Toxicity evaluation of Meiliang Bay, Lake Taihu, China—a drinking water source

In this study, potential toxicity in organic pollutants in Meiliang Bay, Lake Taihu, a drinking water source, was investigated using the comet assay and zebrafish embryo test, and two control sites in Lake Changdang and the Changjiang (Yangtze) River were established. For the genotoxicity assay, results showed that organic extracts from water samples induce DNA damage on human lymphocytes and mouse testicular cells. A statistically significant difference (p < 0.01) was observed versus the solvent control, as shown by multiple comparisons at a dose of 100 ml tube⁻¹. The degree of DNA damage caused by Meiliang Bay water was most serious (human lymphocytes cells—184 arbitrary units (AU); 234 AU on mouse testicular cells). Organic extracts also affected zebrafish embryo development. Embryo coagulation, axis abnormality, slow absorbability of vitellicle, and multi-edema related to teratogenesis at 96 hpf were observed. In the high dose group, there was obvious edema in the hearts and vitellicles for most dysplastic embryos. Toxic potential in organic pollutants in drinking water sources from the Yangtze River and Lake Changdang were less serious than Meiliang Bay. Therefore, the drinking water source in Meiliang Bay was unsafe compared to the Changjiang River and Lake Changdang, and it is exigent that Meiliang Bay water quality should be ameliorated further.     

Genotoxic potential of marine sediments from the North Sea

The alkaline comet assay is a method for detecting DNA strand breaks and alkali labile sites in individual cells. An in vitro system was used to investigate the genotoxic potential of complex mixtures such as organic extracts of marine sediments. DNA damage was induced in leukocytes isolated from carp (Cyprius carpio) by exposure to organic sediment extracts from the North Sea or hydrogen peroxide as positive control, respectively. The minimum concentration for significant effects ranged from 1 to 40 mg sediment dry weight per milliliter assay volume. The sensitivity of the method was enhanced by using the DNA repair inhibitor, 1-beta-D-arabinofuranosylcytosine (ara C). From the results, it can be suggested that total organic carbon (TOC) as well as the different compositions of contaminants present in the sediment extracts may contribute to the genotoxic effects observed. The comet assay can be applied successfully as an in vitro bioassay for investigations on genotoxicity of marine sediment extracts.     

Comparison of two extraction procedures for the assessment of sediment genotoxicity: implication of polar organic compounds

Four sediment samples (Va?ne Airport VA, Va?ne Center VC, Va?ne North VN and Reference North RN) were collected in the Berre lagoon (France). Sediments were analyzed for polycyclic aromatic hydrocarbons (PAHs) by use of pressurized fluid extraction with a mixture of hexane/dichloromethane followed by HPLC with fluorescence detection analysis. Organic pollutants were also extracted with two solvents for subsequent evaluation of their genotoxicity: a hexane/dichloromethane mixture intended to select non-polar compounds such as PAHs, and 2-propanol intended to select polar contaminants. Sediment extracts were assessed by the Salmonella/microsome mutagenicity test with Salmonella typhimurium TA98+S9 mix and YG1041±S9 mix. Extracts were also assessed for their DNA-damaging activity and their clastogenic/aneugenic properties by the comet assay and the micronucleus test with Chinese Hamster ovary (CHO) cells. The PAH concentrations were 611ngg(-1)dw, 1341ngg(-1) dw, 613ngg(-1)dw and 482ngg(-1)dw for VA, VC, VN and RN, respectively. Two genotoxic profiles were observed, depending on the extraction procedure. All the non-polar extracts were mutagenic for TA98+S9 mix, and VA, VC, VN sediment samples exerted a significant DNA-damaging and clastogenic activity in the presence of S9 mix. All the polar extracts appeared mutagenic for TA98+S9 mix and YG104±S9 mix, and VA, VC, VN were genotoxic and clastogenic both with and without S9 mix. These results indicate that the genotoxic and mutagenic activities mainly originated from PAHs in the non-polar extracts, while these activities came from other genotoxic contaminants, such as aromatic amines and nitroarenes, in the polar extracts. This study focused on the important role of uncharacterized polar contaminants such as nitro-PAHs or aromatic amines in the global mutagenicity of sediments. The necessity to use appropriate extraction solvents to accurately evaluate the genotoxic hazard of aquatic sediments is also highlighted.     

Oxygen requirements of zebrafish (Danio rerio) embryos in embryo toxicity tests with environmental samples

The zebrafish embryo test is a widely used bioassay for the testing of chemicals, effluents and other types of environmental samples. Oxygen depletion in the testing of sediments and effluents is especially important and may be a confounding factor in the interpretation of apparent toxicity. In order to identify oxygen levels critical to early developmental stages of zebrafish, oxygen consumption of zebrafish embryos between 0 and 96h post-fertilization, minimum oxygen levels required by the embryos for survival as well as the effects of oxygen depletion following exposure to model sediments were determined. No significant effects on zebrafish embryo development were observed for oxygen concentrations between 7.15 and 3.33mg/L, whereas at concentrations between 3.0and 2.0mg/L minor developmental retardations were observed, yet without any pathological consequences. Oxygen concentrations lower than 0.88mg/L were 100% lethal. In the sediment contact tests with zebrafish embryos, native sediments rich in organic materials rapidly developed strongly hypoxic conditions, particularly at the sediment-water interface (0 to 500μm distance to the sediment).     

Effects of sediment eluates and extracts from differently polluted small rivers on zebrafish embryos and larvae

The effects on newly fertilized eggs, embryos and larvae of zebrafish Danio rerio following exposure to sediment samples from the more heavily contaminated River Körsch, southern Germany, occurred earlier and were more prominent than in samples from the less contaminated Krähenbach. Dose- and time-related effects following exposure to Körsch sediment eluates and extracts included: (1) hatching failure and subsequent death of larvae exposed to undiluted aqueous sediment eluates and reduced hatching rates at sediment extract concentrations 0·0125%; (2) increased mortality after exposure to 25 and 50% dilutions of aqueous sediment eluates, and dilutions of 0·00625% sediment extracts; (3) reduction of heart beat frequency for 50% dilutions of sediment eluates and concentrations of 0·025% extracts; (4) increased frequency of heart and yolk sac oedema after exposure to 0·0125% sediment extracts. Since adverse effects of sediment extracts observed in zebrafish laboratory tests correlated with reproductive failure in natural populations of brown trout Salmo trutta f. fario in the severely polluted River Körsch, early life stages tests with zebrafish appear to be a suitable tool to assess the contamination rate of natural sediments.     

Meristic and morphometric variation in the silver perch, Leiopotherapon plumbeus (Kner, 1864), from three lakes in the Philippines

A number of researchers have applied multivariate methods to elucidate the population structure of fishes. In this study, we also used multivariate techniques to examine meristic and morphological variations in the silver perch, Leiopotherapon plumbeus (Kner, 1864), from three Philippine lakes, namely, Laguna de Bay, Sampaloc Lake, and Taal Lake. We also aimed to determine whether or not there are meristic and morphological differences among the populations of silver perch from the three lakes, considering that this fish species was introduced from Laguna de Bay into Sampaloc Lake and Taal Lake in the late 1950s and early 1970s, respectively. A total of 710 specimens from four different sites were used in the study: 155 each from Binangonan and Tanay areas of Laguna de Bay and 200 each from Sampaloc Lake and Taal Lake. Based on analysis of variance, eight meristic and 26 transformed morphometric characters were selected for subsequent analyses. Nineteen (19) principal components extracted from the 34 significant variables accounted for 82.3% of the variation in the original variables. Factor analysis using varimax rotation produced four factors: factor 1 was dominated by fin measurements while the highest loadings for factor 2 were gill raker counts. Factors 3 and 4 were dominated by various body and head measurements. Cluster analysis showed specimens from Sampaloc Lake and Taal Lake in one group, while majority of the specimens from Binangonan and Tanay are in another cluster. This suggests a closer morphological similarity between specimens from Sampaloc and Taal. Discriminant analysis gave relatively high correct classification rates (76.13–95.50%). Lower gill raker count was the most discriminating variable. Since both the silver perch from Sampaloc Lake and Taal Lake were introduced from Laguna de Bay, the observed clustering and morphological variation could be attributed to similarities and differences in the lake environments. Laguna de Bay is a shallow eutrophic lake, while Sampaloc Lake and Taal Lake are deep lakes. Further studies, however, are needed to determine which of the myriad of biological and/or physico–chemical factors might have the greatest influence on the observed morphological divergence between the source population and transplanted populations that we found in our study.     

Chromosome aberrations in cultured central mudminnow heart cells and Chinese hamster ovary cells exposed to polycyclic aromatic hydrocarbons and sediment extracts

1. Genotoxicity experiments were conducted with cultured fish cells to determine if the high frequency of epidermal papillomas observed in lemon sole from Sturgeon Bank, where a sewage treatment plant discharges, could be correlated with contamination of the sediments with chemicals such as 3,4-benzopyrene. 2. The frequency of chromosome aberrations was measured in cultured Umbra limi heart (U1-H) and Chinese hamster ovary (CHO) cells following exposure to the polycyclic aromatic hydrocarbons (PAH) 3,4-benzopyrene (BP), 1,2,5,6-dibenzanthracene (DBA), 1,2-benzanthracene (BA), and pyrene (PY), activated using S9 prepared from rainbow trout liver. 3. An increase in the chromosome aberration frequency was only observed following exposure to fish S9-activated BP in both cell lines. 4. Following exposure of the cells to both Sturgeon Bank and Spanish Bank sediment extracts, it was determined that a higher level of toxic and genotoxic activity was associated with the Sturgeon Bank sediments. 5. Since the detection of PAH genotoxicity requires the presence of S9, and since a higher level of genotoxic activity was noted following sediment extract exposures with no S9 present, this suggests that the extracts contain a complex mix of chemicals, some of which express genotoxic activity. 6. An assessment using the micronucleus test failed to indicate in vivo genotoxicity in fish collected from Sturgeon and Spanish Banks. 7. It was, therefore, difficult to associate the observed sediment genotoxicity with the previously noted high incidence of epidermal papillomas in lemon sole from this area.     

Genotoxic damage in Solea senegalensis exposed to sediments from the Sado Estuary (Portugal): effects of metallic and organic contaminants

Juvenile Solea senegalensis (Senegalese sole) were exposed to freshly collected sediments from three sites of the Sado Estuary (West-Portuguese coast) in 28-day laboratory assays in order to assess the ecological risk from sediment contaminants, by measuring two genotoxicity biomarkers in peripheral blood: the percentage of Erythrocyte Nuclear Abnormalities (ENA) by use of an adaptation of the micronucleus test, and the percentage of DNA strand-breakage (DNA-SB) with the Comet assay. Sediments were surveyed for metallic (Cr, Ni, Cu, Zn, As, Cd and Pb) and organic (PAHs (polycyclic aromatic hydrocarbons), PCBs (polychlorinated biphenyls) and DDTs (dichloro-diphenyl-trichloroethane)) contaminants. Sediments from site A (farthest from hotspots of contamination) were found to be the least contaminated and weaker inducers of genotoxic damage, whereas sediments from sites B (urban influence) and C (affected by industrial effluents and agricultural runoffs) were responsible for a very significant increase in both ENA and DNA-SB, site B being most contaminated with metals and site C mainly with organic pollutants, especially PAHs and PCBs . Analysis of genotoxic effects showed a strong correlation between the concentrations of PAHs and PCBs and both biomarkers at sampling times T(14) and T(28), while the amounts of Cu, As, Cd and Pb were less strongly correlated, and at T(28) only, with ENA and DNA-SB. These results show that organic contaminants in sediment are stronger and faster acting genotoxic stressors. The results also suggest that metals may have an inhibitory effect on genotoxicity when interacting with organic contaminants, at least during early exposure. ENA and DNA-SB do not show a linear relationship, but a strong correlation exists between the overall increase in genotoxicity caused by exposure to sediment, confirming that they are different, and possibly non-linked effects that respond similarly to exposure. Although the Comet assay showed enhanced sensitivity, the two analyses are complementary and suitable for the biomonitoring of sediment contaminants in a benthic species like S. senegalensis.     

Genotoxic damage in Solea senegalensis exposed to sediments from the Sado Estuary (Portugal): Effects of metallic and organic contaminants

Juvenile Solea senegalensis (Senegalese sole) were exposed to freshly collected sediments from three sites of the Sado Estuary (West-Portuguese coast) in 28-day laboratory assays in order to assess the ecological risk from sediment contaminants, by measuring two genotoxicity biomarkers in peripheral blood: the percentage of Erythrocyte Nuclear Abnormalities (ENA) by use of an adaptation of the micronucleus test, and the percentage of DNA strand-breakage (DNA-SB) with the Comet assay. Sediments were surveyed for metallic (Cr, Ni, Cu, Zn, As, Cd and Pb) and organic (PAHs (polycyclic aromatic hydrocarbons), PCBs (polychlorinated biphenyls) and DDTs (dichloro-diphenyl-trichloroethane)) contaminants. Sediments from site A (farthest from hotspots of contamination) were found to be the least contaminated and weaker inducers of genotoxic damage, whereas sediments from sites B (urban influence) and C (affected by industrial effluents and agricultural runoffs) were responsible for a very significant increase in both ENA and DNA-SB, site B being most contaminated with metals and site C mainly with organic pollutants, especially PAHs and PCBs . Analysis of genotoxic effects showed a strong correlation between the concentrations of PAHs and PCBs and both biomarkers at sampling times T₁₄ and T₂₈, while the amounts of Cu, As, Cd and Pb were less strongly correlated, and at T₂₈ only, with ENA and DNA-SB. These results show that organic contaminants in sediment are stronger and faster acting genotoxic stressors. The results also suggest that metals may have an inhibitory effect on genotoxicity when interacting with organic contaminants, at least during early exposure. ENA and DNA-SB do not show a linear relationship, but a strong correlation exists between the overall increase in genotoxicity caused by exposure to sediment, confirming that they are different, and possibly non-linked effects that respond similarly to exposure. Although the Comet assay showed enhanced sensitivity, the two analyses are complementary and suitable for the biomonitoring of sediment contaminants in a benthic species like S. senegalensis.     

Effects of a sewage treatment plant outlet pipe extension on the distribution of contaminants in the sediments of the Bay of Vidy, Lake Geneva, Switzerland

In 2001, the municipality of Lausanne extended the outlet pipe of the sewage treatment plant into the Bay of Vidy (Lake Geneva, Switzerland) as a measure to reduce bacterial water pollution and sediment contamination close to the lake beaches. The aim of the present study was to assess the impact of this measure. Lake bottom sediments were collected and analyzed for grain size, organic matter, organic carbon, nitrogen, phosphorus, heavy metals and hydrophobic organic compounds to evaluate their concentration and spatial distribution. Our results demonstrate that, compared to results obtained before the outlet pipe prolongation, the surface area of highly contaminated sediments was reduced by one third after the pipe extension. However, contaminant concentrations are still high and the accumulation of pollutants may represent a significant source of toxicity for benthic organisms. One concludes that contaminant reduction at the source will be necessary for a further improvement.     

A novel statistical approach for the evaluation of comet assay data

The present study forms part of a weight-of-evidence framework including genotoxicological studies in the upper Danube River basin, which aim at elucidating the reasons for the decline in fish catch. The major focus of this paper is the assessment of genotoxicity of sediments from the Danube River basin by use of the comet assay with RTL-W1 cells and with embryos of zebrafish (Danio rerio). A frequently discussed question in this type of approach is how to aggregate and compare the data obtained from genotoxicity testing. There is a need to develop mathematical method combining the information from dose-response curves and level of effectiveness (maximum genotoxic effect). For comparison and ranking of the genotoxic potential of samples from different locations along the Danube River, several methods based on EC50, Lowest Observed Effect Concentration (LOEC), and maximum induction factor were compared with respect to their validity. An evaluation system termed the "3-step analysis" was developed to facilitate consideration of a maximum number of aspects of the raw data. The so-called "concentration-dependent induction factor" (CDI) introduces an index for a straightforward, precise and realistic assessment of the genotoxic potential of any kind of field sample or genotoxic agent.     

Surficial sediment phosphorus fractions along a biogeochemical gradient in Nyanza (Winam) Gulf, northeastern Lake Victoria and their possible role in phosphorus recycling and internal loading

Different phosphorus fractions and metal element composition of surficial sediments were measured on three occasions in 2005 and 2006 along a transect between Nyanza Gulf and offshore Lake Victoria, in order to assess the potential for sediments to contribute to the water column P concentrations in Lake Victoria. Total phosphorus (TP), apatite phosphorus (AP), inorganic phosphorus (IP) and organic phosphorus (OP) increased in sediments along the gulf towards the main lake while the non-apatite inorganic phosphorus (NAIP) increases were less defined. The longitudinal gradient of sediment TP and its fractions in Nyanza Gulf is a result of high rates of terrigenous input and resuspension and transport of the light, phosphorus rich inorganic and organic matter towards the main lake. TP in the sediment ranged from 812.7 to 1,738 mg/kg dry weight (DW) and was highest in the Rusinga Channel, the exchange zone between the gulf and the main lake. AP was the most important TP fraction, contributing between 35 and 57.3% of TP. Ca content in the sediment was strongly associated with TP and AP in the sediment (r² = 0.92 and 0.98, respectively) in the gulf and the channel, indicating the importance of apatite in controlling P availability in these zones. In the gulf and the Rusinga Channel, the less bioavailable apatite phosphorus dominated, whereas in the deeper main lake OP was the major fraction illustrating the importance of anaerobic release of P from sediments and acceleration of internal P loading in the main lake.     

A permanent fish cell line (EPC) for genotoxicity testing of marine sediments with the comet assay.

Genotoxicity and cytotoxicity were evaluated in an in vitro system with a permanent cell line Epithelioma papulosum cyprini (EPC) derived from a skin tumour of carp (Cyprinus carpio L.). EPC cells were exposed to different concentrations of organic sediment extracts from the North Sea for 24h. After incubation the cells were analysed for viability and DNA strand breaks with the comet assay or single cell gel electrophoresis (SCGE). The results confirm the sensitivity of this assay. Out of 10 marine sediment samples from the North Sea, 9 showed a dose-dependent genotoxic effect. The EC(50) of sediment extracts ranged from 7 to 307 mg sediment dry weight/ml assay volume. Hepatic microsomal enzymes from dab (Limanda limanda L.) was proposed for enzymatic activation of benzo[a]pyrene (BAP) or sediment extracts, respectively. The suitability of this in vitro test system for assessing genotoxic and cytotoxic effects of marine sediment extracts on EPC cells could be demonstrated.     

Phosphorus fractions and alkaline phosphatase activity in sediments of a large eutrophic Chinese lake (Lake Taihu)

Spatial, vertical, and seasonal variations in phosphorus fractions and in alkaline phosphatase activity (APA) were investigated in sediments in a large-shallow eutrophic Chinese lake (Lake Taihu) in 2003–2004. The phosphorus content was highest in the most seriously polluted lake area. Iron-bound phosphorus (Fe(OOH)～P) dominated (47% on average) among the phosphorus fractions determined according to Golterman (Hydrobiologia 335:87–95, 1996). Notably, organically-bound P comprised a further significant additional portion (acid-soluble + hot NaOH-extractable organic P = 25%), which was highest at the most polluted sites. The Fe(OOH)～P content was the lowest in spring (April, 2004), suggesting that degradation of organic matter led to the release of iron-bound phosphates. Sediment APA showed a significant positive relationship with both organically-bound P and Fe(OOH)～P. Consequently, organically-bound P is an important portion of the sediment phosphorus in Lake Taihu. It is mainly derived from freshly-settled autochthonous particles and from external discharges. Organically-bound P induces APA and may lead to the release of bioavailable phosphates from the organic sediments, thereby accelerating lake eutrophication.     

The Nicotine-Evoked Locomotor Response: A Behavioral Paradigm for Toxicity Screening in Zebrafish (Danio rerio) Embryos and Eleutheroembryos Exposed to Methylmercury

This study is an adaptation of the nicotine-evoked locomotor response (NLR) assay, which was originally utilized for phenotype-based neurotoxicity screening in zebrafish embryos. Zebrafish embryos do not exhibit spontaneous swimming until roughly 4 days post-fertilization (dpf), however, a robust swimming response can be induced as early as 36 hours post-fertilization (hpf) by means of acute nicotine exposure (30–240μM). Here, the NLR was tested as a tool for early detection of locomotor phenotypes in 36, 48 and 72 hpf mutant zebrafish embryos of the non-touch-responsive maco strain; this assay successfully discriminated mutant embryos from their non-mutant siblings. Then, methylmercury (MeHg) was used as a proof-of-concept neurotoxicant to test the effectiveness of the NLR assay as a screening tool in toxicology. The locomotor effects of MeHg were evaluated in 6 dpf wild type eleutheroembryos exposed to waterborne MeHg (0, 0.01, 0.03 and 0.1μM). Afterwards, the NLR assay was tested in 48 hpf embryos subjected to the same MeHg exposure regimes. Embryos exposed to 0.01 and 0.03μM of MeHg exhibited significant increases in locomotion in both scenarios. These findings suggest that similar locomotor phenotypes observed in free swimming fish can be detected as early as 48 hpf, when locomotion is induced with nicotine.     

Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment

The development of comet assay for aquatic organisms is of particular relevance in light of the importance of coastal fisheries to several countries around the world. Two of the most common fish species native to southern Brazil are the gray mullet (Mugil sp.) and sea catfish (Netuma sp.) for which we have produced a standardized comet assay using whole erythrocytes taken from samples of these fish. We investigated the potential of the comet assay for monitoring genotoxicity in mullet and sea catfish and made a preliminary investigation of the baseline levels of DNA damage in the erythrocytes of samples of these fish from non-polluted areas as well as assessing the in vitro sensitivity of erythrocyte exposed to 2, 4 and 8 x 10(-5) M of methyl methanesulfonate (MMS) for 1, 2, 6 and 24h at 25 and 37 degrees C. Our results show that there was an increase in baseline DNA damage at higher temperatures and that the amount of MMS-induced DNA damage also increased at higher temperatures and that there was a clear dose/time response to treatment with MMS. To assess the possibility of using fish for environmental biomonitoring we also used the comet assay to investigate the in vitro genotoxic effect of MMS on whole blood cells from human donors and found a clear concentration-related effect at all exposure times, findings which agree with those of other workers. This study demonstrates the potential application of the comet assay to erythrocytes of mullets and sea catfish. However, these findings also suggest that temperature could alter both baseline DNA damage in untreated animals and in vitro cell sensitivity towards genotoxic pollutants.     

A bioassay to assess the potential effects of sediment resuspension on phytoplankton community composition

A laboratory assay (SAGA or Sediment Algal Growth Assay) was developed to assess the potential impact of sediment resuspension on the structure of phytoplankton communities, and to evaluate the effectiveness of various sediment treatments in decreasing the abundance of blue-green algae in the event of sediment resuspension during storms. In assays with sediment from eutrophic Akanoi Bay, Lake Biwa, Japan, 7–11 species of phytoplankton seeded from the sediments grew during the 3-week assay indicating that sediment resuspension has the potential to increase both phytoplankton biomass and species diversity. Treatment of sediments with Ca(NO3)2 substantially decreased phytoplankton biomass (measured as chlorophyll concentration) in assays with sediments from Akanoi Bay and the North Basin of Lake Biwa. Further, among various oxidation treatments of sediments, Ca(NO3)2 was most effective in decreasing or preventing filamentous blue-green algal growth in N- and P-replete media. In contrast, when sediments were added to P-limited phytoplankton dominated by green algae and diatoms, no growth of blue-green algae occurred regardless of sediment treatment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Recent variations in sediment organic carbon content in Lake Soyang (Korea)

Four gravity cores were taken from Lake Soyang, which was created by the construction of an artificial dam in 1973. The variation of organic carbon content of the bottom sediments since the construction was interpreted using a variety of sedimentological and geochemical measurements. Based on the textural properties of sediments and the total organic carbon (TOC) content, it is clear that sediment organic carbon has been deposited very consistently throughout the postconstruction period, except for local sudden inputs caused by summer flooding events near fish farms or farmland areas. The autochthonous input resulting from the enhanced primary production led by phytoplanktons each year is likely to become a major problem at downstream sites within Lake Soyang. The vertical variation of δ¹³C values of sediment organic matter also reflects the fact that the level of autochthonous supply of organic carbon becomes high downstream.An erratum to this article can be found at .