DIATOMS AS INDICATORS OF WATER POLLUTION IN RIVERS

Contents: Abstract.2 Introduction...3 Aims..6 Methods.7 Sites description.7 Map of sites...9 Field sampling..10 Digestion..10 Preparation of permanent slides...11 TDI calculation.12 Biodiversity calculation13 Statistics description.13 Results..14 Testing for normality17 Testing for correlation..18 Testing for significant differences24 Independent Pooled T-test25 Discussion.26 Reference..29 Appendix...30

Abstract: Benthic diatoms are used for assessment of eutrophication of water systems, but their relationship to water quality is not always definite. This study assessed the correlation of TDI to some physico-chemical factors of rivers. Diatoms were sampled from eight sites of four different water qualities assigned by the EPA, based on the Q-scheme value. Each site was tested for Dissolved Oxygen (mg/L), Temperature (C), Conductivity (S/cm) and pH. TDI for each site was calculated and assessed for correlation to these physico-chemical factors and to calculated biodiversity (Shannon Wiener index) of each site. No relationship of TDI to any of the above was found. More samples would have to be obtained from the sites over longer period in order to be able to assign representative TDI values. It is also highly recommended to assess other communities (macrophytes, invertebrates, grazers pressure) and physical factors (type of sediment, light intensity, flow, etc.) and take holistic approach of the assessment.

Keywords: Diatoms, TDI, Trophic Diatom Index, Physico-chemical factors, River Eutrophication.

Introduction: Diatoms or Bacillariophyceae are Class of Division Heterokontrophyta (brown algae). They are unicellular representatives of brown algae which can form colonies, but are mainly attached to substrate by stalks or glide on mud with aid of mucilage they produce. Their presence can be seen as thin biofilm layer present on surfaces of stones, macrophytes or other macroalgae. They are cosmopolitan species inhabiting freshwater or marine environment and can occur in terrestrial habitat also. Around 12,000 species is known of more than 250 genera, but there may be over 100,000 species in total. In freshwaters, around 80 genera of diatoms are known. In oceans, diatoms form major part of photosynthetic phytoplankton and it is estimated that themselves, they contribute about 25 35 % of the worlds productivity in terms of carbon fixation (Kelly, 2005). Diatom body consists of protoplasma enclosed in a cell wall (frustule) made of silica. This consists of two overlapping, interlocking halves epitheca and hypotheca, joined together by girdle bands. Girdle holds the two valves together by cementing organic substance (Kumar & Singh, 1979). Identification of diatoms relies almost exclusively on frustules visible in light microscope. Figure 1: Exploded pennate diatom:
(Kelly,200)

Valves are usually identical for one species. They vary between species in shape and symmetry, as well as in the type of arrangement of pores, slits and other features of the silica wall. These differences formed the basis of diatom identification (Kelly, 2005). Diatoms are divided into two major groups depending on the shape of the cell. Centric diatoms are circular and pennate diatoms are elongated in valve view. Some diatoms have a raphe paired longitudinal slit system associated with motility. Raphe secretes mucilage which aids in locomotion or can be used for attachment of diatoms. Mucilage can be also secreted by rimoportula, a tube-like slit found in most centric diatoms and some pennales. Diatoms can be prostrate Cocconeis sp. lying on substrate stalked - Achnanthidium sp. attached on stalk arborescent Gomphonema sp. on branched stalks These different growth forms lead to different assemblages depending upon the type of substrate (e.g. stable rock is preferred by Achnanthidium sp. and Gomphonema sp. and fine sediments are favoured by motile species ( Nitzschia sp.)). Silica wall of diatoms is relatively insoluble, their remains can accumulate and preserve in sediments of rivers and lakes. These fossil records in conjunction with knowledge of diatoms ecological specificity have been used to infer lake or ocean histories, in particular pH changes and nutrient status as well as climate change (Kelly, 2005). Scientists have noticed that certain diatom species thrive in certain environmental conditions and reflect influence of physical and chemical factor of site. These observations have been developed into indices used for routine water quality monitoring. TDI (Trophic Diatom Index) was designed to monitor eutrophication in rivers (Kelly, 2000). Diatoms are being used in most EU Member States as cost-effective proxies for phytobenthos one of the biological elements required by Water Framework Directive from Member States to be included in ecological status assessment. Rapid

response of diatoms to environmental change makes them very useful indicators of water quality, but it also means that their assemblages are variable in their composition. The sample therefore provides information on the condition of the biota at a point of time (Kelly, 2009). Most of the methods developed for water quality monitoring involve models that relate ecological status to community composition (Kelly, 2009). Assessment of community composition has several advantages over physico chemical measurements of water quality. Most commonly used systems of water quality monitoring have been based on benthic macroinvertebrates (Atazadeh, 2007) (e.g. Qvalue system or BMWP). These are assessing organic pollution, whereas TDI is a measure of eutrophication. Q value system and BMWP index were developed from Trent Biotic Index, which is based on effects of organic pollution, e.g. reduction in community diversity and loss of macroinvertebrate groups. Q value divides invertebrates into five broad groups (A E), A being most sensitive and E most tolerant. It is based on the relative proportions of the characteristic indicator groups of the taxa. River status is then determined by the proportion of each those groups in the sample. EPA rivers can be classified as bad (Q1, Q1-2, Q2), poor (Q3), moderate (Q3-4), good (Q4) or excellent (Q4-5, Q5). For TDI, each taxon is allocated a score based on its sensitivity to phosphorus, because TDI is a measure of the effect of nutrients phosphorus predominantly on stream communities (Kelly, 2000). The score then plays role in calculating the TDI. There are other physico chemical factors of aquatic environment being tested if they influence diatom populations and if so, how. These factors are mostly conductivity (S/L), dissolved oxygen (mg/L), temperature (C), pH and nutrients, mainly phosphorus and nitrates. Relationships between diatoms and the chemical environment are well established, but because diatoms are just one part of phytobenthos, diatom-based assessments are subject to an additional uncertainty (Kelly & King, 2009). Diatom biomass can be influenced by other factors such as grazing, temperature and competition for space with larger macroalgae. Aims of this study: 5

To calculate TDI values of the sites to estimate the level of eutrophication. To compare calculated TDI values to Q-scheme status of those sites designated by EPA. To assess difference between sites of poor/moderate status and good/excellent status. To assess relationship between TDI and physico-chemical factors of the sites. To estimate biodiversity of diatom populations in each site.

Methods:

Diatom samples were obtained from following sites on following dates shown in Figure 2: Terryland 27th January 2010 Lough Kip River 27th January 2010 Grange Abbert Black - 10th February 2010 - 10th February 2010 - 12th March 2010

Ballycuirke 12th March 2010 Owenriff - 18th March 2010 Loughinch River - 18th March 2010

Sites description: Terryland - Originates in Castlegar - Galway Grid reference: M 31306 27263 EPA status: Poor (Q 2-3) Water is derived mainly from calcareous material. Slower river, where sedimentation can occur. Its main part is in Galway city. Lough Kip River - west of Lough Corrib, Co. Galway Grid reference: M 22182 31250 EPA status: Good (Q 4) 2006, 2009 survey High status (Q 4-5) from 1997 - 2003 surveys River flows from Lough Kip to Ballycuirke over acidic soil; relatively fast flowing without sedimentation.

Grange east of Lough Corrib, co. Galway Grid reference: M 47654 47624 EPA status: Good (Q 4) - year 2000 onwards Moderate (Q 3-4) measurement in 1997 Poor (Q 3) 1994 River is on calcareous, medium to high base soil. Farming is frequent in area. Abbert east of Lough Corrib, co. Galway Grid reference: M 55661 42846 EPA status: Good (Q4) from 2006 onwards River flows over calcareous soil region; area highly farmed. Black - east of Lough Corrib, co. Galway Grid reference: M 25519 49076 EPA status: Moderate (Q3-4) 2003 Good (Q 4) 2000 Moderate (Q 3-4) 1989 River flows over calcareous stone and surrounding land is used for farming. Ballycuirke sample was taken from river flowing from this lake, it is on west side of Lough Corrib, co. Galway Grid reference: M 22998 32557 EPA status: Mesotrophic Lake Owenriff river on west side of Lough Corrib, co. Galway Grid reference: M 11486 42604 EPA status: Good (Q 4) - 2006 and 2009 assessments High status (Q 4-5) 2003, 2000 Moderate (Q 3-4) - 1997 River flowing from Glengowla area in Connemara through Oughterard, where the sample was taken. River is fast flowing without much sedimentation. Quite large number of freshwater mussels was noted at the site, implying good water quality. Loughinch River west of Galway city 8

Grid reference: M 18895 23997 EPA status: moderate (Q 3-4) River flowing from Lough Inch over acidic soils into the sea near Furbogh town. This water body was quite stagnant with plenty of sediment present. Figure 2: Site map

Field sampling: Two samples were obtained from River Owenriff and Loughinch River. Other samples were taken by other students as part of their fieldwork. Five cobbles from across each river of about the same size were picked. Each was gently swirled in the river to remove any macroinvertebrates and placed into white tray with little of the river water and its top side was brushed with toothbrush until the water was dark brown. Stone was returned to the river so as it was not picked again for the sampling. This was repeated with other four cobbles. Water from the white tray containing diatom samples was poured into plastic jar labelled accordingly. Toothbrush was rinsed and cleaned against Wellingtons boots. Physico chemical data were obtained. Dissolved oxygen using probe ORION Model 810, pH was measured by Metter Toledo MP120 pH Meter together with conductivity and temperature. On the return to lab, samples were preserved with few drops of Lugols Iodine and stored in dark until needed. Digestion: Large pieces of material were removed from samples, jars shaken and approximately 15 ml of the material were transferred into 30 ml centrifuge tube. Calcareous material wasnt expected, but after previous experience, few drops of HCl were added. Distilled water was added to bring the volume to 25ml. These were then centrifuged until the material settled at the bottom of centrifuge tubes. Supernatant was discarded into waste jar in fume hood and centrifuge tubes placed into a rack submerged in cold water. Concentrated sulphuric acid (5 ml) was added to each tube, then 2ml of saturated solution of potassium permanganate were added and tubes were shaken gently to mix the sample with chemicals. Oxalic acid (10 ml) was added slowly to each sample, which caused effervescence. If the mixture didnt become clear, 5 more ml were added. After 10 minutes, all reactions in samples stopped and cooled down. Distilled water was added to the neck of tubes and samples were centrifuged until they settled. Supernatant was discarded into waste jar and material transferred into smaller test tubes with distilled water. These were centrifuged until material settled and 10

supernatant was discarded to waste jar. This was repeated until the sample reached neutral pH. As soon as the sample reached neutral pH, sample was transferred into clean, fully labelled vial and stored. Preparation of permanent slides - mounting: Distilled water was added to the samples which were too concentrated until slightly cloudy solutions were obtained. Samples were shaken to obtain homogenous solution and few drops of one sample were placed onto a glass cover slip with clean pipette. Cover slip was dried on low heat on a hot plate. A glass slide was labelled and placed onto heated hot plate placed in fume hood. One drop of Naphrax was placed on the glass slide and cover slip placed onto it with the side containing diatom film down. The slide was left on the hot plate until the Naphrax stopped producing bubbles; cover slip was gently pressed onto slide, so as all the left bubbles were pressed away and slide was taken off the hot plate and left in fume hood to cool down. Slide was then checked for the right diatom density under microscope. This procedure was repeated for all samples. Slides were examined and diatoms identified using identification key: Kelly, Martyn. Identification of common benthic diatoms in rivers. 2000 AIDGAP, and Kelly M.G., Bennion H., Cox. E. J., Goldsmith B., Jamieson J., Juggins S., Mann D.G. + Telford R.J. (2005). Common freshwater diatoms of Britain and Ireland: an interactive key. Environment Agency, Bristol. Slides were examined from right to left side with slide label being placed on the right side. If the slide reached its end and 300 frustules count was not reached, slide moved down one field view and counting continued from left to right.

TDI calculation:

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TDI index is based on the weighted average equation (Zelinka and Marvan 1961 cited in Kelly, 2000): where: aj = abundance (proportion) of valves of species j in sample sj = pollution sensitivity (optimum) of species j in sample (values 1 5) vj = indicator value (tolerance) of species j in sample Sensitivity scores: 1 = diatoms which favour very low nutrient concentrations (sj) 2 = diatoms which favour low nutrient concentrations 3 = diatoms which favour intermediate nutrient concentrations 4 = diatoms which favour high concentrations of nutrients 5 = diatoms which favour very high nutrient concentrations A few taxa have sensitivity values of zero. These include taxa relatively rare in freshwaters and whose ecological preferences are not well defined (Lucid Diatom Key). This index calculates WMS (Weighed Mean Sensitivity) value, which is further used in equation TDI = (WMS x 25) - 25 to calculate TDI. TDI values range from 0- 100, with higher values indicating higher levels of nutrients in the water system.

index =

a
j =1 n j =1

sj vj
j

vj

Biodiversity calculation:

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For estimation of biodiversity, Shannon Wiener index was used. Its formula is as follows:

H = pi log pi
' i =1

where: s = number of species pi = relative abundance of species the i-th species (calculated ni/N) ni = no. of individuals of the i-th species N = total number of individuals in the community

Statistical calculations: All data were tested for normality, using Ryan-Joiner test for normality. If some data were found not to be normal, they were log10-transformed and then worked on those. Physico-chemical data, number of frustules and biodiversity data were tested for correlation to TDI values using Correlation test in Minitab. Poor and moderate status rivers data were joined and the same was done with good and excellent rivers. These two groups were separately tested for normality with Ryan-Joiner normality test. In order to test these two groups for significant difference between them, they had to have proven equal variances and the independent pooled Ttest was then used.

Results:

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Within the eight sites 93 taxa were recorded belonging to 37 genera. The composition of species in each site was different from site to site, even though there were species found to be common and abundant for some sites. The most abundant species for each site and their percentages are noted in Table 1 below. Table 1: Four most abundant species from each river and percentage in which they appear in that river with TDI of each site.
River Site Abbert Species Amphora pediculus Navicula lanceolata Amphora lybica Cocconeis placentula Nitzschia dissipata Encyonema silesiacum Achnanthidium minutissimum Fragilaria capucina Navicula lanceolata Achnanthidium minutissimum Gomphonema parvulum Navicula molestiformis Gomphonema olivaceum Amphora pediculus Navicula lanceolata Nitzschia dissipata Cocconeis placentula Achnanthidium minutissimum Nitzschia dissipata Achnanthes oblongella Cocconeis placentula Fragilaria capucina Gomphonema parvulum Cymbella sp. Achnanthidium minutissimum Achnanthidium minutissimum Sellaphora bacillum Navicula oblonga Rossithidium linearis Achnanthidium minutissimum Rhoicosphenia abbreviata Gomphonema olivaceum Navicula minima Percentage (%) 35.5 17.9 10.2 7.7 32.2 18.8 13.4 6.1 30.7 10.4 9.3 9.3 9.1 42.5 15.8 10.2 8.6 27.1 12.1 8.8 5.9 36.6 11.6 9.2 8.2 47.5 10.6 7.6 5.6 30 17.8 13.8 7.4 TDI value 85.6

Ballycurike

66.6

Black

86.5

Grange

88.4

Lough Kip River

51.6

Owenriff

49.6

Loughinch River

41.5

Terryland

67.4

Achnanthidium sp. was found on six sites and was also found in large proportions. In Lough Kip River, it formed 27.1% of diatom community and in Loughinch River site, it was found to take 47.5% of the community. Navicula sp. was found on five sites,

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being the most abundant in river Black with 30.7% (N. lanceolata) and 9.3% (N.molestiformis). Cocconeis sp., Nitzschia sp and Gomphonema sp. were all found on three sites all in relatively low numbers. Fragilaria sp. and Amphora sp. were found on two sites only with Fragillaria sp. being the most abundant in river Owenriff (36.6 %). Amphora sp. was the most abundant in River Grange (42.5 % of species) and in River Abbert, two Amphora sp. were found - A. pediculus (35.5 %) and A. lybica (10.2 %).

Table 2: Data collected for TDI analysis.

River Site
Ballycuirke Terryland Abbert Loughinch River Grange Lough Kip Black Owenriff

Conducti vity (s/cm)

194.0 653.0 648.0 157.0 122.7 148.6 121.2 109.3

DO (mg O2 dm-3)
9.60 6.35 11.50 8.87 12.42 11.40 10.42 9.98

pH
7.73 7.07 7.75 7.68 8.02 6.83 7.76 6.30

TDI 66.6 67.4 85.6 41.5 88.4 51.6 86.5 49.6

EPA Status
poor poor moderate moderate good good excellent excellent

No. spp.
21 30 19 31 27 31 17 22

No. frustules Identifi Non ed - ID

314 484 352 301 266 306 450 404 0 64 45 30 26 73 15 39

H' 2.2 2.3 2.1 2.2 2.1 2.7 2.3 2.2

Conductivity (S/cm) had two outlier values in rivers Terryland and Abbert. Conductivity is a measure of dissolved salts or dissolved ions in solution. It is controlled by geology, watershed size and other sources of ions (e.g. pollution). Therefore higher conductivity values ( > 500 S/cm) can indicate hard water, whilst low conductivity values ( < 100 S/cm) can indicate soft water rivers. DO (mg O2/dm3) was highest in rivers Grange, Abbert and Lough Kip. The pH values were smallest at Owenriff (6.3) and Lough Kip River ( 6.83). TDI values are mixed for different EPA status values, which were based on Q-scheme system. Rivers described as excellent based on Q-scheme were found to have both low and high TDI values. Same pattern can be seen in other sites of good and moderate status. One site from each pair has much lower TDI value than the other site of same Q-system status. Rivers Ballycuirke and Terryland are classified as poor water quality sites and their TDI values are very close to each other of TDI 66.6 and 67.4 respectively. TDI values around 60 are classified as moderately eutrophic and values around 80 are fairly eutrophic (N Chathin 2008).

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Biodiversity indices of diatoms in each site were found to be quite high, 2.1 to 2.7 with the lowest value being of rivers Grange (2.1) and Abbert (2.1) and the highest of Lough Kip River (2.7). Figure 3: Graphical representation of collected data on different sites.
Physico-chemical factors and TDI of rivers
Conductivity (s/cm) 700 600 500 400 Value 300 200 100 0 Terryland Ballyqurike Loughkip Owenriff Grange Spiddal Abbert Black DO (mg O2 dm-3) pH TDI No. spp.

River site

Apart from two conductivity measurements outliers, it seems that when conductivity is lower (sites Grange and Black), TDI values are higher and in raised conductivity values (sites Loughinch River and Lough Kip River) the TDI is lower. These factors dont appear to be in correlation with any other physico-chemical properties or biodiversity. All samples were tested for normality by performing Ryan-Joiner test for normality. Conductivity data were found to be not normal, therefore had to be log- transformed. All other data were found to be normal.

Testing for normality H0: Data do not deviate from straight line (Data are normal). HA: Data do deviate from straight line (Data are not normal). Accept H0 if RJcrit< RJcalc. or if p-value >/=0.05 (p=0.05, n-2df); n=8

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Conductivity RJ calc. = 0.819 RJ crit. = 0.8804 RJ crit. > RJ calc. also p value < 0.05 Accept HA - Data are not normal. Log-transformed conductivity RJ calc. = 0.881 RJ crit. = 0.8804 RJ crit. < RJ calc, also p value > 0.05 Accept H0- Log-transformed conductivity data are normal. DO RJ calc. = 0.965 RJ crit. = 0.8804 RJ crit. < RJ calc, also p value > 0.05 Accept H0- DO data are normal. pH RJ calc. = 0.928 RJ crit. = 0.8804 RJ crit. < RJ calc, also p value > 0.05 Accept H0- pH data are normal. p-value > 1.000 p-value > 1.000 p-value = 0.028 p-value < 0.010

TDI RJ calc. = 0.958 RJ crit. = 0.8804 RJ crit. < RJ calc, also p value > 0.05 Accept H0- TDI data are normal. p-value > 1.000

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No. of Frustules RJ calc. = 0.965 RJ crit. = 0.8804 RJ crit. < RJ calc, also p value > 0.05 Accept H0- frustule count data are normal. In calculation of biodiversity normality p-value was calculated 0.045 therefore less than probability of 0.05, but after calculating critical RJ and calculated RJ it was decided the data are normal also. Correlation of separate physico-chemical data to TDI was tested both by generating plots and by hypothesis testing. Biodiversity RJcalc. = 0.900 RJcrit. = 0.8804 RJcrit. < RJcalc. p - value > 0.045 Accept H0- Biodiversity data are normal. p-value > 1.000

Testing for correlation: H0: There is no association between TDI and conductivity. HA: There is association between TDI and conductivity. rcritical at p=0.05; n-2 df Decision: Accept H0 if rcalculated < rcritical also if p value > or = 0.05

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Correlation of TD I w ith c on du ctivity

90

80

70 TDI 60 50 40 2.0 2.1 2.2 2.3 2.4 2.5 log-cond. 2.6 2.7 2.8 2.9

Figure 4: Exploration of correlation of TDI to conductivity (S/cm). rcalculated = 0.247 pvalue = 0.556 rcritical = 0.707 n =8 rcalculated < rcritical Accept H0 there is no association between TDI and conductivity.

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Correlation of TD I w ith D O
90

80

70 TDI 60 50 40 6 7 8 9 DO 10 11 12 13

Figure 5: Exploration of correlation of TDI to DO (mg O2/L). H0: There is no association between TDI and DO. HA: There is association between TDI and DO. rcalculated = 0.386 pvalue = 0.344 rcritical = 0.707 n =8 rcalculated < rcritical Accept H0 there is no association between TDI and DO.

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Correlation of TD I w ith pH
90

80

70 TDI 60 50 40 6.5 7.0 pH 7.5 8.0

Figure 6: Exploration of correlation of TDI to pH. H0: There is no association between TDI and pH. HA: There is association between TDI and pH. rcalculated = 0.618 pvalue = 0.102 rcritical = 0.707 n =8 rcalculated < rcritical Accept H0 there is no statistically significant association between TDI and pH. From the plot, we could say that there is some positive correlation between TDI and pH, even though it is not statistically significant correlation.

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Correlation of TD I w ith n o.of fru stu les

90

80

70 TDI 60 50 40 300 350 400 No.fr ust ules 450 500

Figure 7: Exploration of correlation of TDI to number of frustules counted. H0: There is no association between TDI and No. of frustules counted. HA: There is association between TDI and No. of frustules counted. rcalculated = 0.122 pvalue = 0.773 rcritical = 0.707 n =8 rcalculated < rcritical Accept H0 there is no association between TDI and no. of frustules.

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Correlation of TD I to D iversity
90

80

70 TDI 60 50 40 2.1 2.2 2.3 2.4 H 2.5 2.6 2.7

Figure 8: Exploration of correlation of TDI to diversity (H). H0: There is no association between TDI and diversity. HA: There is association between TDI and diversity. rcalculated = -0.395 pvalue = 0.333 rcritical = 0.707 n =8 rcalculated < rcritical Accept H0 there is no association between TDI and biodiversity. There was no significant correlation found between any of the physical factors to TDI. Number of frustules counted was found not to be significantly correlated to TDI either. There is no statistically significant correlation of TDI to biodiversity. The strongest relationship was found between TDI and pH. TDI and no. of frustules had the smallest correlation. All relationships were positive except TDI against biodiversity, which was quite strong, but negative.

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Test for significant difference between poor/moderate status rivers and good/excellent status rivers: Ryan-Joiner test for normality was performed with positive result. In order to assess significance in difference between the sites, F-test for equal variances was also done as follows: Test for equal variances: H0: There is no difference in group variances. HA: There is difference in the two group variances. Fcritical at p=0.05; (n1 - 1 df; n2 1 df) Decision: Accept H0 if Fcalculated < Fcritical Fcalculated = 1.38 pvalue = 0.796 Fcritical = 9.2766 n =4 Fcalculated < Fcritical Accept H0 there is no significant difference in variances between poor/moderate and good/excellent rivers. also if p value > or =0.05

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Independent pooled T test: Performed for two independent, random, small samples which are normal and their variances are equal. H0- there's no difference between the two samples of different water qualities. HA - there's a difference between the two samples of different water qualities. Accept H0 if t-calc < t-crit. (p = 0.05, n1+n2 -2df)

R elation sh ip of tw o rivers qu ality grou ps

90

80

70 TDI 60 50 40 poor/moderate good/excell

Figure 9: Boxplot to explore relationship and similarity of poor/moderate and good/excellent rivers. Estimate for difference: -3.8 95% CI for difference: (-38.0, 30.5) TValue = -0.27 p-Value = 0.798 DF = 6 CIs span 0 therefore there is no difference between the two sample means. Also pvalue > 0.05 also accept H0. Therefore the TDI values for poor/moderate rivers are not significantly different from good/excellent rivers TDI values.

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Discussion During the diatom identification, ninety-three taxa belonging to thirty-three genera were found. The most abundant species within all sites was Achnanthidium minutissimum and the next most abundant were motile Navicula sp. TDI calculated from sites at given dates were 66.6 for Ballycuirke river, 67.4 for Terryland both being classified as poor status rivers. For Abbert and Loughinch rivers both of moderate status, the TDIs were 85.6 and 41.5 respectively. Good status rivers Grange and Lough Kip River had TDI 88.4 and 52.6 respectively and the excellent rivers Black and Owenriff had TDIs calculated to be 86.5.and 49.6 respectively. TDI values greatly vary between sites of the same water quality status based upon the Qscheme index. Dissolved oxygen (mg O2/L) had highest values in rivers Grange, Lough Kip River both being good status rivers and river Abbert, being classed as moderate water quality site. Correlation of this factor to TDI wasnt proven in statistical testing. Conductivity readings were also found not to be correlated to TDI and also included two outlier data of higher values if compared to the rest (653 S/cm and 648 S/cm in rivers Terryland and Abbert respectively). Figure 3 in results section suggests some relationship between TDI and conductivity, e.g. when TDI is low, conductivity values raise (Loughinch River and Lough Kip River) and the opposite when the TDI values are higher, conductivity readings are lower (rivers Grange and Black). pH data were found not to have statistically significant correlation to TDI, but after visual exploration of the result (Figure 6) it seems that there is some positive relationship. The pH correlation suggests possible correlation of pH to TDI, but the sample size is too small to confirm this theory with statistical significance. The differences in TDIs within the sites of same Q-value could have been caused by myriads of factors which act upon any river community. These factors are temperature, amount of light received, nature of substrate, nutrient availability (nitrates and phosphates), competition for space, natural succession processes, grazing pressure and hydromorphological regime (Kelly & Bennion, 2009). Assessments of water quality based on Q-scheme system of TDI should also take these factors into an account and assess the site as a whole. Q-scheme Index is based on macroinvertebrate 26

communities, which may not react to physico-chemical changes as fast as diatoms. Qscheme Index is also assessing level of organic pollution of water body, not eutrophication as TDI does. Taking only one sample for the water quality analysis cant represent accurately the ecological status of the water system, because diatom assemblages are dynamic. For example Achnanthidium minutissimum is usually the dominant species during early succession, but as its resources become limiting in the biofilm, longer stalked taxa such as Gomphonema acuminatum and loosely attached and tangled taxa as Fragilaria capucina (Kelly & King, 2009). Based on this pattern, Rivers Lough Kip and Lough Inch river communities are quite new, river Black has these two species abundant also, together with motile diatom Navicula sp. River Owenriff has most abundant taxon Fragilaria capucina and Cymbella sp. is also present, which suggests that this community is older than the previous rivers. Also higher percentage of motile species indicates that factors other than water quality, such as sedimentation, may be influencing the diatom assemblages (N Chathin, 2008). Again many factors are affecting the community structure and therefore the TDI value. In statistical analyses, Q-scheme based poor/moderate and good/excellent sites didnt show significant difference in TDI values because both high and low TDI values occur in rivers assigned same Q-value; again more samples would be needed to confirm this result. There was no correlation of TDI of sampled sites to any of the physicochemical factors. Leira and Sabater (2004) study confirms this as follows: Chemical analyses of water is a good indicator of the chemical quality of the aquatic systems, but do not integrate ecological factors and dont necessarily reflect the ecological state of the system. Although not significant, there was some correlation found of TDI to pH. pH was used in several analyses of sediment cores of diatom fossils. These were used as estimation of previous conditions on Earth and ocean acidity. Based upon this, I would agree with the association of pH to TDI values and diatom community structure, and would suggest that bigger, more representative samples were taken in future analyses. Samples should be taken repeatedly at same sites for two to three years, with three samples spread over a year to obtain consistent data. I would also 27

suggest that sampling of nutrients (phosphates and nitrates) are added to the analyses, as they play an important role in phytoplankton growth and their biodiversity. A holistic view on the assessment of communities in sites should be developed rather than focusing on the biological elements separately (Kelly, Haig 2009), because the stream ecosystem assemblages closely interact within.

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Reference:
Atazadeh I., Sharifi M., Kelly M.G. 2007 Evaluation of the Trophic Diatom Index for assessing water quality in River Gharasou, western Iran. Springer Science + Business Media B.V. Kelly M., Bennion H., Burgess A., Ellis J., Juggins S., Gurthie R., Jamieason J., Adriaenssens V., Yallop M. 2009 Uncertainty in ecological status assessments of lakes and rivers using diatoms. Springer Science + Business Media B.V. Kelly M.G., Bennion H., Cox. E. J., Goldsmith B., Jamieson J., Juggins S., Mann D.G. + Telford R.J. (2005). Common freshwater diatoms of Britain and Ireland: an interactive key. Environment Agency, Bristol. Kelly M. G., Haigh A., Colette J. & Zgrundo A. 2009. Effect of environmental improvements on the diatoms of the River Axe, southern England. Kelly M., King L., N Chathin B. 2009. The Conceptual Basis of Ecological-Status Assessments using Diatoms. Biology and Environment: Proceedings of the Royal Irish Academy. Kelly Martyn; Identification of common benthic diatoms in rivers. 2000, by AIDGAP

Kumar H.D. and Singh H. N.; 1979 A Textbook on Algae. Macmillan Tropical Biology Series; Macmillan International College Editions. Leira M., Sabater S. 2004 Diatom assemblages distribution in catalan rivers, NE Spain, in relation to chemical and physiographical factors. Elsevier Ltd. N Chathin B. and Harrington T. J. 2008 Benthic Diatoms of the River Deel: Diversity and Community Structure. Royal Irish Academy.

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Appendix: Examples of diatoms present in sites sampled

Achnanthes oblongella

Achnanthidium minutissimum

Amphora lybica

Amphora pediculus

Cocconeis placentula

Cymbella sp.

Encyonema silesiacum

Fragilaria capucina

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Gomphonema olivaceum

Gomphonema parvulum

Navicula lanceolata

Navicula minima

Navicula molestiformis

Navicula oblonga

Nitzschia dissipata

Rhoicosphenia abbreviata

Rossithidium sp.

Sellaphora bacillum

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