Genotoxicity of marine sediments in the fish hepatoma cell line PLHC-1 as assessed by the Comet assay

The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest observed genotoxic potential was additionally extracted using various solvents in order to test which class of compounds, according to their polarity, is most responsible for the observed genotoxic effect. Non-polar solvents (cyclohexane and dichloromethane) yielded stronger genotoxic effect but the highest level of DNA damage was determined after exposure to sediment extract obtained with the solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and encourage the use of fish cell lines as versatile tools in ecogenotoxicology.     

Biological effects of marine contaminated sediments on Sparus aurata juveniles

Chemical analysis of the compounds present in sediment, although informative, often is not indicative of the downstream biological effects that these contaminants exert on resident aquatic organisms. More direct molecular methods are needed to determine if marine life is affected by exposure to sediments. In this study, we used an aquatic multi-species microarray and q-PCR to investigate the effects on gene expression in juvenile sea bream (Sparus aurata) of two contaminated sediments defined as sediment 1 and 2, respectively, from marine areas in Northern Italy. Both sediments affected gene expression as evidenced by aquatic multi-species microarray analysis and q-PCR. Exposure of S. aurata juveniles to sediment 1 and sediment 2 altered expression of genes that are biomarkers for endocrine disruption. There were differences between the effects of sediment 1 and sediment 2 on gene expression in S. aurata juveniles indicating that the chemicals in the two sediments had different physiological targets. These results suggest that the classification of sediment solely on the basis of specific chemical profiles is inadequate, and not a true indicator of its potential to cause harmful effects. Our data also indicate that integration of physiochemical analysis and bioassays for monitoring the downstream harmful effects on aquatic organisms are required to gain a complete understanding of the effects of sediment on aquatic life.     

Ecotoxicological effects at contaminated sites

Contamination sites pose significant environmental hazards for terrestrial and aquatic ecosystems. They are important sources of pollution and may result in ecotoxicological effects on terrestrial, groundwater and aquatic ecosystems. At severely contaminated sites, acute effects occur, but the core problem lies in long-term chronic effects. Ecotoxicological effects occur at all levels of biological organization, from the molecular to the ecosystem level. Not only certain organisms may be affected, but the ecosystems as a whole, both terrestrial and aquatic, in its function and structure. Contaminants at large contaminated sites often share critical properties such as high acute and/or chronic toxicity, high environmental persistence, often high mobility leading to contamination of groundwater, and high lipophilicity leading to bioaccumulation in food webs. Contaminants present at polluted sites occur as mixtures, therefore interactions between individual compounds are of importance. The bioavailability is a key factor for ecotoxicological effects of contaminants. This is demonstrated by a case study on organotins. Organotins belong to the most toxic pollutants known so far for aquatic life. Widespread contamination of harbor sediments occurs globally due to the ongoing use of organotins in antifouling paints in large ships. In lake sediments, tributyl- and triphenyltin are very persistent and bioavailable to biota even after a long time. Bioavailability of these compounds is dependent on pH and organic matter. Organotins are accumulated in sediments, but remobilization occurs when contaminated sediments are disturbed and dredged. A key question in dealing with contaminated sites is the assessment and evaluation of the toxicity of contaminants to the environment. Usually, established OECD tests and whole effluent toxicity tests are performed for an ecotoxicological evaluation and for hazard assessment. However, these assays are often expensive, laborious and sometimes not sensitive enough. As a consequence, we have used fast and reliable in vitro systems such as fish cell lines for the evaluation of sediments and landfill leachates contaminated by polychlorinated hydrocarbons (PAH). Determination of cytotoxicity as a measure for acute toxicity, and induction of cytochrome P4501A (CYP1A) as a biomarker of exposure and toxicity were found to be important measures, which can be used for hazard and risk assessment. We have developed a concept for the ecotoxicological evaluation of PAH contamination based on induction equivalents, which can be applied for aquatic and terrestrial ecosystems. One of the key question and present gaps, however, includes the long-term chronic ecotoxicological effects on soil and aquatic biota, which are largely unknown.     

Evaluation of the genotoxicity of environmental contaminants in sediments to rainbow trout hepatocytes

Rainbow trout hepatocytes were used as an in vitro bioassay to assess the genotoxic potential of single chemicals and marine sediment extracts. Freshly prepared trout hepatocytes were exposed to either benzo[a]pyrene, N-methyl-N′-nitro-N-nitrosoguanidine, β-naphtoflavone, or organic extracts of marine sediments for 24 h at 15°C. Genotoxicity was assayed using the nick translation assay, which makes use of a nonradioactive nucleotide (biotin-dUTP), and the DNA alkaline precipitation assay followed by fluorometric detection of DNA strands. Exposure to benzo[a]pyrene or methyl-N′-nitro-N-nitrosoguanidine, known indirect-and direct-acting genotoxins respectively, produced genotoxicity to rainbow trout hepatocytes with both assays. β-Naphtoflavone displayed genotoxic activity in trout hepatocytes. Sediment extracts and reference sediment extracts displayed high toxicity and genotoxicity to trout hepatocytes. Chemical analyses showed that these sediments contained significant amounts of organochlorine pesticides, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons. Cell toxicity was correlated with total levels of organochlorine pesticides and polychlorinated biphenyls but not total levels of polycyclic aromatic hydrocarbons. No positive correlation was found with the nick translation assay between total levels of chemicals and genotoxicity in marine sediments. Genotoxicity obtained with the alkaline precipitation assay was correlated with levels of the organochlorine pesticide DDT. However, more tests would be required to further substantiate possible links with other specific chemicals. © by John Wiley & Sons, Inc.     

Dietary accumulation of PCBs from a contaminated sediment source by a demersal fish (Leiostomus xanthurus)

Accumulation and dietary transfer of PCBs from contaminated harbor sediments were studied in a laboratory food chain consisting of sediments, polychaetes (Nereis virens) and a predatory fish (Leiostomus xanthurus). The study was conducted in two phases to distinguish dietary uptake from PCB accumulation resulting from sediment exposure alone. In phase I fish and polychaetes were separately exposed to field-collected PCB contaminated sediments (5.2 μg/g dry weight as Aroclor 1242 and 1254) in flow-through sea-water systems for 40 days to allow organisms to attain steady state concentrations. In Phase II the dietary fraction of PCB accumulation was determined by selectively feeding exposed and control groups of fish polychaetes having a known PCB body burden. In addition the effect of direct sediment contact on PCB accumulation by L. xanthurus was investigated. Results demonstrate that contaminated sediments can serve as a source of PCBs for uptake and trophic transfer in marine systems. Fish exposed to PCB-contaminated sediments and fed a daily diet of polychaetes from the same sediment accumulated more than twice the PCB whole-body residues than fish exposed to similar conditions but fed uncontaminated polychaetes. The dietary contribution of PCBs accounted for 53% of the total body burden measured in fish fed for 20 days, and this percentage appeared to be increasing. Results also indicate that fish isolated from direct contact with PCB-contaminated sediment do not significantly (P ≤ 0.05) accumulate PCB residues when compared with fish allowed contact with sediment.     

Toxicity assessment of urban marine sediments from Western Norway using a battery of stress-activated receptors and cell-based bioassays from fish

A luciferase reporter gene-based bioassay battery consisting of stress-activated receptors from fish, complemented with traditional fish cell-based bioassays, were used to assess the toxicity of marine sediment samples from the Byfjorden area around the city of Bergen (Norway). The reporter assays covered a wide range of cellular signalling and metabolic pathways, representing different molecular initiating events in the adverse outcome pathway framework. Cytotoxicity, generation of reactive oxygen-species, and induction of 7-ethoxyresorufin-O-deethylase activity were analysed using fish liver and gill cell lines. Chemical analyses of the sediment extracts revealed complex contamination profiles, especially at the innermost stations, which contained a wide array of persistent organic pollutants, polycyclic aromatic hydrocarbons, and metals. Sediment extracts from these sites were more potent in activating the stress-activated receptors than the other extracts, reflecting their toxicant profiles. Importantly, receptor- and cell-based bioassays complemented the chemical analyses and provided important data for future environmental risk assessments of urban marine sediments.     

Endocrine effects of contaminated sediments on the freshwater snail Potamopyrgus antipodarum in vivo and in the cell bioassays in vitro

Lake Pilnok located in the black coal-mining region Ostrava-Karvina, Czech Republic, contains sediments highly contaminated with powdered waste coal. Moreover, population of the endangered species of narrow-clawed crayfish Pontastacus leptodactylus with high proportion of intersex individuals (18%) was observed at this site. These findings motivated our work that aimed to evaluate contamination, endocrine disruptive potency using in vitro assays and in vivo effects of contaminated sediments on reproduction of sediment-dwelling invertebrates. Chemical analyses revealed low concentrations of persistent chlorinated compounds and heavy metals but concentrations of polycyclic aromatic hydrocarbons (PAH) were high (sum of 16 PAHs 10mug/gdw). Organic extracts from sediments caused significant in vitro AhR-mediated activity in the bioassay with H4IIE-luc cells, estrogenicity in MVLN cells and anti-androgenicity in recombinant yeast assay, and these effects could be attributed to non-persistent compounds derived from the waste coal. We have also observed significant in vivo effects of the sediments in laboratory experiments with the Prosobranchian euryhaline mud snail Potamopyrgus antipodarum. Sediments from Lake Pilnok as well as organic extracts of the sediments (externally added to the control sediment) significantly affected fecundity during 8 weeks of exposure. The effects were stimulations of fecundity at lower concentrations at the beginning of the experiment followed by inhibitions of fecundity and general toxicity. Our study indicates presence of chemicals that affected endocrine balance in invertebrates, and emphasizes the need for integrated approaches combining in vitro and in vivo bioassays with identification of chemicals to elucidate ecotoxicogical impacts of contaminated sediment samples.     

In vitro biomonitoring in polar extracts of solid phase matrices reveals the presence of unknown compounds with estrogenic activity

Determination of estrogenic activity has so far mainly concentrated on the assessment of compounds in surface water and effluent. This study is one of the first to biomonitor (xeno-)estrogens in sediment, suspended particulate matter and aquatic organisms. The relatively polar acetone extracts from these solid phase matrices do not contain the well-known estrogenic compounds such as hormones, alkylphenols and phthalates. An in vitro 'estrogen receptor-mediated chemical activated luciferase gene expression' (ER-CALUX) assay was applied to samples from various locations in the Netherlands. Estrogenic activity measured in polar fractions of particulate matter and sediment extracts ranged from below detection limit to up to 4.5 pmol estradiol equivalents (EEQ)/g dry weight. Estrogenic activity in freshwater river sediments was up to five times higher compared to sediments from large lakes and coastal locations. Tissue extracts EEQs were determined in bream (Abramis brama), flounder (Platichthysflesus), freshwater mussels (Dreissena polymorpha) and marine mussels (Mytilus edulis). The highest biota EEQ levels were found in the freshwater zebra mussel (30 pmol EEQ/g lipid). One sample site showed greatly elevated EEQs in sediment and biota, which correlated with effects found in the wild populations of bream. The EEQ activity of the unknown compounds in the polar fraction mostly was much higher than the calculated EEQ levels based on known estrogens in the non-polar fraction (previously published data).     

Improved flatfish health following remediation of a PAH-contaminated site in Eagle Harbor, Washington

Eagle Harbor in Puget Sound, WA became a Superfund site in 1987 due to polycyclic aromatic hydrocarbons (PAHs) released chronically from a nearby creosoting facility. Early studies here (1983-1986) demonstrated up to an approximately 80% prevalence of toxicopathic liver lesions, including neoplasms, in resident English sole (Parophrys vetulus). These lesions in English sole are consistently associated with PAH exposure in multiple field studies, and one laboratory study. Later studies (1986-1988) incorporated biomarkers of PAH exposure and effect, including hepatic CYP1A expression and xenobiotic-DNA adducts, and biliary fluorescent aromatic compounds (FACs). Before site remediation, lesion prevalences and other biomarker values in this species from Eagle Harbor were among the highest compared to other sites in Puget Sound and the US Pacific Coast. To sequester PAH-contaminated sediments, in 1993-1994, a primary cap of clean sediment was placed over the most-contaminated 54acres, with a 15-acre secondary cap added from 2000-2002. Lesion prevalences and biomarker values before primary capping were reduced compared to 1983-1986, consistent with facility closure in 1988 and shore-based source controls begun in 1990. Liver lesion risk, hepatic CYP1A activities, and levels of biliary FACs from fish collected immediately after and at regular intervals up to 2 years after primary capping were variable relative to pre-capping. Over the entire monitoring period since primary capping (128 months), but particularly after 3 years, there was a significantly decreasing trend in biliary FACs, hepatic DNA adducts and lesion risk in English sole. In particular, lesion risk has been consistently low (<0.20) compared to primary cap initiation (set at 1.0), from approximately 4 years after primary capping through April 2004. These results show that the sediment capping process has been effective in reducing PAH exposure and associated deleterious biological effects in a resident flatfish, and that longer term monitoring of pollutant responses in biological resources, such as resident fish, is needed in order to demonstrate the efficacy of this type of remediation.     

Toxicity of elemental sulfur in sediments

Elemental sulfur occurs naturally in marine and limnic sediments. Elemental sulfur, brought in solution in aqueous media by using organic solvents such as methanol as carrier solvent, was toxic in a bacterial luminescence test, known as the Microtox test. Previously, it has been shown that the toxicity in the luminescence test of whole sediments also was correlated to i.a. elemental sulfur using multivariate statistical analysis. Organic solvent extracts of sediments obtained in receiving waters of effluents from a pulp and paper mill was toxic in the luminescence test, and using a toxicity evaluation procedure, the toxic substance was identified as octameric cyclic sulfur, S₈. The substance dominated the toxicity in extracts of both a contaminated sediment and a sediment from a control area. Since the toxicity in the Microtox test of aqueous solutions of S₈ decreased upon storage, a conversion process of the toxic form was indicated. Acute toxicity of S₈ was not limited to the luminescent bacteria in the Microtox test, but was observed in tests with fish larvae if tested with the transient form of elemental sulfur. Tests of acute toxicity with zebra fish and perch larvae were responsive to elemental sulfur. Probably, the toxic form of elemental sulfur is the single cyclic octamer, that due to low aqueous solubility, binding to particulate sediment material or aggregation is converted into a nontoxic form. Acute toxic effects may occur in sulfur containing sediments of varying redox potentials or where elemental sulfur deposits are turbated. © 1998 John Wiley & Sons, Inc. Environ Toxicol Water Qual 13: 217–224, 1998     

Comparison between two bivalve species as tools for the assessment of pollution levels in an estuarian environment

Estuaries are semi-enclosed marine areas with water short residence times. Estuary ecosystems show a higher susceptibility to contamination, as historically these sites are linked to urban and industrial development. Polycyclic aromatic hydrocarbons (PAH) are ubiquitous contaminants present in high quantities in these marine environments. Chemical analyses of sediments provides information regarding PAH pollution levels but not a direct measure of the toxicological effects attributed to these contaminants. Samples of sediments and of two bivalve species, Cerastoderma edule and Mytilus galloprovincialis, were collected from two locations (Corcubión and A Concha) in an estuary from northwestern Spain. The PAH levels in sediment and bivalve species and possible sources were determined. A moderate level and a low level of pyrogenic PAH contamination were observed in Corcubión and in A Concha, respectively. Genotoxic damage was evaluated in gills and hemocytes from mussels and cockles by means of the comet assay. DNA damage measured as DNAt values showed a reliable relationship with pollution load levels of the two sampling sites. The higher sensitivity of C. edule compared to M. galloprovincialis enables one to recommend including another species coupled with mussels for biomonitoring estuarine environments.     

Embryonic Fundulus heteroclitus responses to sediment extracts from differentially contaminated sites in the Elizabeth River,VA

Sites along the Elizabeth River are contaminated with polycyclic aromatic hydrocarbons (PAHs) from historical creosote production and other industrial processes. Previous studies have demonstrated that Atlantic killifish collected from sites throughout the Elizabeth River display resistance to the teratogenic effects of PAH-exposure in a manner commensurate with sediment PAH concentrations. The current study characterized various chemical pollutants in sediment and investigated the effects of aqueous sediment extracts from sites along the Elizabeth River to the cardiac development of Atlantic killifish embryos from fish collected from an uncontaminated reference site. Embryonic cardiac deformities were more prevalent after exposure to extracts from sites with high PAH loads. However, activation of cytochrome P4501A, a gene up-regulated by PAH-induction of the aryl hydrocarbon receptor and measured using an in ovo EROD assay, did not consistently increase with PAH concentrations. This work further characterizes sediments in the Elizabeth River, as well as provides insight into the evolutionary pressures at each ER site.

     

Relationships between microtox test results,extraction methods,and physical and chemical compositions of marine sediment samples

Samples of marine subtidal sediments collected in 1985 and 1986 from Elliott Bay, Washington, were analyzed at the Environmental Laboratories of the Municipality of Metropolitan Seattle as part of a larger study. Interstitial water and solvent extracts of subtidal sediments were analyzed by the Microtox procedure to determine toxicity. Particle size distribution, and concentration of oil and grease, metals, and polynuclear aromatic hydrocarbons, were compared to toxicity of interstitial water and solvent extracts. Physical and chemical compositions of sediments affects the toxicities of solvent extracts and interstitial water extracts differently. Samples with smaller particle sizes had relatively high concentrations of oil and grease and metals, and less toxic interstitial water. Samples with larger particles had less oil and grease and metals, and more toxic interstitial water. The opposite was true for the solvent extracts: samples with smaller particle sizes with relatively higher concentrations of oil and grease and metals had more toxic solvent extracts. Samples containing larger particles with relatively lower concentrations of oil and grease and metals had less toxic solvent extracts. No correlations were found between the toxicity of the solvent extracts and polynuclear aromatic hydrocarbons. The Microtox procedure has the potential to estimate toxicity of both water-soluble and solvent-soluble compounds in marine sediments if two different extraction procedures are used.     

Histological, growth and 7-ethoxyresorufin O-deethylase (EROD) activity responses of greenback flounder Rhombosolea tapirina to contaminated marine sediment and diet

Pathological abnormalities and mixed function oxygenase (MFO) enzyme changes are frequently used as indicators of anthropogenic contaminant exposure and effect. However, there is a paucity of research investigating the effects of contaminated sediment on native Australian benthic teleosts. As part of an ecotoxicological assessment of contaminated marine sediments in northern Tasmania, CYP1A induction, histological and growth response of the greenback flounder, Rhombosolea tapirina, exposed to contaminated marine sediments were examined. Hatchery reared flounder were exposed to reference sediment, contaminated sediment or contaminated sediment and diet for 6 weeks. CYP1A induction, using the ethoxyresorufin-O-deethylase (EROD) assay, and the histological and growth response in the flounder were examined on cessation of the exposure trial. Significant differences were found between treatments in histological, growth and EROD response. Exposure to contaminated sediment and diet elicited a multi-organ histological response: principally partial and total epidermal erosion and multifocal necrosis of the liver. The prevalence of total epidermal erosion was greatest with exposure to disturbed contaminated sediment (66.65+/-16.65%). The prevalence of multifocal necrosis of the liver was greatest with exposure to contaminated sediment and diet (66.65+/-16.65%). Growth reduction, measured as percentage growth inhibition, was evident in flounder exposed to contaminated sediment and diet (18.2+/-11.99%). Additionally, exposure to contaminated sediment and diet elicited elevated induction of the EROD liver detoxification enzyme (139.65+/-24.22 pmol/min/mg protein) compared to exposure to contaminated sediment and non-contaminated diet (6.25+/-0.81 pmol/min/mg) indicating the presence and potential bioavailability of xenobiotics via food. Further, more inhibited growth and histological alteration associated with exposure to contaminated sediment and diet suggest contaminants in Deceitful Cove sediment are cytotoxic.     

Assessment of polynuclear aromatic hydrocarbons ecotoxicity threshold in marine sediments through in situ input/decay measurements

The ability of decomposers to process variable amounts of xenobiotics in the marine sediment is a useful aggregate indicator of their capacity to prevent their accumulation and eventual ecotoxic effects. Since decomposition processes depend on environmental factors at the sediment which are difficult to mimic in laboratory systems, in situ evaluations in undisturbed sediments are of great interest. A method and its results are presented to evaluate the decomposition rates of PAHs (polynuclear aromatic hydrocarbons) in coastal undisturbed marine sediments at different levels of pollution input. The method is based on the application of pulse chromatography concepts to interpret trap and bed sediment monitoring data obtained at regular time intervals, using models of the water column as an anisotropic carrying medium. The results are for a 14 month data series from moderately polluted sediments near an urban site and at a more distant nearly pristine site on the south Atlantic coast. QSAR (quantitative structure activity relations) indicate that decay rates increase with higher UV absorption and lipidic solubility. At low levels of total PAH input to the sediments (<0.05 g day^–1 g^–1), decomposition mechanisms effectively process these compounds within a few days. At higher input levels (up to 0.12 g day^–1 g^–1), decomposition lags behind the inputs by approximately 25% and PAHs accumulate in the sediment. In situ estimates of the PAH input/decay ratios provide reliable ecosystem indicators of a safe threshold for anthropogenic inputs of PAHs to the marine environment and a basis for receptor-based standards aimed at their regulation.     

In Vitro Biomonitoring in Polar Extracts of Solid Phase Matrices Reveals the Presence of Unknown Compounds with Estrogenic Activity

Determination of estrogenic activity has so far mainly concentrated on the assessment of compounds in surface water and effluent. This study is one of the first to biomonitor (xeno-)estrogens in sediment, suspended particulate matter and aquatic organisms. The relatively polar acetone extracts from these solid phase matrices do not contain the well-known estrogenic compounds such as hormones, alkylphenols and phthalates. An in vitro estrogen receptor-mediated chemical activated luciferase gene expression (ER-CALUX) assay was applied to samples from various locations in the Netherlands. Estrogenic activity measured in polar fractions of particulate matter and sediment extracts ranged from below detection limit to up to 4.5 pmol estradiol equivalents (EEQ)/g dry weight. Estrogenic activity in freshwater river sediments was up to five times higher compared to sediments from large lakes and coastal locations. Tissue extracts EEQs were determined in bream (Abramis brama), flounder (Platichthys flesus), freshwater mussels (Dreissena polymorpha) and marine mussels (Mytilus edulis). The highest biota EEQ levels were found in the freshwater zebra mussel (30 pmol EEQ/g lipid). One sample site showed greatly elevated EEQs in sediment and biota, which correlated with effects found in the wild populations of bream. The EEQ activity of the unknown compounds in the polar fraction mostly was much higher than the calculated EEQ levels based on known estrogens in the non-polar fraction (previously published data).     

CYP1A induction potential and the concentration of priority pollutants in marine sediment samples -In vitro evaluation using the PLHC-1 fish hepatoma cell line

The use of in vitro biotests in combination with chemical determination of priority pollutants is considered a promising approach in environmental risk assessment. The main goal of this study was to evaluate the relationship between the CYP1A induction potential and the concentration of priority pollutants (PAHs, PCBs and heavy metals) in contaminated marine sediments. Six sediment samples characterized by different types of pollution were collected from the Bay of Kvarner, Croatia. CYP1A induction potency was determined in vitro by the measurement of ethoxyresorufin-O-deethylase (EROD) activity in PLHC-1 fish hepatoma cells. The results were compared to the potency of the model CYP1A inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and expressed in 2,3,7,8-TCDD equivalents. All of the tested sediment samples were able to induce CYP1A activity in a dose-dependent manner. On a general scale, there was a good correlation between CYP1A induction and the concentration of priority pollutants in the tested samples. However, some samples, which had relatively low levels of priority pollutants, exhibited a strong CYP1A induction response. Therefore, apart from the confirmed usability and sensitivity of the EROD determination in the PLHC-1 cells as a suitable in vitro model in ecotoxicology, the results of this study indicate that the list of priority pollutants usually determined in the attempt to evaluate the risk of adverse effects to marine wildlife should be reconsidered.     

Evaluation of cytotoxicity,genotoxicity and teratogenicity of marine sediments from Qingdao coastal areas using in vitro fish cell assay,comet assay and zebrafish embryo test

Marine sediments are often a final sink for numerous anthropogenic contaminants and may impose serious effects on benthic organisms and ecosystem. An in vitro cell assay using a cell line derived from flounder gill (FG) cells, an in vitro comet assay in FG cells, and an in vitro zebrafish embryo assay were used to evaluate the in vitro cytotoxicity (measured by MTT reduction), genotoxicity and teratogenicity of crude sediment extracts of Li Cang (LC), Zhan Qiao (ZQ) and Olympic Sailing Center (OSC) from Qingdao coastal area. Sediments from the three sites displayed different cytotoxicity, genotoxicity and teratogenicity potencies; however, all three assays yielded similar LOECs (lowest observed effect concentration) for each site, suggesting that the assays were equally sensitive to and suitable for initial screening of the LOECs of marine sediments. The cytotoxicity, genotoxicity and teratogenicity for these three sampling sites were in the same order of LC > ZQ > OSC, indicating different degrees of contamination. Interestingly, trials with the three sediment extracts at the doses inducing a similar cytotoxicity as evaluated with MTT reduction did not produce similar genotoxicity and teratogenicity, with the genotoxic and teratogenic activities of LC and ZQ extracts being markedly higher than those of OSC sediments. These findings indicate that cytotoxicity does not form a fully equivalent toxicity index with that of genotoxicity and teratogenicity. Therefore, in order to assess the true toxic potential of marine sediments, all three assays should be performed. Analysis of 16 EPA (US Environmental Protection Agency) priority PAHs in these three sediment samples showed a clear correlation between PAH concentrations and sediment toxicities, with a higher PAH content corresponding to higher toxicity although PAHs are surely not the only cause.     

Evidence of population genetic effects of long-term exposure to contaminated sediments-a multi-endpoint study with copepods

In the environment, pollution generally acts over long time scales and exerts exposure of multiple toxicants on the organisms living there. Recent findings show that pollution can alter the genetics of populations. However, few of these studies have focused on long-term exposure of mixtures of substances. The relatively short generation time (ca. 4-5 weeks in sediments) of the harpacticoid copepod Attheyella crassa makes it suitable for multigenerational exposure studies. Here, A. crassa copepods were exposed for 60 and 120 days to naturally contaminated sediments (i.e., Svindersviken and Trosa; each in a concentration series including 50% contaminated sediment mixed with 50% control sediment and 100% contaminated sediment), and for 120 days to control sediment spiked with copper. We assayed changes in F(ST) (fixation index), which indicates if there is any population subdivision (i.e., structure) between the samples, expected heterozygosity, percent polymorphic loci, as well as abundance. There was a significant decrease in total abundance after 60 days in both of the 100% naturally contaminated sediments. This abundance bottleneck recovered in the Trosa treatment after 120 days but not in the Svindersviken treatment. After 120 days, there were fewer males in the 100% naturally contaminated sediments compared to the control, possibly caused by smaller size of males resulting in higher surface: body volume ratio in contact with toxic chemicals. In the copper treatment there was a significant decrease in genetic diversity after 120 days, although abundance remained unchanged. Neither of the naturally contaminated sediments (50 and 100%) affected genetic diversity after 120 days but they all had high within treatment F(ST) values, with highest F(ST) in both 100% treatments. This indicates differentiation between the replicates and seems to be a consequence of multi-toxicant exposure, which likely caused selective mortality against highly sensitive genotypes. We further assayed two growth-related measures, i.e., RNA content and cephalothorax length, but none of these endpoints differed between any of the treatments and the control. In conclusion, the results of the present study support the hypothesis that toxicant exposure can reduce genetic diversity and cause population differentiation. Loss of genetic diversity is of great concern since it implies reduced adaptive potential of populations in the face of future environmental change.     

A whole sample toxicity assessment to evaluate the sub-lethal toxicity of water and sediment elutriates from a lake exposed to diffuse pollution

The impact of diffuse pollution in aquatic systems is of great concern due to the difficult to measure and regulate it. As part of an ecological risk assessment (ERA), this study aims to use a whole sample toxicity assessment to evaluate the toxicity of water and sediment from Lake Vela, a lake that has been exposed to diffuse pollution. In this way, standard (algae: Pseudokirchneriella subcapitata; cladoceran: Daphnia magna) and local species (algae: Aphanizomenon flos-aquae; cladoceran: Daphnia longispina) were exposed to surface water, and sediment elutriates were collected seasonally from two sites at Lake Vela: one near the east bank (ES), surrounded by agricultural lands; and the other near the west bank (WS), surrounded by a forest. The results confirmed the seasonal contamination of both environmental compartments by pesticides, including organochlorine pesticides, and the presence of high concentrations of nutrients. Although both sites were contaminated, higher levels of pesticides and nutrients were detected in ES, particularly in the sediments. Bioassays showed that water samples (100% concentration) collected in summer and autumn significantly affected the growth rate of P. subcapitata, which could be attributed to the presence of pesticides. Likewise, they revealed an apparent toxicity of elutriates for P. subcapitata and for both daphnids, in summer and autumn. In fact, although pesticides were not detected in elutriates, high levels of un-ionized ammonia were recorded, which is considered highly toxic to aquatic life. By comparing the several species, P. subcapitata was revealed to be the most sensitive one, followed by the daphnids, and then by A. flos-aquae. Results obtained in this study underlined the importance of whole samples toxicity assessment for characterizing the ecological effects of complex mixtures from diffuse inputs, in the ERA processes.