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ABSTRACT
Maize grains are important source of food for human and animal population. A common method
for the protection of grains from pest infection is the use of aluminum phosphide (Alp) pesticide
sold under the brand JustoxinR in Nigeria. A simple and suitable HPLC technique for the
determination of Alp pesticide applied on maize was developed and validated. The HPLC system
achieved a good separation on a Zorbax Eclipsed XDB C18 reversed-phase Column (5µm, 150
mm x 4.6 mm), maintained at room temperature (25ºC) using isocratic mobile phase consisting of
methanol: milliQ water (50:50 v/v) at a flow rate of 1.0 mL/ min. 20 mL of injection volume was
used at a run time of 15 min and a detection wavelength of 195 nm. The chromatographic
conditions indicated a precision, RSD of 0.911-0.932%. The method showed a good linear
relationship at the working standard range of 5 – 50 µg/mL. The coefficient of determination was
0.995. The LOQ and LOD were determined as 7.47 µg/mL and 2.46 µg/mL respectively. The
recoveries after fumigation were between 77.4% and 120.1%. The study showed increase in the
amount of Alp pesticide residue applied on maize grain stored for 7, 10 and 14 days respectively.
Key words: Aluminium phosphide (Alp), pesticide residues, maize grain, high performance liquid
chromatography
Introduction
Food crops are essential to keep the stability of population in country by providing able work
force, feedstock for animal feeds and foreign exchange. The human populations are therefore
threatened by pest and rodents who continuously feed on food crops to the verge of total
destruction if not properly controlled. Pesticide is often applied in order to prevent the destructive
activities of pests. Maize, a commonly grown and one of the most staple food crops in Nigeria, is
usually protected from pests by the application of Aluminium phosphide (Alp) pesticide normally
considered to be cheap and effective in the control of pests (Bond, 1989; Musshoff et al., 2008;
USEPA, 2001). Alp is a synthetic compound which produces a toxic gas, phosphine, when in
contact with the moisture in the atmosphere (WHO, 2007). Alp pesticide contains Alp and suitable
additives to control the evolution of hydrogen phosphide (phosphine gas) and to prevent self-
ignition (APVMA, 2004). The use of Alp involves direct application of the solid in the form of
tablets, pellets or paper bag in an air tight container (Bond, 1989; Moghhadamnia, 2012).
Application by hand is allowed and the effective dosage for bulk fumigation in upright storage is
1-2 g/m3 for minimum exposure of 5 days at 21-25 oC or 4 days at 26oC above (Bond, 1989). The
most important factors are the aeration and fumigation time (Ali et al., 2010).
Alp is highly poisonous; though, self poisoning is the mostly reported case but accidental
poisoning is uncommon. Farmers are exposed occupationally during fumigation but poisoning is
rare (Anger et al., 2000; Mehrpour et al., 2012; Musshoff et al., 2008; WHO, 2007). There is an
increasing concern on the environmental risk of Alp pesticide in food. Food is wholly implicated
as a source of Alp since it is insoluble in water. USEPA (2001) has rated it as one of the highest
risk posing chemicals and recommended a limit of 0.0113 mg/kg/day for a chronic reference dose
spectroscopic tests (IR spectrum, NMR, mass spectra), Chromatography (HPLC or GC retention
time with reference compound) or any other suitable test method. Determination of Alp in wheat
kernels by spectrometric method has been reported. The phosphine gas was generated to the
headspace and determined spectrophotometrically by reaction with silver nitrate (Longobardi et al,
2008). The phosphine gas was also determined with micro-gas chromatography (Longobardi et al,
2008). Zheng et al. (2014) determined phosphine gas from Alp applied on agaric by gas
rice, Zhang et al. (2011) dissolved the phosphine gas in toluene and then analysed Alp using flame
photometric detector. Headspace Gas Chromatography was also employed by Graver et al. (2001)
and Misumi et al. (2008). Carlson and Thompson (1998) converted phosphine gas into
orthophosphate and then isolated the phosphate by ion chromatography with eluent conductivity
detection. Reports on HPLC methods are rare and most of these methods are technical and also
require set of equipment which are not commonly found. This study introduces a simple technique
for the determination of Alp in fumigated maize grain. The storage capacity of Alp pesticide in
Sample location
The samples were collected from a local farmer at Funbi-Fagun Crescent along Ondo-West
Local Government Secretariat, Ondo State, Nigeria (Fig. 1). Ondo State has a vast farmland
and well known for agricultural production. The location was carefully selected based on the fact
Aluminium phosphide pesticide (Alp) under the brand JustoxinR was purchased from a retailed
Aluminium phosphide standard, HPLC grade methanol and milliQ water were supplied by a
Methodology
The study was structured in three stages. The first stage was aimed at evaluating the presence of
Alp pesticide residue in a maize grown in a natural farmland devoid of pesticides or any related
chemicals. In the second stage, sub samples of the maize were stored in an air-tight container (Fig.
2) for fourteen days in a way to mimic the common storage pattern by the farmers. The contents
were then treated with the Alp (Justoxin R). Alp pesticide residue was determined at predetermined
days. In the third stage, rather than whole storage in a container, the maize was treated separately
and stored in three different containers. Alp pesticide residue was determined in each of the
Sample collection
Dried yellow maize (Zea mays) ears were purchased immediately after harvesting from local
farmers. Some monitoring and consistent inspections were done to ascertain the integrity of no
pesticide usage on the maize plants. The kernels were carefully removed from their cobs; the
cleaned ones were completely picked out from the damaged grains, silk and the other associated
foreign materials.
About 8 g of the maize kernel was further dried and crushed to a fine powder with pre-cleaned
pestle and mortar. The moisture content was then determined by weighing 2 g of milled grain and
dried in an oven at 105ºC for 2 hours. The grain moisture content was recorded as 11 %, this
Five (5) air-tight containers with 700 g maize capacity were each filled with 500 g maize grains
and covered. Then, 0.005 g Alp pesticide, properly wrapped in an envelope, was placed in the
centre of each of four containers while the secluded container was used for method blank. Each
container was properly labeled. The first container was kept and opened on 7 th, 10th and 14th day
for sub-sampling. The second, third and fourth containers were kept and opened individually on
1mg/mL (1000 ppm) of the stock solution of Alp pesticide was prepared by dissolving 20 mg of
the commercial pesticide in 20 ml of methanol. The solution obtained was then sonicated for 10
mins and later centrifuged to give the clear portion. Aliquot working standards of 5, 10, 20, 40 and
Sample preparation
Maize kernel was picked randomly from the stored samples. Alp pesticide residues were extracted
with 10 mL methanol in approximately 1.0 g of the maize kernel by mechanical agitation for about
20 mins. The extract was filtered and kept for HPLC analysis. The procedure was repeated for all
1g of untreated stored maize was also weighed. The maize kernel was then sonicated with 10 mL
of methanol for 20 mins to extract Alp pesticide residue in a similar procedure as described for the
standard and the treated samples. The extract was filtered and analysis was performed with HPLC
The HPLC system involves water Alliance 2695 of SM4 4267A auto sampler and UV 2489
detector. The chromatographic elution was performed on a zorbax eclipse XDB C 18 reversed phase
column (150 mm x 4.6 mm, 5 µm). Different ratios of solvents were prepared and used as mobile
phase at different flow rates with the aim of getting a better peak resolution with an optimum flow
rate. All solvents used were filtered through 0.45µm membrane and degassed prior to use. The
best isocratic condition was obtained at the injection volume 20 µl, solvent mixture of methanol:
milliQ water (50:50 v/v), a constant flow rate of 1.0 ml/min and run time of approximately 15
mins. The wavelength of the system after adjustment was monitored at 195 nm. The column was
Method Validation
Accuracy
After storage for 7 days, approximately 1 g of the maize kernel was weighed and dozed with 15
mg/L of the standard solution. The recovery of the standard used was then determined after the
Precision
Three replicate analyses were done on 1g of the treated samples after 7 days. The procedure was
repeated for samples left for 10 and 14 days. The intra-day precision was determined as RSD%
Three triplicate determinations of another 1 g of the 7-day treated sample was determined at 2 days
interval to establish the inter-day precision. The results were calculated in RSD%.
Linearity Range
The area response of the working standard was plotted against the concentration using Microsoft
office excel 2007 software to generate a calibration curve. The linearity is shown in Fig.4.
The limit of detection (LOD) and the limit of quantification (LOQ) of Alp pesticide residue were
determined as 3.3 σ / S and 10 σ / S respectively, where σ is the standard deviation of the response
Quality Control/Assurance
Reagents used were of analytical and HPLC grades. A solvent blank was run at the end of
chromatographic determination and a needle wash was done to ensure that there was no carry over
Alp pesticide was not detected in the method blank indicating that there was minimal level of
contamination in the procedure used and insignificant level of pesticide in the maize kernel (Table
1). The accuracy as indicated by the recovery studies was between 77.4 and 120.1 % (Table 2).
This was close to the 83.4%-112.6% value in agaric (Zheng et al., 2014), 76.8 to 106.5% values in
rice (Zhang et al., 2011) and 85.0 – 100.0% in wheat (Rangaswamy, 1984). The method indicated a
good linear relationship of the area response to the working standard within a linear range of 5 to
50 µg/mL. This was better in range than the 0.012–1.2 mg/L value reported by Zhang et al. (2011).
Similar correlation coefficient, 0.995, was obtained (Fig. 4) compare to the 0.999 in Zhang et al.
(2011), and 0.995 reported in (Zheng et al., 2014) at a working range of 0.05-50 mg. The LOQ and
LOD determined were 7.47 µg/mL and 2.46 µg/mL respectively. The method showed good intra-
day and inter-day precisions, the RSD% determined in both cases were less than 1. The proposed
method is simple, accurate and suitable for the determination of Alp pesticide on maize grain.
The treated maize kernels contain Alp pesticide residue level which generally increased
over the days of storage (Table 1). This is expected as the pesticide is released into the grain
gradually to prevent infection of pest and hence preserving the integrity of maize grain. Bond
(1989) reported that the increase was supposed to have reduced after about 5 days. The continuous
increase with the number of days as observed could pose serious health challenges in the
environment. The amount of residue determined when the kernels were opened intermittently,
were lower than when they were stored separately and opened at once at the specific days (Table
1) as well shown in ( Fig. 5 and Fig. 6). This was probably due to the disturbance of phosphine-air
layer and escape of pesticide into the atmosphere when the containers were opened. There could
be a significant level of exposure risk to workers who are in charge of grain storage especially
Phosphine gas is poisonous, but Alp pesticide is still preferred because of the short
life span of the gas in air and the residue left is not toxic (USEP, 2001). This study revealed that
there could be a substantial amount of the Alp pesticide residue on maize crops capable of posing
serious health risks to human and animal population who feed on them. Local farmers, who
indulge in the fumigation of maize grain, could also be exposed to the pesticide and the exposure
is further compounded by the fact that most of the local farmers show low level of education and
incompetency in the application of pesticides as they lack the basic protective equipment (Dahiru
et al., 2014; Ugwu et al., 2015). There is need for regulation and close monitoring of pesticide
usage in local and modern agricultural practices. Farmers should be educated and trained on the
proper usage and the effect of wrong pesticide practices to themselves and the general community.
The study further buttressed the recommendation of USEPA (2001) that the application of Alp
pesticide must be done with nose mask and other necessary gadgets. Consumers need to avoid
eating raw and treated agricultural commodity before washing or cooking. There exists some data
gaps on the current trend or levels of alp pesticide in agriculture produce in our markets. There is
therefore a need for investigation of alp pesticide residue on the produce sold in market and the
Conclusion
The proposed HPLC method is simple and suitable for a routine determination of Aluminium
phosphide (Alp) pesticide applied on maize grain. Farmers and other handlers of pesticides should
be trained and educated on the toxicity implications of Alp pesticide on crops. There is need for
consistent and adequate monitoring of pesticide producers claim on labels and the usage on food
crops to prevent health risks of the pesticides on human and animal populations. There is also need
for further investigation of Alp pesticide application on raw and cooked food in the market.
Fig 2: Treated maize grains
90000
f(x) = 1710.93x + 2755.67
80000 R² = 1
70000
60000
Peak Area
50000
40000 Linear ()
30000
20000
10000
0
0 10 20 30 40 50 60
Conc. (ppm)
30
f(x) = 2.39 exp( 0.18 x )
25 R² = 0.99
20
Conc. (ppm)
15
10
0
6 7 8 9 10 11 12 13 14 15
Days of exposure
Fig 5: Alp pesticide residue of treated samples open in the same container
35
30
f(x) = 4.83 exp( 0.13 x )
Concentraton (ppm)
25 R² = 1
20
15
10
5
0
6 7 8 9 10 11 12 13 14 15
Days of storage
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