Passive samplers for chemical substance monitoring and associated toxicity assessment in water.

The European legislation, and in particular the Water Framework Directive requires the development of cost efficient monitoring tools that can provide the required information for the assessment of water contamination. Passive sampling methods represent one of the novel tools that have a potential to be used in various regulatory monitoring programmes aimed at assessing the levels of chemical pollutants. These methods are particularly interesting for sampling polar organic pollutants in water because they provide representative information of the water quality over extended time periods (days to weeks) in environments with fluctuating contaminant concentrations. This is achieved by integrative sampling of pollutants over the whole sampler deployment period. These tools can be coupled to toxicity testing using bioassays that give information on toxic and ecotoxic hazards associated to substances that are present, these substances being identified or not. In this study the polar organic chemical integrative sampler (POCIS) was used in surface water to evaluate the water contamination by polar organic compounds and their potential toxicity.

Assessment of the effects of discontinuous sources of contamination through biomarker analyses on caged mussels.

The present study analysed potential adverse effects of discontinuous sources of contamination, namely the discharge of a combined sewer overflow (CSO) and of runoff in an urban area, the Bay of Santander (North Iberian Peninsula). Water samples and caged mussels were used to analyse concentrations of contaminants and biological responses. Mussels (Mytilus galloprovincialis) were transplanted to a marina receiving runoff from a petrol station and to a CSO discharge site. Samples were collected in synchrony with heavy rains along 62days. Lysosomal membrane stability (LMS) and acyl-CoA oxidase (AOX) activity were measured as core biomarkers and were analysed at all sampling times. Histopathology of digestive gland and gonads, transcription levels of vitellogenin gene, volume density of black silver deposits and micronuclei formation were measured at initial and final stages of the transplant. Chemical analyses of metals, polycyclic aromatic hydrocarbons (PAHs) and endocrine disruptors were performed in water samples and mussel flesh. Mussels accumulated low concentrations of contaminants, which is in accordance with results obtained from exposure biomarkers. AOX activity decreased in all transplanted mussels after the first heavy rain, but this change seems to be related to the seasonal pattern of the enzyme activity. Mussels located close to the CSO discharge site showed a reduction in LMS after the first rain event, when compared to mussels before the transplant and to mussels from the reference location. However, this was attributable to natural environmental changes rather than to pollution. Values of the rest of analysed biomarkers were below threshold values reported for the study area.

Evidence of genotoxicity related to high PAH content of sediments in the upper part of the Seine estuary (Normandy, France).

In order to characterize the genotoxicity in the Seine estuary and Seine bay, chemical and toxicological analyses were performed on 17 sediments collected in June 2001 and June 2003. Many potent mutagenic and/or carcinogenic compounds - including polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and metals - were detected. Those compounds were found to be at relatively high concentrations in the upper part of the Seine estuary but were barely detectable at sites outside the plume from the Seine. The levels of pollution did not vary significantly between the two sampling periods, except that PAH concentrations in sediments collected at Oissel and Le Havre showed a marked increase in June 2003. The toxicity of organic extracts from sediments was evaluated by both embryotoxicity and in vitro genotoxicity (SOS Chromotest) assays. Organic extracts from sediments taken from the Seine estuary appeared significantly more embryotoxic than those from the Seine bay. In addition, the sediment extracts from the upper part of the Seine estuary exhibited higher genotoxicity than those from the lower part, and no genotoxicity was reported for sediments from the Seine bay. The genotoxic activity was detected only after adding an S9 microsomal fraction, suggesting the preponderant involvement of pro-genotoxic organic compounds. In addition, SOS Chromotest responses obtained with purified organic fractions revealed that PAH and, to a lesser extent, unknown polar organic compounds were probably responsible for this genotoxicity. Altogether, these results suggest that sediments from the upper Seine estuary are genotoxic and embryotoxic, and therefore, could be potentially hazardous for species living or feeding in the area.

DNA adduct measurements in zebra mussels, Dreissena polymorpha, Pallas. Potential use for genotoxicant biomonitoring of fresh water ecosystems.

The purpose of this study was to examine PAH accumulation and bulky DNA adduct formation in the digestive gland of zebra mussels exposed in their habitat or in controlled laboratory conditions to complex mixture of PAH. DNA adducts were measured using a 32P-postlabelling protocol with nuclease P1 enrichment adapted from Reddy and Randerath [Reddy, M.V., Randerath, K., 1986. Nuclease P1-mediated enhancement of sensitivity of 32P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551]. Specimens collected in the upper part of the Seine estuary were shown to accumulate higher levels of PAH (up to 1.6 microg g(-1) dry weight) in comparison to individuals from the reference site (0.053 microg g(-1) dry weight). The former exhibited elevated levels of DNA adducts (up to 4.0/10(8) nucleotides) and higher diversity of individual adducts with five distinct spots being specifically detected in individuals originating from the Seine estuary. Zebra mussels exposed for 5 days to 0.01% (v/v) of organic extract of sediment from the Seine estuary were shown to accumulate high amounts of PAH (up to 138 microg g(-1) dry weight) but exhibited relatively low levels of DNA adducts. Exposure to benzo[a]pyrene led to a dose-dependent accumulation of B[a]P (up to 7063 microg g(-1) dry weight) and a clear induction of DNA adduct formation in the digestive gland of mussels (up to 1.13/10(8) nucleotides). Comparisons with other bivalves exposed to the same model PAH, revealed similar levels of adducts and comparable adduct profiles with a main adduct spot and a second faint one. This study clearly demonstrated that zebra mussels are able to biotransform B[a]P and probably other PAH into reactive metabolites with DNA-binding activity. This work also demonstrated the applicability of the nuclease P1 enhanced 32P-postlabelling method for bulky adduct detection in the digestive gland of zebra mussels. DNA adduct measurement in zebra mussels could be a suitable biomarker to monitor PAH-exposure and evaluate genotoxicity in fresh water ecosystems.

Genotoxicant accumulation and cellular defence activation in bivalves chronically exposed to waterborne contaminants from the Seine River.

The aim of the present work was to investigate genotoxicant accumulation and biological responses of zebra mussels and blue mussels collected along a pollution gradient in the Seine estuary and in the Seine Bay. The sampling area included three contaminated and one reference sites for each species. The study focused on polyaromatic hydrocarbons (PAH), lindane, polychlorobiphenyls (PCB) and metals known to be potential genotoxicants and/or reactive oxygen species (ROS) inducers. Enzymatic activities related to cellular defence systems including the phase II enzyme glutathione S-transferase (GST) and three antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were measured in gills. DNA adducts and DNA strand breaks (Comet assay) were measured in digestive gland and hemocytes, respectively. Species differences were observed in metal accumulation (As and Pb), GPx activity and DNA adduct formation. A marked upstream-downstream gradient was reported for PAH body burden and to a lesser extent for PCB and metals with the highest values measured just downstream the industrialized area of Rouen. GST and SOD activities in gills of bivalves were positively related to PAH and metals body burden, respectively. Activation of those cellular defences may prevent accumulation of electrophilic metabolites and free radicals and thus may protect DNA and others macromolecules against oxidation and adduction. Although DNA strand breaks and bulky adducts were detected in both species, levels were relatively low and no significant site differences were observed in June 2003. Our results indicate a clear relationship between genotoxicant accumulation and positive activation of detoxification and antioxidant systems but poor consequences in term of DNA damage for wild population of mussels inhabiting the Seine estuary.

A multibiomarker approach to the assessment of pollution impacts in two Baltic Sea coastal areas in Sweden using caged mussels (Mytilus trossulus).

Blue mussels (Mytilus trossulus) were transplanted in cages for three months in two Swedish coastal areas in the Bothnian Sea (northern Baltic Sea) to investigate the interactions between analysed environmental chemicals and biological responses. A wide array of biological parameters (biomarkers) including antioxidant and biotransformation activity, geno-, cyto- and neurotoxic effects, phagocytosis, bioenergetic status and heart rate were measured to detect the possible effects of contaminants. Integrated Biomarker Response index and Principal Component Analysis performed on the individual biological response data were able to discriminate between the two study areas as well as the contaminated sites from their respective local reference sites. The two contaminated sites outside the cities of Sundsvall (station S1) and Gävle (station G1) were characterised by different biomarker response patterns. Mussels at station S1 showed a low condition index, increased heart rate recovery time and phagocytosis activity coinciding with the highest tissue concentrations of some trace metals, polycyclic aromatic hydrocarbons and organotins. At station G1 the highest organochlorine pesticide concentration was recorded as well as elevations in glutathione S-transferase activity, thiamine content and low lysosomal membrane stability. Significant variability in the geno- and cytotoxic responses and bioenergetic status was also observed at the different caging stations. The results obtained suggest that different chemical mixtures present in the study areas cause variable biological response patterns in organisms.

Analysis of hormonal steroids in fish plasma and bile by coupling solid-phase extraction to GC/MS.

An analytical procedure for the simultaneous determination of twelve endogenous steroids (testosterone, androstenedione, 17beta-estradiol, estrone, pregnenolone, progesterone, dihydroandrostenedione, dihydrotestosterone, 11alpha-ketotestosterone, 17alpha-hydroxyprogesterone, 17alpha-hydroxypregnenolone, 17alpha,20beta-dihydroxy-4-pregnen-3-one) in plasma and bile samples by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC-MS) has been developed. After enzymatic hydrolysis for bile samples only, samples were concentrated and purified using two successive SPE (C(18) and NH(2)) cartridges. Analytes were derivatized with a mixture of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) / mercaptoethanol / ammonium iodide (NH(4)I) and determined by GC-MS in selective ion monitoring mode. For most of the steroids monitored, recoveries were in the range 90-120% in plasma and in the range 60-70% in bile, and the reproducibility was below 10% for the complete procedure. Limits of detection obtained ranged from 0.1 to 0.4 ng/g in fish plasma and from 1.6 to 14 ng/g in fish bile. The developed method was successfully applied to the determination of plasma steroids in flounders (Platichthys flesus) collected from two French estuaries.