Studies on antioxidant status in Mugil cephalus in response to heavy metal pollution at Ennore estuary

Estuaries, the important component of natural environment are under pressure nowadays due to pollution from different sources like industries, agricultural fields etc. Ennore estuary one of the highly polluted estuary situated in Chennai, Tamilnadu, India, due to heavy industrialization surrounding this site poses serious threat to its inhabitants. The present paper focuses on studying the response of the fish Mugil cephalus with reference to its antioxidants during their exposure to metals like iron and chromium present in the industrial effluents that are discharged into the Ennore estuary. The results on comparison with unpolluted Kovalam estuary showed that fish from Ennore experiences severe oxidative stress with significant alteration being observed with antioxidant enzyme activities. Since these results were also found to vary with seasons, the determination of oxidative stress biomarkers in M. cephalus along with seasonal variations may serve as a convenient approach during pollution biomonitoring programme.     

Imposex and butyltin concentrations in Bolinus brandaris (Gastropoda: Muricidae) from the northern Tunisian coast

The imposex incidence and butyltin concentration i.e. tributyltin with its di- and mono-substituted metabolites were investigated in the muricid Bolinus brandaris sampled from two sites on the northern Tunisian coast (the Lagoon of Bizerta and the small Gulf of Tunis). Both populations had imposex, with stages of imposex development varying between VDS 1 and VDS 4.3. All imposex indices (imposex frequency (I %), female penis length, female vas deferens length, vas deferens sequence index, relative penis length index, and vas deferens length index) were significantly higher in snails from the Bizerta lagoon. Butyltins were detected in the whole tissues of both sexes from the two sites. TBT levels were higher in gastropods collected from the lagoon of Bizerta (12.65 ±1.48 ng Sn g^???1 dw in female and 15.21 ±1.13 ng Sn g^???1 dw in male) than in individuals from the Gulf of Tunis (10.71 ±1.26 ng Sn g^???1 dw in female and 11.65 ±1.63 ng Sn g^???1 dw in male), corroborating the data of imposex analysis. These results confirmed that B. brandaris could be used as a bioindicator species of butyltin pollution in the studied areas. In addition, this study provided baseline data that could serve for long-term monitoring of TBT pollution in Tunisia, since legislation to reduce the use of TBT-based antifouling paints has not been introduced yet.     

Seasonal variation of oxidative stress biomarkers in clams Ruditapes decussatus sampled from Tunisian coastal areas

Seven (7) sites from to the Tunisian coastal area were monitored through four seasons using several oxidative stress biomarkers including lipid peroxidation. Catalase (CAT) specific activity was determined in clam Ruditapes decussatus digestive gland. Lipid peroxidation was assessed in the same tissues using malondialdehyde (MDA) concentration, neutral lipid (NL) and lipofuscin accumulation. Results show that catalase specific activity varies between sites and fluctuates in time. The highest CAT activities were recorded in sites from Bizerta Lagoon and sites neighbouring Sfax city. Malondialdehyde accumulation in digestive gland cells seems to be very pronounced in animals sampled in summer and autumn. Digestive gland sections of clams sampled from reported heavy metal polluted sites exhibited higher volume density of residual bodies. Our results showed that the neutral lipids and lipofuscin content in clam's digestive gland are more sensitive to general stress as compared to other biomarkers, and could be used successfully in clams as early warning tools in field biomonitoring programs.     

Indexing method for assessment of pollution potential of leachate from non-engineered landfill sites and its effect on ground water quality

Dumping of solid waste in a non-engineered landfill site often leads to contamination of ground water due to leachate percolation into ground water. The present paper assesses the pollution potential of leachate generated from three non-engineered landfill sites located in the Tricity region (one each in cities of Chandigarh, Mohali and Panchkula) of Northern India and its possible effects of contamination of groundwater. Analysis of physico-chemical properties of leachate from all the three landfill sites and the surrounding groundwater samples from five different downwind distances from each of the landfill sites were collected and tested to determine the leachate pollution index (LPI) and the water quality index (WQI). The Leachate Pollution Index values of 26.1, 27 and 27.8 respectively for landfill sites of Chandigarh (CHD), Mohali (MOH) and Panchkula (PKL) cities showed that the leachate generated are contaminated. The average pH values of the leachate samples over the sampling period (9.2 for CHD, 8.97 for MOH and 8.9 for PKL) show an alkaline nature indicating that all the three landfill sites could be classified as mature to old stage. The WQI calculated over the different downwind distances from the contamination sites showed that the quality of the groundwater improved with an increase in the downwind distance. Principal component analysis (PCA) carried out established major components mainly from natural and anthropogenic sources with cumulative variance of 88% for Chandigarh, 87.1% for Mohali and 87.8% for Panchkula. Hierarchical cluster analysis (HCA) identifies three distinct cluster types for the groundwater samples. These clusters corresponds to a relatively low pollution, moderate pollution and high pollution regions. It is suggested that all the three non-engineered landfill sites be converted to engineered landfill sites to prevent groundwater contamination and also new sites be considered for construction of these engineered landfill sites as the present dumpsites are nearing the end of their lifespan capacity.     

Biochemical and Ultrastructural Changes in Plant Foliage Exposed to Auto-Pollution

Auto-pollution is the by-product of our mechanized mobility, which adversely affects both plant and human life. However, plants growing in the urban locations provide a great respite to us from the brunt of auto-pollution by absorbing the pollutants at their foliar surface. Foliar surface configuration and biochemical changes in two selected plant species, namely Ficus religiosa L. and Thevetia nerifolia L., growing at IT crossing (highly polluted sites), Picup bhawan crossing (moderately polluted site) and Kukrail Forest Picnic Spot (Low polluted site) were investigated. It was observed that auto-exhaust pollution showed marked alterations in photosynthetic pigments, protein and cysteine contents and also in leaf area and foliar surface architecture of plants growing at HP site as compared to LP site. The changes in the foliar configuration reveal that these plants can be used as biomarkers of auto-pollution.     

Genotoxic effects of water pollution on two fish species living in Karasu River,Erzurum, Turkey

Karasu River, which is the only river in the Erzurum plain, is the source of the Euphrates River (Eastern Anatolia of Turkey). The river is in a serious environmental situation as a result of pollution by agricultural and industrial sewage and domestic discharges. The present study aims to evaluate genotoxic effects of toxic metals in chub, Leuciscus cephalus, and transcaucasian barb, Capoeta capoeta, collected from contaminated site of the Karasu River, in comparison with fish from an unpolluted reference site. Heavy metal concentrations in surface water of the river were determined. The condition factor (CF) was taken as a general biomarker of the health of the fish, and genotoxicity assays such as micronucleus (MN) and other nuclear abnormalities (NA) were carried out on the fish species studied. MN and NA such as kidney-shaped nucleus, notched nucleus, binucleated, lobed nucleus, and blebbed nucleus were assessed in peripheral blood erythrocytes, gill epithelial cells, and liver cells of the fish. A significant decrease in CF values associated with a significant elevation in MN and NA frequencies was observed in fish collected from the polluted sites compared with those from the reference site. Results of the current study show the significance of integrating a set of biomarkers to identify the effects of anthropogenic pollution. High concentrations of heavy metals have a potential genotoxic effects, and the toxicity is possibly related to industrial, agricultural, and domestic activities.     

Use of the land snail Helix aspersa for monitoring heavy metal soil contamination in Northeast Algeria

The objective of this study was to investigate the impact of anthropogenic activities on soil quality using the land snail Helix aspersa as a bioindicator. Soil samples and snails were collected from several sites in Northeast Algeria during the summer and winter of 2010. All of the sites were chosen due to their proximity to industrial factories—a potential source of soil pollution via heavy metal contamination. The concentration of heavy metals (Pb, Cd, Mn, and Fe) in soil samples was analyzed using atomic absorption spectrophotometry. Activity levels of glutathione S-transferase (GST) and acetylcholinesterase (AChE), indicators of oxidative stress and neurotoxicity, respectively, were measured in snails collected from each site. GST and AChE activity were found to vary between sites and by season. The highest levels of GST activity were registered during the summer at sites closest to potential sources of pollution. AChE activity levels also peaked during the summer with the highest values recorded at the site in El Hadjar. These increased levels of bioindicative stress response correlated with increasing metal concentration in soil samples collected at each site.     

Integrated survey of water pollution in the Suquía River basin (Córdoba, Argentina)

We report a combined two-year seasonal monitoring of Suquía River basin using both chemical parameters and biomarkers measured in Jenynsia multidentata, aiming to correlate external levels of contaminants with the response of oxidative stress biomarkers in this fish. Identified pollution sources correspond to city sewage as well as agricultural and small industry activities downstream from Córdoba city. Physicochemical parameters integrated into a water quality index (WQI) were measured in Suquía River during dry and wet seasons. Ag, Mn, Cu, Cr, Ni, Fe, Pb and Zn were also monitored in water and sediment samples. Biomarkers include detoxication and antioxidant enzymes: catalase (CAT), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR). Enzymes showed a pollution dependent response, with increased activities in fish collected close to the sewage exit and progressive drop further downstream, matching changes in the Water Quality index. The combined use of biomarkers with water quality parameters allowed both the identification of pollution sources and the evaluation of effects of contaminants on the aquatic biota.     

Evaluation of air pollution phytotoxicity in a seasonally dry tropical urban environment

This study was conducted in the urban environment of Varanasi, India, to evaluate the plant responses to urban air pollution. Twenty sites were selected in four different zones of the city. At each site, seven woody perennials of same age classes were selected. Out of the four zones (I, II, III and IV), zone IV was used as a reference (control) zone as it received the minimum pollution input. Plant species growing in polluted and control areas were compared with respect to foliar dust load, per cent leaf area injury, leaf area, specific leaf weight and chlorophyll, ascorbic acid, SO ₄ ^2– S and total N concentration in the leaves. Results indicated that the air pollution level in Varanasi causes leaf damage, reduces leaf area, specific leaf weight and chlorophyll, ascorbic acid and total N concentrations in the leaves. Sulphur concentration in leaves increased with increasing level of SO₂ in the ambient air. The magnitude of such changes was maximum at the zone receiving maximum pollution load. Carissa carandas was found to be the most sensitive species and Bougainvillea spectabilis, the least. The study shows that the urban air pollution level in Varanasi is detrimental for the growth of plants involved in this study.     

Shell disturbances and butyltins burden in commercial bivalves collected from the Bizerta lagoon (northern Tunisia)

Shell disturbances and soft tissues butyltin burden were investigated in commercial bivalves Lithophaga lithophaga, Mytilus galloprovincialis, Solen marginatus and Crassostrea gigas from the Bizerta lagoon. Shell disturbances were found in all bivalves, being scarce in S. marginatus. In the internal valve of L. lithophaga, burrowing annelids and sipunculids living inside galleries were observed, while in the external valve, brown-blackish or white stains were found. In M. galloprovincialis, a yellowish mass located at the shell anterior side was found fixed firmly to the pearly layer by a hard brownish structure covering some annelid elliptic eggs. In the internal shell layer of some specimens collected in April, embryos belonging to tubiculous annelids at various developmental stages were observed. In C. gigas, shell thickening was revealed in some specimens corresponding to white doughy deposits at the internal valve and between shell layers. In S. marginatus, only one specimen showing a cavity at the posterior site was found. Total butyltin concentrations in the studied bivalves varied between 30 and 245?ng/g dry weight with tributyltin (TBT) being the predominant compound. The highest concentration was recorded in L. lithophaga collected from the Bizerta Bay and the lowest concentration in S. marginatus from Maghraoua. This study provided baseline data that could serve for long-term monitoring of TBT pollution in Tunisia, since legislation to reduce the use of TBT-based antifouling paints has not been introduced yet.     

Biochemical responses in tree foliage exposed to coal-fired power plant emission in seasonally dry tropical environment

A biomonitoring study was conducted to investigate the responses of plants exposed to power plant emission in a dry tropical environment. For this purpose, five sampling sites were selected in the prevailing wind direction (NE) at different distance to thermal power plant (TPP) within 8.0 km range and a reference site was selected in eastern direction at a distance of 22.0 km. The two most common tree species, Ficus benghalensis L. (Evergreen tree) and Dalbergia sisso Roxb. (deciduous tree) were selected as test plants. Ambient sulphur dioxide (SO(2)), nitrogen dioxide (NO(2)), suspended particulate matter (SPM), respirable suspended particulate matter (RSPM), dust-fall rate (DFR) and plant responses such as leaf pigments (chlorophyll a, chlorophyll b and carotenoids), ascorbic acid, sugar and sulphate-sulphur (SO4(2-)-S) contents were measured. Ambient SO(2), NO(2), SPM, RSPM and DFR showed significant spatial and temporal variation at different sites. Considerable reduction in pigment (chlorophyll a, chlorophyll b and carotenoids) and sugar contents were observed at sites receiving higher pollution load. Ascorbic acid exhibited significant positive correlation with pollution load. Accumulation of SO4(2-)-S in leaf tissue showed significant positive correlation with ambient SO(2) concentration at all the sites. At the same time, SO4(2-)-S showed significant negative correlation with pigment and sugar content. D. sisso Roxb. tree was found to be more sensitive as compared to F. benghalensis L. tree.     

Quantitative assessment of eutrophication: A scoring system for characterising water quality in coastal marine ecosystems

A scoring system based on nutrient concentrations was developed to assess coastal water quality according to the trophic level. Three nutrient data sets from eutrophic, mesotrophic and oligotrophic waters were used as the reference information for setting up a semi quantitative water quality scale (from 0 to 5) to express different nutrient loadings. The validity and sensitivity of the method was applied to a number of stations spaced out along the coastal area of Rhodes (Greece). A score for each nutrient/sampling site was calculated and the scorecard formed, was the data matrix used for numerical classification of the stations. The results showed (a) good discrimination between cutrophic, mesotrophic and oligotrophic waters (b) nitrate among the nutrients showed the maximum sensitivity in characterising pollution levels. The reference data sets used for assessing eutrophication levels ensured the objectivity of the method. The proposed method is described step-by-step and it is suggested that the method can be further adapted to describe other forms of pollution becoming a useful quantitative technique in coastal management practices.     

Evaluating the power of surface attendance counts to detect long-term trends in populations of crevice-nesting auklets

This study assessed metals in water and different tissues of Labeo rohita and the impact of these metals on DNA and proteins as biomarkers of gills and muscles of these fish from three different polluted sites (reference or low = KW, medium = CH and high = SK) of the Indus River, Pakistan. The Mn, Pb, Cu, Zn, Hg, and Cr levels in water, gills, liver, muscles, and skin of these fish were compared with the international permissible levels. All metals except Pb and Hg in water were within the acceptable limits of drinking water. In contrast, the Mn, Hg, and Cr levels in the fish tissues were higher than their permissible limits for fish as a human food. Here, the gills contained higher metals than the other tissues. Different patterns of biomarkers were found in fish from these sites. While the gills did not show four protein bands (55, 30, 18.4, and 16.4 kDa), the muscles showed four new protein bands (100, 85, 45, and 20 kDa) for fish from the medium and high polluted sites as compared to the reference or low polluted site. The fish from the CH and SK sites of the Indus River contained low molecular weight DNA in their gills but high molecular weight DNA in their muscles when compared with the KW site. This study suggests that the proteins and DNA profiles of L. rohita could be used as biomarkers to assess the impact of potential environmental stressors such as metals on the freshwater systems.     

Environmental quality assessment combining sediment metal levels, biomarkers and macrobenthic communities: application to the óbidos coastal lagoon (Portugal)

Macroinvertebrate benthic communities are one of the key biological components considered for the assessment of benthic integrity in the context of the Water Framework Directive (WFD). However, under moderate contamination scenarios, the assessment of macrobenthic alterations at community level alone could be insufficient to discriminate the environmental quality of coastal and transitional waters. Keeping this in view, sediment quality of moderately contaminated sites in a coastal lagoon (óbidos lagoon, Portugal) was assessed by the combination of sediment metal levels, Carcinus maenas biomarkers (accumulated metals and oxidative stress responses) and macrobenthic communities. Two sites were selected in confined inner branches (BS and BB) and a third one in the middle lagoon (ML). The site BB presented slightly higher levels of metals in sediment but biological variables calculated for macrobenthic data were not significantly different between sites. The biotic index M-AMBI that is being applied to assess environmental quality of transitional waters in the scope of the WFD pointed either to high (site ML) or good quality status (BS and BB) in the selected sites. However, crabs from BB site presented significantly higher levels of Ni in hepatopancreas than those from ML and macrobenthic community structure was significantly different between BB and ML. Additionally, spatial differences were obtained for oxidative stress parameters suggesting that BB site presented stressors for crabs (higher GST and lower GSH_t at BB site). Factor analysis (PCA) integrating sediment contamination, biomarkers in crabs and macrobenthic data also distinguished BB site as the most environmentally disturbed. On the other hand, at ML site, some macrobenthic variables (equitability and polychaetes’ diversity) were found to be enhanced by current environmental conditions, suggesting the existence of a better sediment quality. Current results pointed to the usefulness of integrating macrobenthic community alterations with responses at organism level (bioaccumulation and biochemical endpoints) in order to increase the accuracy of environmental quality assessment in lagoon systems. Moreover, the application of different statistical methods was also found to be recommendable.     

An assessment of acute biomarker responses in the demersal catfish Cathorops spixii after the Vicuña Oil Spill in a harbour estuarine area in Southern Brazil

The Vicuña oil tanker exploded in Paranaguá Bay (South of Brazil), during methanol unloading operations in front of Paranaguá Harbour, on November 15th, 2004, releasing a large amount of bunker oil and methanol. Two weeks after the accident, the acute effects of the Vicuña Oil Spill (VOS) were evaluated in the demersal catfish Cathorops spixii, comparing a contaminated (at the spill site) and a reference site inside the Bay. Data were compared to previous measurements, taken before the accident, in the same species, from the same sites. The physiological biomarkers were the ones that best reflected acute effects of the spill: plasma osmolality, chloride, calcium, magnesium, and potassium. Morphological (liver and gill histopathology) and genetic (piscine micronucleus and DNA strand breaks) biomarkers revealed that damage was already present in fishes from both reference and contaminated sites inside the Bay. Thus, the reference site is not devoid of contamination, as water circulation tends to spread the contaminants released into other areas of the Bay. Acute field surveys of oil spill effects in harbour areas with a long history of contamination should thus be viewed with caution, and whenever possible previous evaluations should be considered for proper appraisal of biomarker sensitivity, especially in mobile bioindicators such as fish.     

The responses of the hepatosomatic index (HSI), 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione-S-transferase (GST) activity in sea bass (Dicentrarchus labrax, Linnaeus 1758) caged at a polluted site: implications for their use in environmental risk assessment

The present study investigates the response of three hepatic biomarkers in adult sea bass (Dicentrarchus labrax, Linnaeus 1758) caged at a wastewater outlet of an oil refinery with fish caged at a pristine site used as controls. The biomarkers that were investigated were the hepatosomatic index (HSI), 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione-S-transferase (GST) activity. In addition, we have measured the levels of polycyclic aromatic hydrocarbons (PAHs) and selected heavy metals (lead, cadmium, mercury, copper and zinc) in sediment samples at the polluted site. Although the polluted site had high environmental levels of PAHs and heavy metals, there was no difference in hepatic EROD activity and HSI between fish caged at the polluted site and controls. On the other hand, GST activity was significantly lower in fish caged at the polluted site compared to controls. Our results point out that the studied biomarkers have limited use in environmental risk assessment studies, at least when caged adult sea bass is used as the sentinel species and complex toxicant mixtures are involved.     

Assessment of trace metals using lichen transplant from automobile mechanic workshop in Ile-Ife metropolis,Nigeria

The level of air pollution around the automobile mechanic workshops has been generally overlooked. This study, examined the level of trace metals in automobile mechanic workshops and the suitability of using transplanted lichen thalli of Lepraria incana for measuring air pollution in such areas. Samples of the lichen thalli were transplanted into seven different sites and were attached to the bark of trees at each site. The samples were harvested from the sites after 3-month exposure. Concentrations of Pb, Cu, Cd, Fe, Zn, and S content were determined using an atomic absorption spectrophotometer. Results showed that there was a significant difference in the trace metals concentrations across the sites (p?<?0.05). The analyzed lichen samples showed a range of 91.26–119.35 ppm for Fe, 30.23–61.32 ppm for Zn, 1.25–2.45 ppm for Cu, 0.017–0.043 ppm for Cd, 0.018–0.051 ppm, and 0.37–0.42 ppm for S. From the study, sites 6 and 7 presented higher concentrations of Cd, Pb, and Zn than other sites. The enrichment factor calculated showed that Zn, Cd, and Pb were greatly enriched from the workshops. The trend in the concentration of these heavy metals suggests that activities in these workshops might become a major source of certain heavy metals in the environment and if the pollution activities persist, it might become worrisome over time.     

Use of biomarkers in Nile tilapia (Oreochromis niloticus) to assess the impacts of pollution in Bolgoda Lake, an urban water body in Sri Lanka

The present study reports the first analysis of water pollutants in Sri Lankan waters using a suite of biomarkers in Nile tilapia (Oreochromis niloticus) residing in Bolgoda Lake which receives urban, industrial and domestic wastes from multiple sources. The fish were collected from the lake in the dry period (April 2005) and wet periods (September 2005, October 2006) and the levels of biomarkers viz. hepatic ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST), metallothioneins, biliary fluorescent aromatic compounds, brain and muscle cholinesterases (ChE) were compared with those of the laboratory reared control fish and the fish obtained from a less polluted water body, Bathalagoda reservoir (reference site). The results revealed that biomarker levels of the fish collected from the reference site were not significantly different from the controls. Hepatic EROD and GST activities in fish from Bolgoda Lake were induced 4.2-16.6 folds and 1.4-3.3 folds respectively compared with the control fish. Analysis of bile in the lake fish revealed recent uptake of naphthalene, pyrene and benzo(a)pyrene type polycyclic aromatic hydrocarbons (PAHs). The induction of EROD activities in feral fish reflects the exposure of fish to aryl hydrocarbon receptor agonists including PAHs present as pollutants in the Bolgoda Lake. Cholinesterase activity in the fish inhabiting one sampling site of Bolgoda Lake was lower (22-40% inhibition) than the activity measured in the control fish indicating the presence of anticholinesterase pollutants in the area. Hepatic metallothionein levels in the lake fish were higher (1.9-3.2 folds) in comparison to the controls indicating metal exposure. The results support the potential use of these biomarkers in Nile tilapia in assessing pollution in tropical water bodies.     

Impact of Hazardous Exposure to Metals in the Nile and Delta Lakes on the Catfish, Clarias lazera

The impact of metal pollution in the Nile and Delta lakes was studied in Clarias lazera. Lake Edku is grouped as the site highest in metal concentrations. Compared to Lakes Maryût and Edku, the Nile stations displayed lower levels of metal contamination signifying their selection as reference sites. Metals were accumulated most by the liver, less by muscles and least by serum. Iron showed the highest bioconcentration factors (BCF) in liver, muscles or serum compared to other metals. Even after the refinement of the surrounding water, fish seem to uphold the metals that have been previously ingested. Mercury, in particular, seems to eventually reside in muscles rather than in liver. The positive relationship between total serum protein and metal contents may indicate the formation of specific metal-binding proteins. Meanwhile, serum hyperalbuminemia in polluted fish is probably a shock response towards the high stress index induced by metals. The hypothesis that most of serum copper is bound to ceruloplasmin is documented also in C. lazera. Moderate, but not extennsive, exposure to metals was quite stimulatory to liver alkaline phosphatase (AP) biosynthesis. AP could, thus, be considered as a promising bioindicator for assessing sublethal water intoxicosis.     

APPLICATION OF BIOLOGICAL MEASURES FOR STREAM INTEGRITY ASSESSMENT IN SOUTH-EAST BRAZIL

The sensitivity of eleven metrics using macroinvertebrate assemblages were evaluated in an environmental gradient in a tropical river in south-east Brazil. Eight sites were sampled in an altitudinal range of 160–650 m.a.s.l. during 1999 (April and August) and 2000 (February). Four substrates were sampled at each site: riffle litter, pool litter, stony substrates and sediment. Correspondence Analysis indicated that assemblages were primarily more influenced by physical changes (like deforestation and erosion processes) than the water chemistry. The sensitivity of each metric was evaluated through the application of box-and-whisker plot method by its power to assess impairment (metrics should be able to discriminate reference sites from impaired sites) and natural variability (metrics should not discriminate two reference sites). Metrics that failed in at least one of the above premises were not considered as sensitive. In this study, the most sensitive metrics were Shannon index, BMWP-ASPT, %_EPT, and relative abundance of EPT to Chironomidae.