisture energy
levels and fertilisation rates for different years.
Practically-reasonable crop yield. This ecotechnology offers the practical opportunity
to create an agroecosystem water-nutrient status, which is scientifically-based and
necessary to obtain reasonable amount and quality of crop yield and saves energy,
fuel, organic and mineral nutrients, and human labour in agricultural practices, which
is also for the first time.
Table 1 shows the recommended soil-moisture energy levels both L = 5 and 15
J /kg1/2, which are necessary to obtain the both maximum genetically-possible amount
1/2
of yield and the practically-reasonable one, respectively. Under the mentioned soil
and regional conditions, the reasonable yield amount of grain is on average equal to
11.91 t/ha for the H 708 maize hybrid, creating the plant density of 65 000 plants
per ha. All amounts of yield are calculated for 14% of standard grain moisture. The
farmer should take into account the losses due to a reduced plant density and other
factors in agricultural field, and those during harvesting and transport of the grain.
370
Table 2. Yield (t/ha) of the Н 708 maize grain depending on L (J1/2/kg1/2) of water status of Calcareous
Chernozem soil in the Complex Experimental Station, near the town of Lom, Bulgaria, which is obtained
experimentally under different rates (kg/ha) of fertilisation
Year Created energy levels (J1/2/kg1/2) of water status
under appropriate irrigation schedule no irrigation
L=5 L = 10 L = 15 Le = 22 Le = 26
Nutrient rates: N340 P450 (3) K160
1986 16.36 14.24 13.72 9.90 ─
1987 16.22 14.70 12.48 ─ 5.58*
1988 16.07 14.91 12.68 ─ 6.17*
Mean 16.21 14.61 12.96 9.90 5.87*
Nutrient rates: N280 P320 (3) K120
1986 15.85 13.39 12.96 9.71 ─
1987 16.09 13.77 11.07 ─ 5.44*
1988 14.95 13.98 11.71 ─ 5.76*
Mean 15.63 13.71 11.91 9.71 5.60*
Nutrient rates: N220P230 (3) K80
1986 15.54 13.12 12.79 8.70 ─
1987 14.55 10.86 9.52 ─ 7.33
1988 14.93 13.71 12.07 ─ 6.21
Mean 15.00 12.56 11.46 8.70 6.77
No nutrients added: N0P0 (3) K0
1986 12.95 10.25 9.81 7.54 ─
1987 12.14 10.38 9.62 ─ 6.52
1988 11.15 9.48 8.95 ─ 5.42
Mean 12.08 10.03 9.46 7.54 5.97
The yield values marked with asterisk (*) show that the maize plants have been depressed by an water
status of energy level, which is lower than the necessary one for the two high rates (N340P450 (3) K160 and
N280P320 (3) K120). The phosphorus P (3) is supplied in soil for three years13.
The farm, which is well furnished with agricultural and irrigation equipment, is
able to reach the reasonable amount of yield, creating the necessary water status with
the energy level L = 15 J1/2/kg1/2 of soil moisture, and the nutrient status correspond-
ing to this level. This can be done using the offered ecotechnology for monitoring
and estimating, creating the necessary schedule for irrigation and executing it. The
application of this ecotechnology will save energy and fuels, water and nutrients,
and human labour. Moreover, the exact execution of the schedules using appropriate
irrigation equipment by the farmers will minimize or stop the pollution of surface
and underground water.
Protection of environment and benefits. The offered ecotechnology for monitoring,
estimating and managing the water status (EMEMWS) creates modern information
possibilities for farmers to take precise decisions for accomplishing their activities
concerning the regulation of water-and-nutrient status in soil for each crop grown in
different fields. This is a great scientific tool to put into the agricultural practices the
371
ecological principles and requirements, and to help the development of sustainable
agriculture and to produce the safety products. Application of EMEMWS gives pos-
sibilities:
– to determine the necessary amounts of irrigation water and the exact days during
the growing season for correcting the soil water status in order to create the appropriate
energy level L of soil moisture to obtain the crop yield of planned amount and quality;
– to minimise or avoid the deep filtration under the soil root layer, which is a
transport of water and solved substances polluting the underground water, and losses
of water and nutrients for plants. All these need information on: (a) precise scheduling
using the offered ecotechnology; (b) appropriate equipment for watering; (c) filtration
properties of soil profile, and (d) realising of suitable parts of watering norm;
– to minimise or remove the surface water runoff and the linked soil erosion
with it, which is a process of losses for plants of water, nutrients and soil solid phase,
polluting the rivers, lakes and dams located near the agricultural fields. This protects
the surface layer of soil;
– to obtain the reasonably planned crop yield, for which the farmer ensures the
necessary nutrients in soil;
– to avoid the processes of impoverishment or overloading the soil with nutrients.
Both processes lower the soil fertility, depress the growth and development of plants,
reduce the amount of yield, and worsen its quality.
Creating water status with L = 5 J1/2/kg1/2. The ecotechnology gives the scientifically
based decision how to create an energy level from the Class of Biological Optimum
(CBO) in field during the growing season of crops to determine the maximum geneti-
cally possible yield obtained from new crop variety or hybrid (Fig. 1) for the first time
in agricultural science. It should be put into these practices for the first time.
In traditional agriculture, the fixed design of crop watering schedule at 90%
ensuring the permanent total irrigation norm is not related to the biological water
optimum of agroecosystem15.
The great variety of natural agroecosystem water status (under no irrigation)
was estimated with the new index Le of equivalent energy levels of soil moisture and
through the amount of yield obtained in different years.
372
18
16
14
12
Y (t/ha)
10
8
6
4
2
0
0 10 20 30 40
L (J1/2/kg1/2)
Fig. 1. Yield amount (Y, t/ha) of maize grain (H 708) obtained in experimental field under conditions
of appropriate nutrient (N, P and K) status as a function of water status of Calcareous Chernozem soil
(Lom, Bulgaria), which is estimated through the integral L index and created: by means of sprinkling
irrigation in the 1982–1985 period; by furrow irrigation in the 1986–1988 period, and under no irriga-
tion, during the growing season2,13
Table 3 shows the irrigation schedules developed through the EMEMWS and
actually created in the maize agroecosystem to establish the soil-moisture energy
level with L = 5 J1/2/kg1/2 during the growing season of 1981–1988 period2,13. Number
of watering, which is necessary to create this energy level with L = 5 J1/2/kg1/2 in the
different years considered, increases from 5 in 1982 to 11 in 1985. The total irrigation
norm enlarges from 2400 m3/ha in 1982 to 5330 m3/ha in 1985. For the same period,
the mean number of watering is equal to 7.75, and the average total irrigation norm
is 3792 m3/ha.
Creating water status with L = 15 J1/2/kg1/2. For irrigation practices, Zahariev et al.15
recommended the fixed design of maize watering schedule in the considered region,
as follows: 6 times of watering with 600 m3/ha (total norm of 3600 m3/ha) for the
considered Lom region each year, as follows: first and third decades of June; each
decade of July; and second decade of August.
Table 4 shows the irrigation schedules developed through the offered ecotech-
nology and actually created by irrigation in the maize agroecosystem to establish the
soil-moisture energy level with L = 15 J1/2/kg1/2 during the same growing seasons2,13.
373
Table 3. Irrigation schedules developed through the ecotechnology and actually executed in the field to establish the energy level with L = 5 J1/2/kg1/2 during
374
the growing seasons of 1981–1988 period
Year and Le Dates and gross watering norms (m3/ha) Total Total
(J1/2/kg1/2) number of irrigation
May June July August watering norm
(m3/ha)
1981 – 2 June 14 June 2 July 12 July 22 July 7 Aug 21 Aug 7 3570
(19) 320 390 460 540 600 640 620
1982 – 13 June 21 June 30 June 7 July 13 Aug 5 2400
(16) 350 440 480 490 640
1983 14 May 1 June 19 July 31 July 10 Aug 18 Aug 6 3030
(20) 250 310 560 620 650 640
1984 26 May 8 June 22 June 30 June 7 July 18 July 28 July 8 Aug 20 Aug 9 4190
(25) 200 290 380 420 470 550 590 650 640
1985 24 May 7 June 15 June 23 June 1 July 7 July 17 July 26 July 2 Aug 11 Aug 22 Aug 11 5330
(32) 250 320 370 440 450 480 550 580 650 630 620
1986 – 4 June 26 June 25 July 5 Aug 16 Aug 26 Aug 6 3180
(22) 280 410 570 650 630 640
1987 – 11 June 22 June 4 July 17 July 27 July 6 Aug 18 Aug 29 Aug 8 4260
(26) 340 450 470 540 630 620 600 610
1988 20 May 31 May 20 June 6 July 13 July 22 July 1Aug 10Aug 19Aug 28Aug 10 4380
(26) 200 230 340 440 450 510 560 580 540 540
Mean num- 0.625 2.125 2.5 2.5 7.75 3792

ber over
8 years
In the first column are presented the equivalent energy levels Le, J1/2/kg1/2 of natural water status.
Table 4. Irrigation schedules developed through the ecotechnology for monitoring, estimating and manag-
ing and actually executed in the maize agroecosystem to establish the soil-moisture energy level with L
= 15 J1/2/kg1/2 (from Class of Slightly Lowered Levels) during the growing season of 1981–1988 period
Year and Le Dates and gross watering norms (m3/ha) Total Total
(J1/2/kg1/2) number irrigation
May June July August of water- norm
ing (m3/ha)
1981 – 16 June 10 July 3 Aug 3 3050
(19) 750 1100 1200
1982 – 24 June 24 July – 2 1930
(16) 790 1140
1983 – 2 June – 2 Aug 17 Aug 3 2190
(20) 600 1180 410
1984 – 27 June 15 July 4 Aug 3 3000
(25) 780 1000 1220
1985 – 10 June 28 June 14 July 30 July 13 Aug 5 4930
(32) 650 850 1030 1200 1200
1986 – – – 6 Aug 27 Aug 2 1530
(22) 1230 300
1987 – 24 June 27 July 25 Aug 3 2430
(26) 820 1190 420
1988 – 16 June 15 July 31 July 18 Aug 4 3170
(26) 700 970 1120 380
Mean num- 0 1 1 1.125 3.125 2779
ber over 8
years
In the first column are presented the equivalent energy levels Le, J1/2/kg1/2 of natural water status (no
irrigation) for the years considered.
For obtaining on average 11.91 t/ha (Fig. 1), the number of watering, which is
necessary to create this energy level with L = 15 J1/2/kg1/2 in the different years, in-
creases from 2 in 1982 and 1986 to 5 in 1985. The total irrigation norm enlarges from
1530 m3/ha in 1986 to 4930 m3/ha in 1985. For the same period, the mean number of
watering is equal to 3.1, and the average total irrigation norm is 2779 m3/ha.
Obviously, the farmer has to perform not 6 (six) times of watering, but on aver-
age only 3.1 times. He has to use for irrigation not 3600 m3/ha, but on average 2779
m3/ha for the period considered.
Using the fixed design of maize watering schedule15, the farmer should perform
almost 2 times higher number of watering, applying 1.3 times more amount of water
than actually needed amount, both established by us through the application of the
offered ecotechnology. This means that the irrigation practices, in which the farmers
use the fixed design of watering schedule, are not economically effective and cause
great pollution of surface and underground water of the region considered.
We have to emphasise a very important inference. To keep the same energy
level of soil moisture and obtain fixed yield, the farmers have to perform the speci-
375
fied schedule of irrigation during each growing season, which strongly differs by: (a)
number of watering; (b) date of watering; (c) rate of watering, and (d) total irrigation
norm, for the same crop and soil in the region.
The creation of agroecosystem universal water status at the energy level L = 15
J1/2/kg1/2 is accessible for crops grown on all soils under conditions of good irrigation
equipment, available water resources, and put-into-practice computerised Ecotechnol-
ogy for Monitoring, Estimating and Managing the Water Status. We must emphasise
that this energy level correspond to different pre-watering moisture for diverse soil
varieties.
Traditional agriculture and modern technology for managing. Zahariev et al.15 sug-
gested a scheme of Bulgarian regions with fixed design of crop watering schedule at
90% ensuring the permanent total irrigation norm. The scheme is based on a very rough
approach using mean day-and-night air temperature sums averaged over many years.
All designs of crop watering schedule do not take into account: (a) the four other
meteorological factors strongly influencing the agroecosystem water status; (b) the
specific course of soil moisture change in the root layer of crop during the actual
growing season; (c) the different crop stage susceptibility related to available soil
moisture for plants, etc.
Start of drought and degree of its development. The knowledge obtained in agrarian
sciences and practices (soil physics, plant growing, hydro-amelioration), agroecology
and biology up to now did not permit to define precisely the terms: start of drought and
degree of drought thoroughness. The introduction of integral index of energy levels
L of soil moisture and method for their determination2, as well the development of
Ecotechnology for Monitoring, Estimating and Managing (EMEM) the agroecosys-
tem water status13, allowed to determine the first day-and-night, on which the drought
started for crops to be used in agricultural practices. We define the start of drought on
the day when the energy level L = 15 J1/2/kg1/2 is reached in the field. This corresponds
to a decrease in the soil moisture potential down to the value of –225 J/kg averaged
over the soil root layer. The degree of drought thoroughness depends not only on the
five basic meteorological factors, but also on both the properties of soil in each field
and the crop biological features. This degree for each soil and crop can be estimated
using the offered equations2.
CONCLUSIONS
The formation of amount and quality of yield due to water status depends on both
the energy limitation on supplying the plants with soil moisture and the degree of
irreversible biological changes in plant organism caused by the variable water deficit
at the different crop-development stages. The improvement of soil moisture status
adding the necessary water on time implementing the decisions obtained through the
offered ecotechnology for monitoring, estimating and managing is a good agricultural
376
practice for the farmer to save energy, fuel, organic and mineral fertilisers, and hu-
man labour, reaching economically high-effective crop production. Moreover, this
ecotechnology is a powerful control tool to protect the environment.
REFERENCES
1. I. D. CHRISTOV: Energy Levels of Soil Moisture and Bioproductivity. Int Agrophys, 6, 89 (1992).
2. I. D. CHRISTOV: Estimation of Agroecosystem Water Status and Formation of Plant Water Supply
in Soils. Monograph, Ecology Series, PublishScieSet-Eco, Sofia, 2004. 216 p.
3. I. D. CHRISTOV: Effects of Agroecosystem Water Status on Maize Yield Quality. Journal of Balkan
Ecology, 13 (1), 5 (2010).
4. I. D. CHRISTOV: Effects of Soil Moisture Regime on Nutrients Accumulation in Maize. J Hydrol
Hydromech, 41 (6), 333 (1993).
5. I. D. CHRISTOV: Ecological Approach to Crop Irrigation Scheduling and Fertilizing. Journal of
Balkan Ecology, 1 (1), 47 (1998).
6. I. D. CHRISTOV: Scientific Basis for Managing Water and Nutrient Regimes of Soil When Growing
Crops. Soil Science Agrochemistry and Ecology (Sofia), 4–6, 237 (2001).
7. I. D. CHRISTOV: Crop Yield Response to Soil Moisture Deficiency at Separate Stages of Ontogen-
esis. Part 1 and 2. Journal of Hydrology and Hydromechanics, 42 (6), 402 (1994).
8. K. GULUM, E. ATASOY, I. CHRISTOV, E. GALAI: Agrophysical and Agrochemical Scientific
Base for Integrated Management of Agroecosystem Water-nutrients Statuses. Oxid Commun, 38
(1), 210 (2015).
9. I. D. CHRISTOV: Management of Agroecosystem Water Status. Part 1. New Complex Scientific
Base. Journal of Balkan Ecology, 11 (1), 5 (2008).
10. I. D. CHRISTOV: Management of Agroecosystem Water Status. Part 2. Organization of Decision
Support System (DSS) Application and Advantages. Journal of Balkan Ecology, 11 (2), 137 (2008).
11. I. D. CHRISTOV: Management of Agroecosystem Water Status. Part 3. Adequacy of Decision Sup-
port System (DSS). Journal of Balkan Ecology, 11 (3), 229 (2008).
12. I. D. CHRISTOV: Management of Agroecosystem Water Status. Part 4. Relationships among Soil
Moisture Energy Level, Soil Water Properties and Biological Features of Crop. Journal of Balkan
Ecology, 11 (4), 341 (2008).
13. I. D. CHRISTOV: Monitoring and Management of Agroecosystem Water Status for Protecting Envi-
ronment and Establishing Sustainable Agriculture. Monograph, Ecology Series, PublishScieSet-Eco,
Sofia, 2012.
14. B. P. BOYANOV: Hydro-amelioration and Pumping Stations. ABC Technics, Sofia, 2005. 231 p.
15. T. ZAHARIEV, R.. LAZAROV, S. KOLEVA, S. GAYDAROVA, Z. KOYCHEV: Dividing into
Districts with Crop Design Watering Schedule. ZEMIZDAT, Sofia, 1986. 646 p.
377
Overall ecology
THE GREAT SILK ROAD AS A FACTOR OF DEVELOPMENT

OF THE TOURISM INDUSTRY
B. IZENBAYEVa, O. MAZBAYEVa, A. SAIKENa, B. TASBOLATb,

E. ATASOYc*
a
Department of Physical and Economic Geography, Faculty of Natural Sciences,
L. N. Gumilyov Eurasian National University, Astana, Kazakhstan
E-mail: ordenbek@mail.ru; turis@sina.com; izenbaev84@mail.ru
b
Department of Geography, Faculty of Natural Science, Ahmet Yassawi
International Kazakh-Turkish University, Turkistan, Kazakhstan
E-mail: baigeo-55@mail.ru
c
Department of Primary Education and Social Studies, Faculty of Education,
Uludag University, Gorukle Campus, Bursa, Turkey
E-mail: eatasoy@uludag.edu.tr
ABSTRACT
This article discusses the Silk Road as a factor in the development of the tourism
industry in the Kazakhstan section of the Road. The program of study and revival
of the Kazakh section of the route of the Silk Road was repeatedly put forward as a
priority for the development of science, culture and tourism in the country. For its
implementation, the Government of the Republic of Kazakhstan adopted a number
of resolutions. The huge tourist potential creates the conditions for the development
of various types of tourism and economic efficiency of tourism.
Keywords: Silk Road, Kazakhstan, tourism, historical background, cultural heritage,
the development of silk tour, tourist-ethnographic complex, cultural and educational
value.
AIMS AND BACKGROUND

Formation and development of the Great Silk Road constitutes the historical back-
ground of tourism in Kazakhstan. The beginning of its formation dates from the 3rd
millennium BC.
In studying the history of the development of states of Asia and East, history of
wars, diplomacy and travel, no researcher can avoid the theme of the Great Silk Road.
*
For correspondence.
378
It is considered that the Silk Road began to function as a diplomatic and trade
communication only in the middle of the second century BC. The archaeological loca-
tions in the study area indicate that the track had a great importance in the formation
of culture of the peoples of the South1.
The Silk Road served not only for the import of Chinese silk to the countries of
the West and the North, but there was also the exchange of strange and exotic goods
from Rome, Mediterranean, Arab Caliphate, India, Iran and later from the Russian and
European countries. People sold incenses and spices, medicinal drugs and minerals,
fabrics, carpets, precious stones and metals, furs and weapons. Camels from the Moors
and elephants from India, Fergana and Arabian horses, exotic animals and birds from
Africa and India were all sold and exchanged for the royalty and pleasures of khains2.
On the caravan routes, the crop plants and grains, vegetables and fruits, grapes,
spices and herbs were given away. Together with the goods, the Filatov ideas and
religions, architecture and art, household items, jewellery, clothing and shoes were
freely distributed. According to the biographers of famous scientists and royalties,
and chroniclers, many of them travelled to discover other lands and learn the progres-
sive cultures. In the early centuries, a journey undertaken was considered as a sign
of education. People travelled to Egypt, Arab Caliphate, China, Greece, India, Rome
and later to Russia and Europe along the routes of the Post Ways3.

Taking into account the national and international importance of the revival of the
historic centres of the Silk Road in the Republic of Kazakhstan, preservation and
successive development of cultural heritage of the Turkic Speaking peoples, the State
Program of the Republic of Kazakhstan titled ‘Revival of historical centres of the
Silk Road, preservation and successive development of cultural heritage of the Turkic
Speaking States, creation of tourism infrastructure’ were all approved by the Decree
of the President of the Republic of Kazakhstan of February 27, 1998.
In order to implement this Program, the Representative Office of the Republic of
Kazakhstan established Open Joint Stock Company ‘Silk Road – Kazakhstan’ soon
after its approval, the main activity of which were to ensure cooperation between
organisations and individuals and combine the necessary scientific and production
potential in order to revive historic cities and develop the tourism infrastructure of
the Republic of Kazakhstan.
The State Program ‘Revival of historical centres of the Silk Road, preservation
and successive development of cultural heritage of the Turkic Speaking States, crea-
tion of tourism infrastructure’ were developed in Ref. 4.
The program is based on the fundamental researches, analyses and calculations
of the promising areas of economic and social development of the historic cities and
other settlements in the Kazakh section of the route of the Silk Road and the National
Program of Tourism Development in the Republic of Kazakhstan, developed by the
379
Institute of the Committee on Residential and Building Policy of the Ministry of
Energy, Industry and Trade and Ministry of Education and Science – Academy of
Sciences of the Republic of Kazakhstan. The program also takes into account the
proposals for the preservation of historical and cultural monuments and construction
of tourism facilities of the Committee of Culture, Committee of Tourism and Sports,
Ministry of Education and Science, Ministry of Information and Public Accord of the
Republic of Kazakhstan, Akims of Almaty City, Almaty, Zhambyl, South Kazakhstan,
Kyzylorda, Mangistau, Akmola, Karaganda and East Kazakhstan regions5.
The need for the revival of ethnic and cultural identification of habitat landscape,
creation of conditions of social and economic development of the historic centres of
the Silk Road and their inclusion in international tourism system define the main goals
and objectives of this Program.
The main goal of the Program is the revival of the national, socio-economic and
international role of historical urbanisation system of Kazakhstan section of the Silk
Road route (Fig. 1).
Fig. 1. Map of the Great Silk Road through Kazakhstan
Objectives of the Program are as follows:

– Study of nomadic and settled agricultural cultures and aspects of their coop-
eration, immigration and other ethnic and cultural processes that took place in the
territory of Eurasia from ancient times to the present day (including questions of
ideology, religion, philosophy, cults, ancient hymns, music, dance, military rituals,
written sources);
– Archaeological, architectural, town planning researches of facilities of the Silk
Road route. Creating a database of historical and cultural heritage of the Silk Road;
380
– Museumification and restoration of monuments of history and culture;
– Conservation, museumification and improvement the main stages (or frag-
ments) of development of historic towns and other settlements of the Silk Road route,
included in the list of tourist facilities;
– Recreation (regeneration) and successive development of the traditional
building-up of historic centres of the Silk Road. Revival of a true architectural and
spatial and ethnocultural environment in them. Landscaping and engineering and
communication support;
– Creation of the tourism infrastructure on the basis of the traditional develop-
ment of cities and the last Silk Road: Support service and trading enterprises, pilgrim
and tourist complexes, handicrafts workshops, ethnographic museums and theatres,
ritual and cult and historical and religious centres, etc.;
– Development of the tourism industry and pilgrimage at the local, regional and
international levels and in all diversity of its forms (cognitive, business, sport, health,
exotic, etc.):
– Formation of institutions of the Turkic peoples culture on the basis of the folklore
and ethnographic theatres or other ensembles whose activity reflects the traditional
art forms and their successive development;
– Reconstruction of historic landscapes, caravan routes, horse and walking
routes, humanisation of the environment along the Silk Road route, reconstruction
and improvement of ways;
– Stimulating the development of regional economy;
– Providing employment for the local population6.
The scheme of creation of tourism infrastructure in terms of the main objective
of this Program – the revival of the historic centres of the Silk Road and preservation
of the cultural heritage – has two conceptual directions imposed by the historical and
typological and functional differences between monuments of the settled and agri-
cultural – ‘urban’ culture and ‘nomadic’ cultural and ethnographic one. Analyses of
resources and conditions of the tourism organisation in the Kazakhstan section of the
Silk Road route and forecasts of tourist flows performed by the Institute ‘Kazgipro-
grad’ show that both types of monuments have high attractive characteristics and are
objects of attraction of domestic and foreign tourists. The difference between their
state and detention regime requires the organisation of two forms of infrastructure;
the first one is created through the restoration and use of historic buildings of cities
and other settlements, the second one – through the formation of mobile tourist-
ethnographic complexes7.
The first form of infrastructure suggests the revival of historic city centres, resto-
ration of their traditional buildings and allows the placement of tourism, culture and
service facilities; the second form provides the incorporation of the cultural centres
of nomadic civilisation in the list of the pilgrimage and tourism objects, requires
taking measures for the preservation of this unique heritage and historic landscape
and prevents the construction of any permanent structures in the area of these monu-
381
ments. In this connection, there is a proposal for the creation of seasonal ethnographic
museum complexes: dormitory (yurt) settlements, Gipsy-caravan cities, chariots and
other attributes of nomadic life, organically combined with the monuments and their
environment. This decision allows the organisation of conditions for a short stay for
the pilgrims and tourists, fully reveals the cultural traditions of the nomadic way of
life and ensures the effective protection of monuments when they are most visited.
Ensuring the fulfilment of modern sanitary norms by the construction of car service
centres in such a way that will not violate the general perception of monuments and
landscape8.
Thus, the most capacious part of the Program is the restoration of historical
buildings and other centres of the Silk Road route that allows the construction of the
maintenance service and cultural institutions here. As mentioned above, the remains
of numerous monuments of architecture and art buildings are preserved in the terri-
tories of cities and other residential areas of the Silk Road. Among them are castles,
fortifications, caravanserais, commercial and administrative buildings, palaces, reli-
gious and other cult complexes, residential quarters and houses, production facilities,
improvements, communication and other systems, roads and bridges. Restoration of
this Capital Building Fund of cities and settlements of the Silk Road, its use for the
development of traditional forms of crafts, establishment of national schools of art
and ethnographic centres, religious and ritual services, pilgrim and tourist complexes,
shopping and other service enterprises make it possible to solve scientific, cultural
and socio-economic problems for the revival of the historical centres of the Silk Road
and development of the tourism infrastructure9.
Given scope, scientific and specific urban development character of works,
physical deterioration of many monuments that require strengthening and recovery
works, as well as the lack of budgetary allocations, the need to attract private capital,
consolidation of governmental and nongovernmental organisations, the Program
provides the establishment of the Open Joint Stock Company ‘Silk Road – Kazakh-
stan’ to revive the historical cities and other centres of the Silk Road and develop the
tourism infrastructure.
The Program of creation in the sites of nomadic encampments, religious monu-
ments and unique landscape of tourist-ethnographic complex provides the organisa-
tion of special tourist-ethnographic centres, restoration and creation of new bases for
manufacturing dormitories (yurts), nomadic mobile equipment for arranging exotic
tours, furnishings and other household utensils, revival of the national cuisine, cloth-
ing, traditions and rituals, organisation of folk art schools and workshops, training of
personnel in the field of tourist services, etc.10.
CONCLUSIONS
The main scientific and methodological centre of the tourism industry is the Nomadic
Civilisations Museum in Almaty.
382
The Program of study and revival of the Kazakh section of the Silk Road route
has repeatedly been among the priorities for the development of science, culture and
tourism in the country. For its implementation, a number of Resolutions of the Gov-
ernment of the Republic of Kazakhstan has been adopted.
Given the historical, ethnographic and cultural and cognitive value of the objects
in the Kazakhstan section of the Silk Road route with the support of the Governments
of Kazakhstan, the countries of the Central Asian region, as well as international or-
ganisations, a research has been conducted to study the route resources and restore the
historical and cultural objects, and develop the tourism infrastructure in the region11.
This is due, above all, a great tourist potential, which creates prerequisites for the
development of various forms of tourism and consequently, improve the economic
efficiency of tourism in the region.
REFERENCES
1. Economic Integration of Central Asia (Speech Evan Feigenbaum at the Institute of Central Asia and
the Caucasus to Them. Johns Hopkins). February 16, 2007. www.america.gov.
2. Members of the Silk Road Caucus. The Congressional Silk Road Caucus. www.house.gov.
3. HR 1152 – Silk Road Strategy Act of 1999 (Bereuter (R) NE and 5 Cosponsors). August 2, 1999.
Web-site of the President of USA. www.whitehouse.gov.
4. Decree of the President of the Republic of Kazakhstan dated April 30, 1997 No 3476 ‘On the Imple-
mentation of the Tashkent Declaration of Heads of Turkic-speaking Countries, the Draft UNESCO
and the World Tourism Organization on the Development of Tourism Infrastructure on the Silk Road
in the Republic of Kazakhstan’.
5. V. HANSEN: The Silk Road. A New History. Oxford University Press, Beijing, 2015.
6. A. SAIKEN: Travel Opportunities of the Silk Road in Central Asia. In: Proc. of the International
Conference ‘Prospects of Development of the Silk Road in Central Asia’. Urumqi, September, 2015.
7. H. R. 3196 [106th Congress]: Silk Road Strategy Act of 1999. United States House of Representa-
tives. www.house.gov.
8. Silk Road Legislation Passes US Senate Tonight. June 30, 1999. S. Brownbeck Web-site. brownback.
senate.gov.
9. D. SULLIVAN: Economic Integration Is a Key Factor in the Development of Central Asia. Speech
in Ashgabat, Turkmenistan, 14, 2007. www.america.gov.
10. N. A. KUZIEV: Concept of the New Silk Road: the Essence, Specifics and Prospects. Young Scientist,
(2), 406 (2015).
11. A. A. ZHAKUPOV, E. ATASOY, E. GALAY: An Evulation of Recreational Potential of BSNNP in
Order to Increase the Touristic Image of the Pavlodar Region. Oxid Commun, 37 (3), 871 (2014).
383
Overall ecology
ATTITUDES TO GENETICALLY MODIFIED ORGANISMS AND

FOOD AMONG UNIVERSITY STUDENTS
N. UTKUALPa, A. OZDEMIRa, M. BICERb, B. OZDEMIRc*

a
Institute of Health Sciences, University of Uludag, Bursa, Turkey
E-mail: nevinah@mynet.com; ayozdemir@uludag.edu.tr
b
Department of Cardiovascular Surgery, University of Uludag, Bursa, Turkey
E-mail: mbicer23@yahoo.com
c
Department of Cardiology, University of Uludag, Bursa, Turkey
E-mail: buloz2@yahoo.com
ABSTRACT
Genetically modified organisms and food have serious concerns due to their unpre-
dicted nature. The beneficial effects include cost-effectiveness, economical benefits
and endurance of the related product to certain obstacles including infections, and
drought. Many diseases are attributed to genetically modified foods as with environ-
mental hazards. Some of these have not been validated via scientific committees.
In this study we aimed to investigate the attitudes of 85 students of the Institute
of Health Sciences in Uludağ University. The study was a descriptive study. We gath-
ered data via questionnaire focusing on the attitudes, believes, knowledge levels and
socioeconomical characteristics. The mean age of the study group was 21.77 ± 1.21
(range 20–26). Among the group 67 (78.8%) were female. Majority of them mostly
lived in cities. More than half of the students think that genetics of food is changed
to make national governments dependent to foreign countries. Majority of students
think that GMF and GMOs are detrimental for human health. A negative perception
is present of GMOs. The socioeconomic characteristics did not affect the attitudes of
students towards GMOs. 100% of the students think food labels should contain data
related to presence of GMOs.
Keywords: genetically modified organisms, genetically modified food, attitude,
knowledge, University students.
AIMS AND BACKGROUND

Lack of knowledge about biotechnology carries certain risks. Particularly; geneti-
cally modified organisms/food (GMO/GMF) both draws attention and may cause
*
For correspondence.
384
anxious thoughts about the subject. Both type of behaviour deserves attention; first
excessive concern and anxiety completely cause avoidant behaviour and second lack
of knowledge may cause willingly or unwillingly consumption of GMF that may
possess dangers related to health. Genetic engineering in a way is an intervention to
the nature with unpredictable effects on it and the millions of organisms and human.
A genetically altered food not only is consumed by human but also is in interaction
with the plants and animals habituated near or far. The contamination of environment
may cause invasion of the GMO and destruction of flora and fauna.
The beneficial effects of GMO and GMF are the background of these technolo-
gies including cost-effectiveness, economical benefits and endurance of the related
product to certain obstacles including infections, and drought. The population growth
worldwide made such efforts essential. However, the capitalist economical model is
pushing forwards these endeavours. The know-how requires huge budgets which only
can be supplied by global companies which prioritise their profit. Can we be sure that
these companies are not developing the technology towards directions where only
their products will be able to live with intended destruction of the entire natural flora?
Also, do we really need GMO? Urbanisation and industrialisation politics made
the farmers to leave their villages and refrain from cultivations due to increased ex-
penditures for production. All these are the results of economical politics dictated to
the national governments with global organisations like International Monetary Fund.
The cultivation is getting increasingly under the influence of global companies. Na-
tional countries with adequate farmland and wide diversity of products need to import
food from these countries. The hybrid seeds, insecticides and herbicides are getting
more and more derived from global companies. They increase expenditures and the
national governments become more dependent on these global forces.
Favourable effects are also aimed and accomplished by genetic engineering of
organisms. Antioxidant content of the nutrients is important. Studies aiming to increase
antioxidant content of plants are carried. For example potato (Solanum tuberosum) is
cultivated in Europe and the entire world and the tubers of the potato are characterised
by rich starch and protein contents and high concentrations of antioxidants, such as
vitamin C and flavonoids. The phenolic antioxidants in potato are of high importance
as they have health-related properties. In the study of Kostyn et al.1 the most effica-
cious way to increase the content of phenolic antioxidants was the over-expression
of the dihydroflavonol reductase gene. They reported that the produced transgenic
potato plants had been confirmed in their non-toxicity and nutritional benefits on rats.
Flax plants contain 40% fats and moderate amounts of linoleic acid and α-linoleic
acids which are both essential fatty acids required in diet. The content of α-linoleic
acid in flax oil makes it susceptible to oxidation. Adding antioxidants like tocopherols
and carotene are not satisfactory and the oil is not suitable for frying. Zuk et al. via
suppression of the chalcone synthase gene increased hydrolysable tannin accumulation
and thus increased antioxidant status of seeds of the transgenic plant2.
385
Apples rich in polyphenols provide antioxidant properties. In the study of Espley
et al.3 genetically engineered apples with increased flavonoids (MYB10) were com-
pared with non-transformed apples from the same genotype, ‘Royal Gala’ (RG), and
a control diet with no apple. Compared with the RG diet, the MYB10 diet contained
elevated concentrations of the flavonoid subclasses anthocyanins, flavanol monomers
(epicatechin) and oligomers (procyanidin B2), and flavonols (quercetin glycosides), but
other plant secondary metabolites were largely unaltered. In the study, high-flavonoid
apple was associated with decreases in some inflammation markers and changes in
gut micro biota when fed to healthy mice3.
Kim et al.4 stated that that the antioxidant potential of transgenic oil-producing
plants such as soybean, sunflower, and corn may be enhanced by over expressing
homogentisic acid geranylgeranyl transferase during seed germination.
In this study we aimed to investigate the attitudes of 85 students of the Institute
of Health Sciences in Uludag University. The study was a descriptive study. We
gathered data via questionnaire focusing on the attitudes, believes, knowledge levels
and socioeconomical characteristics. We analysed the data with use of SPSS 20.00
software program. The data are given as percentages, mean ± standard deviation.
Significance was assumed in case of a p value <0.05. In the analysis we used the
Pearson chi-square, Yates chi-square, Fisher chi-square and Man Whitney U tests.

The mean age of the study group was 21.77 ± 1.21 (range 20–26). Among the group
67 (78.8%) were female. Majority of them mostly lived in cities. To give a clue of
the parents status of the cases we asked the employment statuses. Of mothers 84.7%
were housewives. Fathers were self-employed in 34.1% of cases. The parents were
living together in 90.6% of cases. The sociodemographic characteristics of the study
group are given in Table 1.
In Turkey GMOs are legally banned. However, lack of importing GMOs and
GMFs cannot be prevented due to lack of enough facilities for detection of GMOs at
the borders or customs. However, the students think that GMOs are banned in Turkey.
The data show that students opinion about GMOs is satisfactory at least in terms of
legal personal rights. The purpose of modifying the genetics of organisms is largely
unknown. Also some speculative questions like biochemical terrorism were asked and
25.9% agreed. More than half of the students think that genetics of food is changed
to make national governments dependent to foreign countries (Table 2).
386
Table 1. Sociodemographic characteristics
Sociodemographic characteristics n %
Age 21.77±1.21
range (20.00–26.00)
Monthly expenditure 519.17±199.53
range (200.00–1150.00)
Gender male 18 21.2
female 67 78.8
Mostly lived in city 46 54.1
town 27 31.8
village 12 14.1
Accommodation house 57 67.1
dormitory 28 32.9
Marital status married 5 5.9
single 80 94.1
Family type nuclear 74 87.1
big 11 12.9
Families economical condition good 17 20.0
moderate 61 71.8
poor 7 8.2
Mothers job house wife 72 84.7
worker 6 7.1
retired 3 3.5
officer 2 2.4
self-employed 2 2.4
Fathers job self-employed 29 34.1
retired 22 25.9
worker 20 23.5
officer 14 16.5
Parents current status of living father and mother 77 90.6
living together
father and/or 3 3.5
mother dead,
divorced 5 5.9
387
Table 2. Data related to knowledge levels of cases about GMO
Knowledge levels n %
1 2 3
How did you first heard about GMO TV-radio 77 90.6
internet 11 12.9
newspaper 7 8.2
friends 8 9.4
academic staff 6 7.1
The purpose in modifying the genetics of organisms
Resistance to pests in agricultural products yes 46 54.1
no 39 45.9
Resistance to herbicides in agricultural products yes 28 32.9
no 57 67.1
Resistance to viral plant diseases in agricultural yes 33 38.8
products no 52 61.2
Improving the quality of vegetable oils in plant yes 7 8.2
products no 78 91.8
Delaying the ripening of herbal products yes 7 8.2
no 78 91.8
Extending the shelf life of plant products yes 68 80.0
no 17 20.0
Increase aroma yes 16 18.8
no 69 81.2
Increase of product quality yes 20 23.5
no 65 76.5
The use of plants as biological weapons yes 20 23.5
no 65 76.5
Biochemical terrorism yes 22 25.9
no 63 74.1
To make our country dependent on agriculture to yes 43 50.6
foreign countries no 42 49.4
Foreign countries want to disrupt the health of people yes 19 22.4
in our country no 66 77.6
Foreign countries want to make more money from yes 40 47.1
our country no 45 52.9
The use of GMO and GMF are hazardous to health yes 78 91.8
no 7 8.2
Harmful effects of the use of genetically modified plants
Have unclear effects on health yes 57 67.1
no 28 32.9
Can cause toxin production in food yes 36 42.4
no 49 57.6
to be continued
388
1 2 3
Leads to antibiotic resistance in humans yes 24 28.2
no 61 71.8
Cause cancer yes 67 78.8
no 18 21.2
Cause infertility yes 43 50.6
no 42 49.4
Cause congenital organ anolamilies yes 50 58.8
no 35 41.2
Cause homosexualism yes 7 8.2
no 78 91.8
Cause dementia yes 21 24.7
no 64 75.3
Cause allergy yes 26 30.6
no 59 69.4
Failure of plant seeds to obtain from GMOs yes 43 50.6
no 42 49.4
There are environmental harm of GM crops yes 36 42.4
no 49 57.6
They cause reduction of agricultural economic power yes 34 40.0
of our country no 51 60.0
Causing the birth of low birth weight infants yes 32 37.6
no 53 62.4
Causing chronic heart failure yes 17 20.0
no 68 80.0
Causes the Alzheimer disease yes 21 24.7
no 64 75.3
Causes rheumatic diseases yes 7 8.2
no 78 91.8
Which of the products sold in your country are GMO as far you know?
corn 73 85.9
tomatoes 63 74.1
soy 71 83.5
peanuts 17 20.0
strawberries 44 51.8
paddy rice 8 9.4
sunflower 15 17.6
rice 23 27.1
potatoes 24 28.2
sweet pepper 28 32.9
389
The attitudes of consumers were investigated towards GMOs in various studies
conducted years before. In a study by Burton et al.5 evaluated consumer attitudes to
GMOs in food and the extent to which these attitudes translated into willingness to
pay to avoid these products. The results indicated that GM food is only one of a num-
ber of concerns of different aspects of the food system in forming food preferences,
and, albeit a significant one. Attitudes towards organic food were useful indicator
of attitudes towards GM technology6. Concerns about GMOs may be reasonable
and unreasonable. GMFs potentially may be allergic. Gene transfers may occur in
the nature and environmental problems may occur. Potential benefits of GMFs are
improved nutritional characteristics and therapeutic effects6.
The data related to attitudes of students about GMOs are given in Table 3.
Table 3. Thoughts and attitudes related to GMO products (n = 85)

Question Answer n %
1 2 3 4
Do you use GMO food? yes 50 58.8
no 35 41.2
Do you consumption of fruits and vegeta- yes 56 65.9
bles out of season? no 29 34.1
When buying food do you check presence yes 37 43.5
of GMO? no 48 56.5
Do you read the content of the foods you yes 54 63.5
buy? no 31 36.5
Does the nutrient content affect your deci- yes 64 75.3
sion of buying? no 21 24.7
Do you prefer the foods with long shelf life? yes 28 32.9
no 57 67.1
Do seasonal fruits and vegetables consumed yes 79 92.9
are GMO crops? no 6 7.1
Do you think GMO food should be allowed yes 1 1.2
in your country? no 84 98.8
What is the current status of GMOs in our allowed unless labelled 1 1.2
country? xan not be sold 84 98.8
What should we do to prevent the harm of ban GMOs 12 14.1
GM crops on human health? audit should be done 8 9.4
the public should be informed 1 1.2
no answer 64 75.3
Should be GMO labelling be present in yes 85 100.0
food? no 0 0.0
Become a legal obligation of the GMO that yes 85 100.0
the information should be on the label no 0 0.0
to be continued
390
1 2 3 4
Should government promote organic farm- yes 84 98.8
ing? no 1 1.2
Should organic products be certified? yes 83 97.6
no 2 2.4
Should organic product sales be audited? yes 83 97.6
no 2 2.4
In Turkey the bans are not adequate to eliminate exposure of public to GMOs
because of certain distinguished regulations. However, GMOs in animal feed can be
imported legally.
We asked this in order to see whether the place where the student was raised af-
fected the attitudes or not. However, this did not affect the use of GMOs or checking
GMOs during buying (p = 0.586 and P = 0.990). Socioeconomic characteristics were
not related to the attitudes of students towards GMOs (Table 4).
Table 4. Association of GMO use and checking food labels for presence of GMO with sociodemographic
characteristics
Use of GMOs Yes No Significance
1 2 3 4 5
Use of GMOs
Gender male 9 9 X2 = 0.345*
female 41 26 P = 0.557
Age 21.64±1.257 21.97±1.150 U = 1072.500; Z = 1.939;
P = 0.053
Mostly lived in city 25 21 X2 = 1.067
town 18 9 P = 0.586
village 7 5
Accommodation house 30 27 X2 = 2.018
dormitory 20 8 P = 0.098
Household family 9 9 X2 = 2.765
members friends 33 24 P = 0.429
relatives 2 1
alone 6 1
Employment unemployed 44 32 X2 = 1.081
whole day 2 2 P = 0.582
half/day certain hours 4 1
Marital status married 4 1 P = 0.644**
divorced 46 34
divorced 52 28
to be continued
391
1 2 3 4 5
Checking presence of GMO during buying food
Gender male 5 13 P = 0.182*
female 32 35
Age 21.67±1.028 21.85±1.352 U = 931.000; Z = 0.419;
P = 0.675
Mostly lived in city 20 26 X2 = 1.533; P = 0.465
town 10 17
village 7 5
Accommodation house 27 30 X2 = 0.618; P = 0.432**
dormitory 10 18
Household mem-family 8 10 X2 = 0.871; P = 0.832
bers friends 26 31
relatives 1 2
alone 2 5
Employment unemployed 32 44 X2 = 1.700; P = 0.427
whole day 3 1
half /day certain hours 2 3
Gender married 3 2 P = 0.649*
divorced 34 46
*The Yates chi-square; **the Fisher chi-square; X2 – chi-square; Z – z-score; U – the Mann–Whitney
U-test; P – p-value.
In a survey examining attitudes of United Arab Emirates University (UAEU)

students towards biotechnology awareness in biotechnology literacy and environ-
mental domains were significantly different according to the enrolled college and the
academic achievement of the student. Aware groups most likely accepted accurate
biotechnology information delivered by reliable sources from internet or lectures;
they grasped their knowledge from surrounding people as a secondary source7. In
our study among the students 90.6% heard about GMO from TV and radio. Among
all students the rate of getting information from internet was 12.9%. Among all the
students the knowledge was derived from academic staff at a rate of 7.1%. Probably
the contents of the lessons will have to be re-assessed in order to cover this topic.
In a paper about GMO, Betts in 1999 (Ref. 8) claiming that contamination of
conventionally produced foods with genetically modified organisms (GMO) could
be more widespread than previously thought and addressed the need for labelling
regulations for GMO in foods in the USA. Foods can never be entirely safe as Borch-
ers et al.9 said. It is threatened by various pathogens, toxins, pollutants, chemicals,
genetically modification of organisms. Of course there are tools available for deter-
mining the GMO for foods. However, the huge number of type of food and widely
used genetically modification puts some risks for food safety. But, event a decade ago
392
easy ways to determine the detection of modified material in common food products
were under investigation10.
Data were collected from 3002 participants through an online survey administered
in Belgium, France, the Netherlands, Spain and the United Kingdom in 2013 revealed
that consumers are willing-to-pay a premium to avoid purchasing rice labelled as GM.
In all countries except Spain, consumers have a significantly higher willing-to-pay to
avoid consuming rice labelled as GM compared to rice labelled as cisgenic, suggesting
that inserting genes from the plant own gene pool is more acceptable to consumers.
The differences between transgenic and cisgenic products are recommended to be
reflected in GMO labelling and trade policies as the authors of the paper said11.
Majority of the Japanese university students are neutral to GMOs and GM food
use12. However, different trends are present among people as reported in a study that
enrolled French consumers. 35% of participants were unwilling to purchase products
made with GMOs, 23% were indifferent or value the presence of GMOs, and 42%
were willing to purchase them if they are sufficiently inexpensive. The study was
in a way reporting results unlike the common studies that generally showed people
attitudes against GMOs (Ref. 13). Finnish students in the study of Maher et al. (n =
3261) were evaluated for their attitudes to GMOs. Genetically modified food attitudes
in this study were mostly negative, and organic food attitudes were positive. The
results of the study indicated that organic food attitudes were associated more with
fundamental personal attributes than genetically modified food14.
Attitudes towards GMOs are largely affected by level of knowledge. This had been
reported in a study that involved education of consumers. After the education, 99% of
the participants correctly indicated that foods from biotech crops were currently on the
market. 90% of trained said that they would consume genetically-modified foods15.
In a sample (n = 750) of university students from three public universities in Costa
Rica a survey was carried out to determine the perception and knowledge about and
GMOs. The study revealed that 88% of the students showed a satisfactory level of
knowledge and 79% reported a favourable opinion and good acceptance of biotech-
nology. Students would accept some risks associated to biotechnology if it represents
an improvement to the competitiveness of Costa Rica. Also the students from social
disciplines showed a higher percentage of negative acceptances to biotechnology and
GMOs when their opinions were compared with those of students from life sciences
and technologies16. Also some herbal oils may have anti-mutagenic effect that needs
to be evaluated as being on the other side of the spectrum17.
A study aimed to assess knowledge connected with GMO and tendency to buy
genetically modified food instead of natural one in 745 medical students, in south
Poland. Terms connected with biotechnology and GMO were well known. Only the
term ‘genetically modified animals’ for 50% of surveyed individuals was unknown.
The majority neither preferred GMO food, nor had positive feelings. They were also
not satisfied with the knowledge about GMO that they had from school or university18.
In a commentary of Xia et al.19 alarm incidents about the safety of GMFs were dis-
393
cussed. In his commentary Xia addresses the prompt reaction of media. The process
of academic evaluation is too slow to counter act the effects of media alerts on a sci-
entific basis. He says ‘the scientific and expert opinions of the scientific community
should be effectively conveyed to the public and information from scientists should
be disseminated as widely and as loudly as that from the media’19.
In our study, majority of students think that GMF and GMOs are detrimental for
human health. A negative perception is present of GMOs. The socioeconomic charac-
teristics did not affect the attitudes of students towards GMOs. 100% of the students
think food labels should contain data related to presence of GMOs.
CONCLUSIONS
Majority of students think that GMF and GMOs are detrimental for human health. A
negative perception is present of GMOs. The socioeconomic characteristics did not
affect the attitudes of students towards GMOs. 100% of the students think food labels
should contain data related to presence of GMOs.
REFERENCES
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Plants with Overexpression of Dihydroflavonol Reductase Can Serve as Efficient Nutrition Sources.
J Agric Food Chem, 61 (27), 6743 (2013).
2. M. ZUK, A. PRESCHA, M. STRYCZEWSKA, J. SZOPA: Engineering Flax Plants to Increase
Their Antioxidant Capacity and Improve Oil Composition and Stability. J Agric Food Chem, 60
(19), 5003 (2012).
3. R. V ESPLEY, C. A. BUTTS, W. A. LAING, S. MARTELL, H. SMITH, T. K. MCGHIE, J. ZHANG,
G. PATURI, D. HEDDERLEY, A. BOVY, H. J. SCHOUTEN, J. PUTTERILL, A. C. ALLAN, R. P.
HELLENS: Dietary Flavonoids from Modified Apple Reduce Inflammation Markers and Modulate
Gut Microbiota in Mice. J Nutr, 144 (2), 146 (2014).
4. Y. H. KIM, Y. Y. LEE, Y. H. KIM, M. S. CHOI, K. H. JEONG, S. K. LEE, M. J. SEO, H. T. YUN,
C. K. LEE, W. H. KIM, S. C. LEE, S. K. PARK, H. M. PARK: Antioxidant Activity and Inhibition
of Lipid Peroxidation in Germinating Seeds of Transgenic Soybean Expressing OsHGGT. J Agric
Food Chem, 59 (2), 584 (2011).
5. M. BURTON, D. RIGBY, T. YOUNG, S. JAMES: Consumer Attitudes to Genetically Modified
Organisms in Food in the UK. Eur Rev Agric Econ, 28 (4), 479 (2001).
6. P. CELEC, M. KUKUCKOVA, V. RENCZESOVA, S. NATARAJAN, R. PALFFY, R. GARDLIK, J.
HODOSY, M. BEHULIAK, B. VLKOVA, G. MINARIK, T. SZEMES, S. STUCHLIK, J. TURNA:
Biological and Biomedical Aspects of Genetically Modified Food. Biomed Pharmacother, 59 (10),
531 (2005).
7. S. ABUQAMAR, Q. ALSHANNAG, A. SARTAWI, R. IRATNI: Educational Awareness of Bio-
technology Issues among Undergraduate Students at the United Arab Emirates University. Biochem
Mol Biol Educ, (2015).
8. K. S. BETTS: Growing Evidence of Widespread GMO Contamination. Environ Sci Technol, 33
(23), 484A (1999).
9. A. BORCHERS, S. S. TEUBER, C. L. KEEN, M. E. GERSHWIN: Food Safety. Clin Rev Allergy
Immunol, 39 (2), 95 (2010).
10. C. BRINEGAR D. LEVEE: A Simple Method for Detecting Genetically Modified Maize in Common
Food Products. Biochem Mol Biol Educ, 32 (1), 35 (2004).
394
11. A.-C. DELWAIDE, L. L. NALLEY, B. L. DIXON, D. M. DANFORTH, R. M. NAYGA, E. J. van
LOO, W. VERBEKE: Revisiting GMOs: Are There Differences in European Consumers’ Acceptance
and Valuation for Cisgenically vs Transgenically Bred Rice? PLoS One, 10 (5), e0126060 (2015).
12. F. MAEKAWA, D. MACER: How Japanese Students Reason about Agricultural Biotechnology. Sci
Eng Ethics, 10 (4), 705 (2004).
13. C. NOUSSAIR, S. ROBIN, B. RUFFIEUX: Do Consumers Really Refuse to Buy Genetically Modi-
fied Food? Econ J, 114 (492), 102 (2004).
14. M. SAHER, M. LINDEMAN, U.-K. K. HURSTI: Attitudes towards Genetically Modified and
Organic Foods. Appetite, 46 (3), 324 (2006).
15. C. R. SANTERRE K. L. MACHTMES: The Impact of Consumer Food Biotechnology Training on
Knowledge and Attitude. J Am Coll Nutr, 21 (3 Suppl), 174S (2002).
16. M. VALDEZ, I. RODRIGUEZ, A. SITTENFELD: Perception about Biotechnology in University
Students in Costa Rica. Rev Biol Trop, 52 (3), 745 (2004).
17. Sh. M. MUHAMMAD: Anti-mutagenic Effect of Ginger sp. and Rosemary sp. Oil on Chromosome
Abnormalities Induced by Colchicines in Pepper Root Tip. Oxid Commun, 1, 262 (2014).
18. J. ZAJAC, M. CHOMONCIK, E. KOLARZYK, D. OGONOWSKA: Controversial Issue in Bio-
technology-Students’ Opinions. Przeglad Lek, 69 (8), 459 (2012) (in Polish).
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vestigations Should Speak Louder during Controversies over Safety. Biosci Trends, 9 (2), 134 (2015).
Received 17 May 2015
Revised 9 July 2015
395
Overall ecology
A FACILE METHOD FOR SYNTHESISING

2-HYDROXY-3,6-DICHLOROBENZOIC ACID
FROM 2,5-DICHLORO-PHENOL
WENHUA OUa, HONG HUANGb*

a
School of Perfume and Technology, Shanghai Institute of Technology,
100 Haiquan Road, 201 418 Shanghai, China
b
College of Marine Science, Shanghai Ocean University, Shanghai, China
E-mail: ouwenhua@sit.edu.cn; hhuang@shou.edu.cn
ABSTRACT
A facile synthesis of 2-hydroxy-3,6-dichlorobenzoic acid was described. 2-Hy-
droxy-3,6-dichlorobenzoic acid was prepared by the Reimer-Tiemann reaction of
2,5-dichloro-phenol with trichloromethane, and then by oxidation with potassium
permanganate. 2-Hydroxy-3,6-dichlorobenzoic acid was obtained easily with a total
yield of 43% after 2 linear steps from 2,5-dichloroaniline as raw material.
Keywords: the Reimer-Tiemann reaction, 2,5-dichloroaniline, oxidation, 2-hydroxy-
3,6-dichlorobenzoic acid.
AIMS AND BACKGROUND

Dicamba is a benzoic acid herbicide used to control noxious and nuisance weeds and
brush. It mimics a plant growth hormone, causing uncontrolled and disorganised plant
growth that leads to plant death. Since dicamba and the similar structure compounds
were introduced by Monsantoin 1996, genetically engineered Roundup Ready® (RR)
crops revolutionised weed management and no-till practices in agronomic cropping
systems. RR crops were resistant to the herbicide glyphosate, which meant that
producers could apply this one herbicide postemergence during the crop season and
achieve excellent, broadspectrum weed control.
Due to the sharp increase in the number of agricultural production, the need for
dicamba to control the growth of weeds in agriculture is growing. 2-Hydroxy-3,6-
dichlorobenzoic acid is a key intermediate in the synthesis of dicamba. Considerable
effort has been spent on developing simple and practical synthetic routes for the
preparation of 2-hydroxy-3,6-dichlorobenzoic acid.
*
For correspondence.
396
Although several synthetic approaches to 2-hydroxy-3,6-dichlorobenzoic acid
have been reported1–9, they require the use of metal-containing catalysts, high pres-
sure and special chemical reaction equipment (the autoclave). Thus, the procedure
is relatively complex, dangerous and the cost of product is too much. The procedure
described by us ensures preparation of 2-hydroxy-3,6-dichlorobenzoic acid in two steps
with good yields with overcoming this disadvantage. 2-Hydroxy-3,6-dichlorobenzal
dehyde was obtained by the Reimer-Tiemann reaction of 2,5-dichloro-phenol with
trichloromethane. 2-Hydroxy-3,6-dichlorobenzaldehyde was oxidised easily by the
similar process reported10. Here we used K4MnO4 and phase-transfer catalyst (Fig. 1).
Cl Cl Cl
OH CHCl 3 OH KMnO4 OH
Ca(OH) 2 H OH
Cl Cl O Cl O
Fig. 1. Synthetic route of 2-hydroxy-3,6-dichlorobenzoic acid
EXPERIMENTAL
Melting points were determined with a SGW X-4 micro melting point apparatus. The
1
H NMR spectra were obtained on a Bruker AM-400 instrument at 400MHz, using
CDCl3 as solvent and tetramethylsilane as internal reference. The elemental analyses
were obtained on a Vario El Cube analyser.
3,6-Dichloro-2-hydroxybenzaldehyde. To an aqueous mixture of calcium hydroxide
(10.5 g, 0.14 mol), sodium carbonate (11.5 g, 0.11 mol) and 2,5-dichlorophenol (5.22 g,
32 mmol), chloroform (12 g, 0.1 mol) was added dropwise with stirring for over 2 h.
Heating and stirring were continued for additional 6 h. The mixture was acidified
with concentrated hydrochloric acid and steam distilled. The distillate was cooled and
the solid that separates was recrystallised from ethanol to afford 1.83 g (yield, 63%)
of 3, 6-dichloro-2-hydroxybenzaldehyde. M.P. 97–99°C (m.p. 97–99°C (Ref. 11)).
1
H NMR (400 MHz, CDCl3): δ 6.92–6.94 (d, J = 8 Hz, 1H), 7.50–7.54 (d, J = 8 Hz,
1H), 10.38 (s, 1H) 12.43 (s, 1H). Found, %: C, 44.35; H, 2.02; Cl, 38.52; O, 15.11.
C7H4Cl2O2. Calculated, %: C, 44.02; H, 2.11; Cl, 37.12; O, 16.75.
3,6-Dichloro-2-hydroxybenzoic acid. Potassium permanganate (8.7 g, 55 mmol) was
added in three portions at 3-hour intervals to a vigorously stirred mixture of aldehyde
(3.4 g, 17.8 mmol), H2O (50 ml), toluene (100 ml), and n-Bu4N+I- (0.5 g) at 20°C.
After the purple mixture was stirred for a total of 8 h, NaHSO3 was added, followed
by acidification with HCl. The light yellow organic layer was separated, washed
with saturated brine, and dried. Evaporation of solvent provided 2.5 g (yield, 68%).
M.P. 194–196°C (m.p. 193–195°C (Ref. 3)). 1H NMR (CDCl3), δ, ppm: 3.90 (s, 1H),
6.98–7.00 (d, J = 8 Hz, 1H), 7.43–7. 45 (d, J = 8 Hz, 1H), 13.90 (s, 1H). Found, %:
C, 41.02; H, 1.87; Cl, 35.62; O, 14.96. C7H4Cl2O3. Calculated, %: C, 40.62; H, 1.95;
Cl, 34.25; O, 21.49.
397
Accessible starting compound for the synthesis of 2-hydroxy-3,6-dichlorobenzoic
acid is 2,5-dichlorophenol which can be prepared easily from 2,5-dichloroaniline
as described in Ref. 11. We made an attempt to synthesise 3,6-dichloro-2-hy-
droxybenzaldehyde by the Reimer-Tiemann reaction of 2,5-dichlorophenol with
trichloromethane under alkaline conditions firstly, and then obtain pure 3,6-dichloro-
2-hydroxybenzaldehyde by steam distilling and recrystallisation. Its processing is
relatively simple and easy. 2-Hydroxy-3,6-dichlorobenzoic acid was obtained by
oxidation of 3,6-dichloro-2-hydroxybenzaldehyde with potassium permanganate.
Compared with the conventional methods, the procedure described by us does not
need expensive metal-containing catalysts, high pressure and using common chemical
reaction equipments. The described reaction is of preparative value for the synthesis
of 2-hydroxy-3,6-dichlorobenzoic acid.
CONCLUSIONS
In conclusion, a facile and practical approach to 2-hydroxy-3,6-dichlorobenzoic acid
has been developed by the Reimer-Tiemann reaction and oxidation. Its ease of work-
up, fairly mild reaction conditions provide an improved access to 2-hydroxy-3,6-
dichlorobenzoic acid. The overall yield of the route is 43%. Other applications of this
process are under investigation in our laboratory.
REFERENCES
1. T. ONOUCHI: Preparation of 3,6-Dichloro-2-hydroxybenzoic Acid and Their Intermediates as
Precursor for Herbicide 3,6-Dichloro-2-methoxybenzoic Acid. JP Patent No 10007615, 1998.
2. Y. ZHANG: Study on the o-alkylation for 3,6-dichlorosalicylic Acid by Chloromeththane. Chemical
Industry Times, 15 (9), 27 (2001).
3. Y. ZHANG, M. LU: The Synthesis of Herbicides Dicamba. Pesticides, 41 (11), 13 (2002).
4. M. Y. MENG, Y. MA, L. Z. TAN, W. D. YANG: Synthesis of 3,6-dichlorosalicylic Acid. Fine and
Specialty Chemicals, 11 (15), 17 (2003).
5. M. Y. MENG, L. Z. TAN, W. D. YANG: New Technology for Synthesis of Aromatic Hydroxycar-
boxylic Acids by Carboxylation in Solvent. Dyes Dyeing, 41 (6), 373 (2004).
6. Z. C. YAN: Study on the Synthesis of 3,6-Dichlorosalicylic Acid. Chem Prod Technol, 10 (2), 8 (2003).
7. X. L. LIU, W. HONG: Supercritical Process for Preparing Herbicide Dicamba with 2,5-Dichloro-
phenol as Raw Material. CN Patent No 102125035, 2011.
8. Z. M. CHENG, X. HE CHU, X. X. YAN, Z. B. YU, Y. W. JIANG, M.W. CHEN, R. H. AI: Prepara-
tion of 3,6-Dichloro-2-hydroxybenzoic Acid with Improved Yield. CN Patent No 102295552, 2011.
9. G. Q. SUN, Y. S. HOU, G. Y. CHEN, Z. Q. LI, Z. J. ZOU, J. ZHANG: Method for Preparing 3,6-Di-
chlorosalicylic Acid. CN Patent No 102838482, 2012.
10. B. L. HIRAN, J. KHUNTWAL, R. K. MALKANI: Pyridinium Fluorochromate Oxidation of Ben-
zaldehyde in N,N-dimethyl Formamide Medium. Kinetics and Mechanism. Oxid Commun, 36 (3),
612 (2013).
11. B. C. LIU, R. Y. JIANG: Improved Process of Diazotization in Preration of Dicamba. CN Patent
No 1830943, 2006.
Received 24 February 2015
398
European legislation
FLOOD RISK MANAGEMENT ANALYSIS FOR REDUCING

HARMFUL EFFECTS ON HUMAN HEALTH, ENVIRONMENT,
CULTURAL HERITAGE AND ECONOMIC ACTIVITY IN THE
REPUBLIC OF SERBIA
M. DUKIC MIJATOVIC*, Z. BJELAJAC, V. KOZAR

Law Faculty of Economics and Justice, University of Business Academy, Novi Sad,
Serbia
E-mail: marijana.dukic.mijatovic@gmail.com; zdjbjelajac@gmail.com;
kozarv@yahoo.com
ABSTRACT
Catastrophic floods are the result of interaction of hydrological phenomena and natural
processes, social and economic environment. Due to influence of climate change, such
extreme hydrological phenomena will be more frequent. Destructive nature of water
effects can cause human casualties, evacuation and damage to the environment; it
can seriously endanger economic development and undermine economic activities.
Therefore, maintaining of natural and economic balance must be primary goal which
requires integrated approach to flood management. The latest flood events in Europe
and especially catastrophic natural disaster in Serbia in 2014 lead us to conclusion
that it is feasible and necessary to reduce risk of flood by practical combinations of
measures in relation to the scope of disaster and degree of exposure to community.
In this regard, one needs clear understanding of existing and potential flood risks in
order to determine preventive reduction measures which represent approach to flood
management. This work, on the occasion of increasingly frequent large scale of floods
in Serbia and neighbouring countries in 2014, suggests that human responsibility and
natural solutions are the best way to reduce the consequences of floods.
Keywords: floods, flood risks, flood risk management, environment, human health.
AIMS AND BACKGROUND

Water is source of life on Earth and it is one of the simplest and most widely used
materials in nature whose physical and chemical characteristics are well known. Some
of these characteristics are unique which is why water is so significant for life suste-
*
For correspondence.
399
nance on our planet. It is usually said that life actually originated in water and exists
thanks to water. Hydrosphere, as only one part of the global ecological system, has
enabled the emergence of biosphere. If we add up all the oceans, seas, rivers and lakes,
water occupies a huge part of the Earth surface. The paradox is that only a small part
of the enormous quantity of water is available to people in terms that it can be used
for drinking and other purposes1. Total amount of water on our planet is estimated at
about 1400 million km3, out of which only 2.5% are fresh water; and even from that
amount only 20% are conducive for human usage with a relatively small repair (clean-
ing and disinfection). Only 2.6 billion people on Earth have minimum sanitary condi-
tions related to water supply, while about half population in developing countries
suffer from diseases caused by defective drinking water2. Except for drinking, people
need water to prepare food, for hygiene, as well as for many processes in industry
and agriculture. In the world water consumption, agriculture has a share of 90%, while
industry and households have 5% (Ref. 3). Chemicals play an important role in
global economy and they are being used in many products4. Groundwater pollution
occurs because of agricultural activities which involve usage of artificial manure5.
Water is far the richest component of all living organisms and it is of fundamental
importance in maintaining both structure and function of all tissues, e.g. cells as basic
units of living matter. Not drinking water leads to death faster than not consuming
food6. Not drinking water leads to death within a few days, after the body loses 10–20%
of its total liquid volume. In case of not eating, life can be held for a few weeks,
despite the loss of all body fat and about 50% of tissue proteins7. Water content in
adult male is 60 ± 15%, and in women it is 55 ± 15%, meaning that water is one of
basic conditions for human survival and life on Earth. Depending on climate condi-
tions, water consumption for human survival ranges from 3–12 l/day. Water is of great
importance for life of animals. Water is primordial environment in which life arose,
so it is understandable that there is mutual relationship, especially for the animals
whose habitat is water. Water is found inside the animals bodies. Water content in
animals bodies varies from 50–93%, while aquatic organisms have the highest water
content8. Water is very important for plants. Aquatic plants are easiest to supply with
water. Many useful substances are dissolved in water, so plants absorb them easily.
However, plants on land can not reach water easily sometimes because they cannot
drain it from the soil9. Integrated water resources management is a complex and dif-
ficult task which includes measures and activities aimed at maintaining and improv-
ing the water regime, providing required quantities of water of different qualities for
various purposes, water pollution protection and protection from damaging effects of
water. Regulation of water and protection from the harmful effects of water is one of
three water activities and activities of general interest. Regulation of water includes
the construction and maintenance of water facilities for water flow regulation (control
objects) and works on the maintenance of stability of shores and riverbeds and main-
taining its bandwidth usage for water, ice and drift. Protection against harmful effects
of water includes works and measures on flood protection from external and internal
400
water and ice, protection from erosion and torrents and works to eliminate the adverse
impact of floods on water facilities and large water troughs10. Risk management from
harmful water effects includes development of a preliminary assessment of flood risks,
elaboration and implementation of flood risk management, general and operational
plans for flood protection, the implementation of regular and emergency flood protec-
tion and protection against erosion and flood11. The thought that man can conquer
nature has always been denied by natural disasters. Recent floods on the territory of
the Republic of Serbia and neighbouring countries tell the opposite. People usually
say that floods caused by rivers are natural phenomena like blood circulation in hu-
mans, and flood damages are humans deed. The economic losses based on cata-
strophic events, from 1970 to the beginning of the 90s of 20th century, increased at
an average rate of 22.3%, which accounted to 22 billion US dollars average per year.
Unlike the average growth rates of economic damages, the average annual growth
rate of damage from the 90s of 20th century to 2013 was 31.2%, and their average
annual amount reached even 147 billion US dollars (Ref. 12). There has been more
than a hundred years of traditional flood defense, billions of dollars spent on techni-
cal approach and floods still continue to bring large-scale disasters. Is it proof of ir-
responsibility and ineffectiveness of modern man in conjunction with the lack of
understanding of complex hydrology and dynamics of rivers, due to the impact of
global climate change? It probably is. That is why people today speak more and more
about water management as one of the most important challenges the world is facing,
including the Western Balkan region, especially after the devastating floods in 2014.
These catastrophic events, especially in Serbia, warn us how essential working on
designing and implementing effective policies and programs for disaster risk reduc-
tion are, by presenting accurate, appropriate and customised information on losses,
hazards, vulnerable aspects and risks. According by the National Strategy for protec-
tion and rescue in emergency situations, in Serbia between 1900–1940 years, 100
natural disasters occurred in a decade alone. The growth rate of these natural disasters
increased from 1960 to 1970, when it was almost seven times higher, and from 1980
to 1990 up to 2000 natural disasters hit Serbia. From 1990 to 2000, the number of
natural disasters in Serbia has increased to 2800 (Ref. 13). The floods were estimated
to have caused effects that are equivalent to 2.7% of GDP in damages and to 2% of
GDP in losses in 2014. The hardest hit economic sectors were energy, mining, and
agriculture but significant damages were also inflicted on transport infrastructure
(roads, bridges and railways)14. Most of the world top scientists do not deny that by
2050, due to warming and sea level rise, rainfalls will be much heavier, winds will
be stronger, and floods in the Old Continent will be more devastating and more often.
There are disagreements only about predicting proportions of natural disasters. It is
no news that by mid-century, waterbeds will pour out more often, but the news is that
floods across the continent will be more connected to one another, as newly published
researches show. So far, studies have been limited to individual river basins, but the
study published in the specialised journal ‘Nature Climate Change’ indicates that the
401
outpouring of riverbed of one basin will hit a lot more other regions and basins across
the Old Continent. Brenden Jongman from the Amsterdam University, the leading
author of this multidisciplinary research indicates that in the analysis of changes in
rain models was concluded that floods will become pan-European problem and that
by the mid-century they will cause third of losses in national companies. For com-
parison, in the first 12 years of this century, as pointed out by this study, the average
cost due to flooding in the EU amounted to about 4.9 billion Euros a year. Based on
projections on the amount of rainfall in the following decades and with existing flood
defense, the costs of the EU will increase to 23.5 billion Euros a year by 2050 (Ref.
15). Therefore it is not surprising that policy of the EU considering flood risk manage-
ment occupies such an important place, based on several significant documents,
whereby we shall refer to Directive 2007/60/EC on the assessment and management
of flood risks. The most important guidelines of the Community European Law refer-
ring to water quality and quantity are those which govern basic waters and their
tributaries, guidelines of European Electronic Network Data on qualitative and quan-
titative waters conditions and Guidelines on Public Participation in the Creation of
Water Plans for Improving Water Conditions. In practice of legally guaranteed subjec-
tive environmental rights, the water rights guidelines are expected to enable judicial
protection, i.e. access not only to administrative, but also legal way. There are numer-
ous European guidelines that apply to all elements of environment, and accordingly
to waters as well. Thus, a specific European guideline requests an opportunity to
review public and private projects from the point of environmental tolerance. The
Guideline on Environmental Liability for Preventing and Remedying Environmental
Damage is of great legal significance. The Regulation on Free Access of Organisations
to the System for Ecological Management and Testing Ecology Functioning is also
important (EMAS, I–III). Guidelines of European law in the field of ecology are
landmarks for the implementation of common environmental objectives into na-
tional law16. Directive 2007/60/EC on Flood Assessment and Management17 aims to
establish a framework for assessing and managing flood risks to reduce the adverse
impact of floods in community for human health, environment, cultural heritage and
economic activity (Article 1). The term ‘flood’ means temporary covering by water
of land not normally covered by water. These include floods from rivers, streams,
torrential streams, and floods from the sea in coastal areas, etc. According to it, the
‘flood risk’ means the combination of probability of flood events and the possible
harmful consequences of flood events on human health, environment, cultural herit-
age and economic activity.
402
Fig. 1. Flood risks management in the EU (Ref. 18)
On the graph shown (Fig. 1) is clear that there are different types of floods, starting
from coastal, over river, rain and other subtypes which depends on its strength in the
short term, fatal injury, economic loss, damage to the environment and the financial
crisis losses, while in the long term it can all be multiplied. Factors that cause it are
numerous, ranging from climate change to the ones directly created by human. Ad-
equate access to prevention, both in the segment of preventing floods, providing funds,
which eliminate the causes of flood, and monitoring of the same by experts; certainly,
harmful consequences of flooding can be reduced, but the emphasis is on a series of
measures such as informing the population, sounding critical points, civil protection
and reconstruction by insurance and social responsibility. Directive 2000/60/EC of
the European Parliament and of the Council of 23rd of October 2000 which lays down
the framework for action in the field of water policies19 requires the management plan
for each river basin in order to achieve good ecological and chemical status, which
will contribute to mitigate the effects of flooding; the more so, because floods are one
of the most widespread climatic hazards and risks to human health, environment and
economy. Given the possibility that the danger of flooding could increase as a result of
climate change, the Directive stipulates that it is necessary to make timely integrated
strategic assessment of flood risk. Solvency II (Directive 2009/138/EC) – as amended
by Directive 2014/51/EU (‘Omnibus II’) – replaces 14 existing directives commonly
known as ‘Solvency I’. The Solvency II Directive is an EU Directive codifying and
harmonising the EU insurance regulation. Primarily this concerns the amount of capital
that EU insurance companies must hold to reduce the risk of insolvency. Often called
‘Basel for insurers’, Solvency II is somewhat similar to the banking regulations of
Basel II (Ref. 20). The Solvency II Directive had to be transposed by Member States
into national law before 31 March 2015. On 1 April 2015, a number of early approval
processes were start, such as the approval process for insurers internal models to cal-
culate their Solvency Capital Requirement. The Solvency II regime will become fully
applicable on 1 January 2016. This timeline – in parallel with EIOPA set of guidelines
403
on preparing for Solvency II – allows supervisors and undertakings to prepare for the
application of the new regime. In addition, Solvency II includes a number of measures
to ensure a smooth transition from Solvency I, mostly: two measures on the valuation
of technical provisions, helping the transition to a market-consistent regime over 16
years; tolerance for insurers breaching the Solvency Capital Requirement within the
first two years; grandfathering of existing hybrid own-fund items that are eligible
under Solvency I, making it easier to meet the new capital requirements and giving
the industry 10 years to adapt the composition of its capital to Solvency II standards
and longer deadlines to report quarterly and annual information to supervisors and to
disclose reports to the public, decreasing gradually from 20 weeks to 14 weeks after
the close of the reporting period over the first 3 financial years21. Only the most senior
tranches may qualify for the favorable capital treatment of high-quality securitisation
positions. These senior tranches provide credit enhancement, in other words, their
credit risk is lower than the credit risk in the entire pool of underlying exposures. It
makes sense from an economic point of view that risk factors for high-quality senior
securitisation positions are no higher than those applicable to the underlying securi-
tised exposures if they were held directly by insurers.

The fact is that because of the dramatic consequences of climate change, we need to
adapt not only to warmer summers, but also to possible natural disasters, major storms
and heavy rains. We are being witnesses that in certain places the amount of rainfalls
in an hour is proportionate to the amount of the same in a month. In such situations,
small streams can be transformed into powerful torrents. The catastrophic floods that
have afflicted Serbia in 2014 probably could not be completely avoided. In fact, there
is no total safety from the dangers and possible damage, and it probably will not exist
in the future either. Despite the experience of human resources and modern equipment,
it may not be successfully synchronised to operate at multiple-hit places. However, it
is definitely possible to regard the timely implementation of precautionary measures,
in order to keep the flood consequences under control. This has been confirmed by
the experiences from recent years, showing that it is very important that the residents
of affected areas estimate the risks and as soon as possible take measures to protect
themselves from the flood risks. Cyclone ‘Tamara’ (as meteorologists named it)
spread in Central and South-Eastern Europe on 13th May 2014. It was spreading on
a large horizontal surface with vertical thicknesses of up to 100 km through the entire
troposphere. Saturation of air masses was about 100% and humidity was increased
due to warm air from south and east. In support to the development of this field of
low pressure was conducive to the physical and geographic specificity of the Balkan
Peninsula. Centre of the cyclonic field was above Serbia and Bosnia and Herzegovina
from 13th to 15th of May where a large amount of rainfall was extracted, the highest
ever recorded since the water meteorological observations started. Weakening and
404
disappearance of cyclone started during the 16th of May (Ref. 22). Obrenovac in
Serbia was mostly hit by the flood; it is estimated that 90% of the settlements were
submerged. The entire settlement of about 8700 inhabitants was evacuated23. The
Power Plant ‘Nikola Tesla’, the largest thermal power plant in Serbia which provides
nearly 50% of electricity for the country, is nearby Obrenovac. However, it remained
afloat thanks to good interventions. The Thermal Power Plant Kostolac which provides
11% of electricity in Serbia was endangered by the outpouring of the river Mlava.
Many other towns were threatened and flooded: Paracin, Petrovac, Bela Palanka,
especially Krupanj, where structures were threatened by floods and landslides. The
flood wave on the river Sava near Sabac reached the corner of 6.6 m, which is the
highest level of the river in the city since records have been kept. The emergency on
the entire territory of the Republic of Serbia was in force from 15th to 23rd of May.
During the nine days of fighting against flood, the water element in the territory of
the country caused total damage estimated at one billion and 532 million Euros. The
floods have inflicted great damage to the energy sector whose repair takes more than
210 million Euros, while the amount of damage in the housing sector is estimated at
227 million Euros. Water and landslides have led to the complete destruction of more
than 400 housing units and nearly 17 000 apartments and housing units suffered partial
damage; 74 health care facilities were damaged, including clinics, health centres and
clinics, as well as 35 nursery, elementary and secondary schools. The Government
of the Republic of Serbia, according to the official methodology for assessing needs
of the EU, the UN and the World Bank, from 10th of June to 10th of July 2014 drew
up an assessment report on the needs for recovery and reconstruction of the flooded
areas24. In Serbia, according to the World Food Program of the UN, about 600 000 of
its citizens out of 7.2 million people were affected by the consequences of flooding
after heavy rains, the hardest in the Balkans in the last 120 years (Ref. 25). Accord-
ing to the Government of the Republic of Slovenia, there were found 51 victims and
an enormous number of birds were killed, therefore large-scale epidemic threatened
because of the hundreds of tons of dead animals and estuary sewage.
Fig. 2. Obrenovac – flooded town26
405
Fig. 3. Consequences of flood in Krupanj 27
Fig. 4. Reflection of the catastrophe which befell Serbia28
Figures 2, 3 and 4 show all flood horrors of biblical proportions, which occurred
in Serbia in May 2014; they should certainly be reminders to take all possible meas-
ures to ensure that this situation does not happen again. The disaster did not bypassed
countries in the region; a similar situation existed in Bosnia and Herzegovina29. Dozens
of cities and villages were cut off from more than 2000 landslides. More than 100 000
people were evacuated, and it was the biggest exodus since the end of the 1992–1995
war. In neighbouring Croatia, thousands of people were evacuated along the Sava
River30. As in many other disastrous situations, quite rightly raises the question if
the human factor is to be blamed, was the flood risk managed adequately? Perhaps
because of the extremely heavy rains caused by the static cyclone above this part of
Europe, the disastrous floods could not be prevented; though if people reacted on
time and invested into prevention, the consequences would have been less. It is sim-
ply incomprehensible that people in Obrenovac, in spite of pledges by the Republic
Hydro-meteorological Service expected the catastrophe without prepared sandbags.
406
In fact, three days before the disaster, namely on 13th of May 2014 were forecasted
heavy rains and rising water levels in the rivers. Serbia which aspires to EU member-
ship must harmonize its legislation with European standards in all spheres, including
water management, development and use of water and water protection. The Law on
Water of the Republic of Serbia31 regulates the legal status of waters, integrated water
management, management of water facilities and water land, resources and funding of
water activities, supervision of the implementation of this law, as well as other issues of
importance to water management. Also, under this Act, the competent Ministry issued
the Ordinance on Determining the Methodology for Making a Preliminary Assess-
ment of Flood Risks32. Article 2 of this Ordinance, a preliminary assessment of flood
risks was made to assess existing or potential flood risk, based on the available data
on floods from the past and analysis of long-term tendencies that affect the level of
flood risk, including climate change. Preliminary assessment of flood risks considered
significant flooding in the past, the likelihood of similar flood events in the future, the
potential adverse consequences of future floods and maps showing the available data.
The potential adverse consequences of future floods to human health, the environment,
cultural heritage and economic activity were estimated based on topography, type
and land use, hydrological characteristics of the channel, the effectiveness of flood
protection, the position of populated areas and areas of economic activity, long-term
development plans and the impact of climate change (Article 7). Harmonisation of
national regulations of the Republic of Serbia with the EU regulations implies the
respect and complies with the provisions of Directive 2007/60/EC on the assessment
and management of flood risks. In accordance with Community legislation, Member
States are obliged to make the preliminary risk available to the public, flood hazard
maps and flood risk management plans, and to encourage the active participation of
stakeholders in the production, review and updating of the flood risk management
plans (Article10). It is indisputable that, as in many other areas, this important seg-
ment is an evident gap between the normative and real, both at the EU level and at
the level of individual countries. Regulations are being made, and one can say that
they adequately reflect the essential content and the real state of things. However, the
problem is in their unenforceability and in high degree of tolerance towards persons
and organisations for the damage they make to the community, the health of people
and the environment, their irresponsibility, whether by their acting or omission of
acting. Since the consequences of catastrophic risk are very large, and can be fatal for
the society, practice introduced two basic models, with more variations, by which the
state struggles with the consequences of catastrophic risks. In the first model the state
does not participate in the coverage of damage incurred as a result of catastrophic
events, but the damages are covered by contractual insurance. This type of model is
applied to countries with highly developed infrastructure and a market economy and
countries without significant natural disasters such as earthquakes, tornadoes, and all
under the condition that the insurance industry is highly developed, what is one of the
main conditions for implementation of Solvency II. In terms of implementation costs,
407
the one-off net cost of implementing Solvency II for the whole EU insurance industry
has been assessed to be around EUR 3 billion to EUR 4 billion, which is relatively
small compared to the annual turnover of the sector (around EUR 1.1 trillion of written
premiums). In terms of capital requirements, taking into account the so-called ‘long-
term guarantees package’ in the Omnibus II Directive, the aggregate available surplus
(free own funds above the capital requirements of each insurer) is likely to be broadly
identical to the aggregate situation under Solvency I. However, the distribution of
capital requirements across undertakings will reflect more accurately individual risks,
leading to a more efficient allocation of capital in the EU (Ref. 34). Unlike the first, in
the second model, the states directly or indirectly participate in the resolution of claims
incurred, despite the existence of contractual insurance. Characteristic of this model is
that the state after the manifestation of catastrophic events and determining the cause
and extent of damage and claims of subjects who suffered damage intervenes from its
budget to reverse the consequences arising from the manifestation of these risks. A
variation on this model involves the establishment of special insurance organisations
that are state-owned through direct insurance or reinsurance; affect the creation of
special funds from which they compensate the damage, as for example in France, in
the USA and Japan33. The main source of the EU law which establishes certain rules
in the field of criminal responsibility is Directive 2008/99/EC on the protection of
the environment through criminal law which lays down the measures that Member
States should undertake in the field of criminal law in order to effectively protect the
environment which in practice almost has no purpose, because the criminal liability
as a rule is absent or only in exceptional circumstances applied.
CONCLUSIONS
The unprecedented water floods in Serbia, in addition to large-scale disasters, brought
a number of important lessons in the context of flood risk management. Firstly, the
management of flood risks should be carried out in a timely and responsible manner,
while the defense systems on the rivers must be strengthened, so the water apocalypse
would not be repeated. It is necessary to restore the existing systems as soon as pos-
sible to the normal state, so cut up levees could not be beaten again by less water,
and in order to make all these activities successfully, they must follow the solutions
of those countries that have shown the most experience in flood protection. Except
the so-called ‘rabbit levees’, they should also use mobile metal barriers as defensive
equipment designed for shorter sections which have several advantages because they
can be installed in very short time to prevent water penetration. Mobile fence can
be practically hung on the previously installed metal posts. It is possible to install
metal wall with a length up to 400 m in a day. When the flood is over, fence can be
uninstalled and put in storage. However, the safest solution is to maintain a stable
defense system. Unfortunately, in some parts of Serbia strong defense systems were
not built because the floods were rare. In many places, the flood protection systems
408
are neglected. It is impossible to move around the dikes in these neglected places.
Weed covers large areas, so the critical points of the embankment are unobservable,
which slows timely response. Likewise, we should consider the type of flooding since
the lowland rivers are easily tamed for their level rises slowly, while the mountain,
torrential rivers rise rapidly and there is no much time for reacting. In the mountain
areas of Serbia we should develop and strengthen active flood system to keep water
in the top part of the course, to build dams followed by the next level of defense –
embankments. However, above all it is necessary to determine the responsibility of
individuals for negligent and unprofessional performance at work, which contributed
to the suffering of people and material damage of huge proportions. Unfortunately,
this kind of reaction in Serbia has failed. Finally, the complete harmonisation with
EU legislation, especially implementation of Solvency II and consistent application
of regulations, preventive measures and investments in systems for flood protection
and flood prevention measures should be a priority task, as well as ensuring public
participation in water plans in terms of network records of quality and quantity of the
state of primary waters. But first of all, our society has to make strong confidence of
our citizens regarding benefits of insurance and to develop their customs to conclude
insurance agreements. All the more so, because it is necessary to invest a lot of money
to repair the damage caused by floods, and many times less to build good defense
systems that would help to significantly limit the losses.
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Canals. Yearbook of the Faculty of Agriculture, 31 (1), page 90–97 (2007).
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Stormy Winds and Hailstorms. J Appl Eng Sci, 12 (2), 137 (2014).
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XII/1.ppt. p.7 visited on 05.11.2015.
13. I. PAVLOVIC: Insurance against Natural Disasters. The World of Insurance, (3), (2012).
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15. http://www.nature.com/nclimate visited on 26.07.2015.
16. J. SALMA: The Legal Instruments for Protection against Waters (Floods and Drought) and Water
Protection. Proceedings of the Faculty of Law, Novi Sad, 47 (3), 27 (2013).
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Assessment and Management of Flood risks (OJL, 288, 6.11.2007, pp.27–34).
18. http://waterissues.eu/tag/floods/ visited on 18.07.2015.
19. OJ L 327, 22.12.2000, p.1 Directive was amended by Decision No 2455/2001/EC (OJ L 331,
15.12.2001, p. 1.
20. http://treasurypeer.com/dummies-2/solvency-iifordummies/#sthash.9pNUl2cw.dpuf, visited on
06.11.2015.
21. https://www.ashurst.com/doc.aspx?id_Content=12078, visited on 06.11.2015.
22. http://serbianmeteo.com/ciklon-koji-je-usao-u-istoriju-ciklon-tamara-13-16-maj-2014/ visited on
18.05.2015.
23. http://www.cnn.com/2014/05/19/world/europe/balkans-flooding/ visited on 20.05.2015.
24. http://www.srbija.gov.rs/vesti/specijal.php%3Fid%3D209591 visited on 20.06.2015.
25. http://www.rs.one.un.org/organizations//UN%2520Floods-FINAL%252010.pdf visited on
01.07.2015.
26. www.rtv.rs/.../baric-evakuisan-u-obrenovcu-spasavanja-i-tok/4/06/2015 visited on 21.07.2015.
27. www.dw.com/23/06/2015. visited on 21.07.2015.
28. http://www.tvbest.rs/52819-samo-da-prezivimo-pas-pliva-u-bujici-maca-mu-na-ledjima/1/06/2015
visited on 22.07.2015.
29. M. NOGIC, M. SCIBAN, M. SAVIC, B. JOLOVIC: Water Quality at the Source in the Municipality
Laktasi, Bosnia and Herzegovina. Oxid Commun, 35 (4), 1065 (2014).
30. http://www.duzs.hr/page.aspx%3FPageID%3D677, visited on 19.07.2015.
31. Law on Waters (‘Official Gazette’ No 30/10 and 93/12).
32. http://www.rdvode.gov.rs/lat/uredjenje-vodotoka-pp-rizika-poplava.php visited on 23.07.2015.
33. http://europa.eu/rapid/press-release_MEMO-15-3120_en.htm, visited on 07.11.2015.
34. http://www.insuranceage.co.uk/insurance-age/news/1184849/european-insurers-expect-modest-cost-
solvency-ii-compliance, visited on 07.11.2015.
410
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ANTIOXIDANT ACTIVITY OF DIOSGENIN FROM Dioscorea

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Liquid-phase oxidation: hydrogen peroxide as oxidiser

EFFECT OF ETO AND HYDROGEN PEROXIDE PLASMA

HUANG HAOa, ZHOU XIAOLIa*, QIN NIANa, HE XIAOYANa,

AIMS AND BACKGROUND

INFLUENCE OF METHANOGENS AND METHANOTROPHS

YANG KEa, JI HONGb,c*, XING ZHIXIANGa, LIU GECHENa, YU SIWEIa,

AIMS AND BACKGROUND

PLANT–SOIL ECOLOGICAL SYSTEM CONSTRUCTION

SAMPLE COLLECTION AND PROCESSING

Table 1. Oligonucleotide probes and relevant characteristics

RESULTS AND DISCUSSION

Fig. 1. Distribution of ORP in soil-plant system

Fig. 2. Comparison of methanogens amounts by different probes

Fig. 3. Effect of plant species on methanogens distribution

amount of methanotroph × 108 (g–1)

Fig. 4. Amounts of type I and type II methanotrophs

Fig. 5. Effect of plant species on Methanotrophs distribution

1–15 3–18 5–17 7–19 9–20 11–21

Fig. 6. Properties of methane emission from a soil plant system

STUDY ON THE OXIDATION CHARACTERISTICS OF

YANG KEa, JI HONGb,c*, XING ZHIXIANGa, LIU GECHENa, YU SIWEIa

AIMS AND BACKGROUND

RESULTS AND DISCUSSION

Table 1. Coal sample components industry analysis results

0.18 y = 0.0324x + 0.0524

Fig. 1. Dry coal sample adsorption curve

0.22 14.5% moisture

Fig. 2. Moisture coal sample adsorption curve

Fig. 3. Isothermal adsorption curve of the coal sample

MINERALOGICAL AND IRON RECOVERY FROM

TIANGUO LIa, JUNQING ANb, XIAOJUN XUa*, SHULI LIUa,

RESULTS AND DISCUSSION

Grinding :P 90% ≤ 48um DDA, CTAB , SBX, ADD

Starch : 100 Cleaner

Concentrate Tailings Tailings Concentrate

Fig. 1. Closed-circuit ﬂowsheet of magnetic separation and reverse ﬂotation experiments

Quartz and glass (1.77%)

Fig. 2. Primary mineral constituents of CCS flotation tails

Fig. 4. Photomicrographs of CCS flotation tails

Table 3. Mineral liberation degree of magnetite and ferroalloy

Embedded characteristics. The mineral interlocking and inclusion relationship of

200 um 200 um 100 um

Magnetic separation studies. In order to achieve the desired grade of concentrate

0.03 0.06 0.09 0.12

Table 5 shows the effects of regrinding fineness of the roughing concentrates on

REVERSE FLOTATION OF IRON SILICATES REMOVAL

Zeta-potential studies. Figure 7 shows the zeta-potential values of magnetite, quartz

Effects of starch on iron silicates removal. Research on magnetite reverse ﬂotation

dosage of collectors (g/t)

H. A. EWAISa, M. A. NAGDYb, S. A. HAMEEDa*

AIMS AND BACKGROUND

Fig. 1. Variation of absorbance as a function of time

RESULTS AND DISCUSSION

Fig. 2. Plots of kobs versus [IO4–] at different temperatures

Fig. 3. Variation of k1 with 1/[H+] at different temperatures

Table 3. Variation of k2 and k3 with temperature

[CoII(ADA)(L)(H2O)]2– + H4IO6– [CoII(ADA)(L)(H4IO6–)]3- + H2O K6 (11)

Effect of acetate ion on the reaction rate. Oxidation reaction of [CoII(ADA)(L)(H2O)]2–

Table 4. Variation of k1 with acetate ion concentration at different temperatures

Fig. 4. Variation of k1 with [CH3CO2–] at different temperatures