Aquatic Toxicology

Alkylphenol-polyethoxylates (APnEO, n = 1-40) are a major group of surfactants and are normally present in raw sewage. Many of the products of the biodegradation of these compounds are both persistent and present in substantial quantities in effluent and in fiver water. We report here on the use of an in vitro bioassay to determine the oestrogenic potencies of these compounds to fish. The bioassay is based on the fact that the synthesis of vitellogenin by hepatocytes is oestrogen dependent. Of the compounds tested, 4-nonylphenol, 4-tert-octylphenol, 4-tert-butylphenol, 4-nonylphenol-diethoxylate, Tergitol-NP9, and 4nonylphenoxycarboxylic acid were all weakly oestrogenic, with potencies between about 1 x 10 -4 to 1 x 10 -6 the activity of 17fl-oestradiol The oestrogenic activity observed appeared to be confined topara or 4 substituted compounds, because 2-tert-butylphenol and 3-tert-butylphenol were inactive. The polyethoxylate compounds became less oestrogenic with increasing lengh of the ethoxy chain. Thus cells exposed to Tergitol NP40EO (with a chain length of 40) did not secrete vitellogenin. Simultaneous exposure of the hepatocytes to Tamoxifen (an oestrogen antagonist) and effective doses of representative compounds caused an inhibition of the oestrogenic effect in all cases, suggesting that the action of these compounds is mediated by the oestradiol receptor.

The literature on metallothioneins (MT) in aquatic invertebrates is large and increasing, and their use as biomarkers in environmental monitoring programmes.This work aims to study at a biochemical level, responses of bivalve commonly found on the North African coasts, Perna perna to metal contaminants (cadmium and copper) and the resulting oxidative stress. Bivalves were exposed in vivo during 7 days to cadmium (200μg/l) and copper (30μg/l). At the end of the experiment, gills and digestive glands were sampled. With regard to the results, a relationship between exposure to metals and the induction of MTs in detoxification processes were drawn and observed in the majority of cases. This study provides important new information that MT may be a suitable biomarker to assess contamination of the marine environment particularly by metals.

The synthetic estrogen, 17␣-ethinylestradiol (EE 2 ), is the active component in oral contraceptive pills. It is excreted from the human body in high amounts and released via sewage treatment plant effluent into aquatic environments. In fish, estrogen receptors have strong binding affinities for EE 2 , and exposure raises the possibility of adverse neuroendocrine responses in aquatic animals. In the present study we explored the effects of dissolved-phase EE 2 on the dynamics of male-male aggression and courtship behaviors in adult zebrafish. Further, we assessed whether the behavioral effects of EE 2 result in changes in male offspring paternity. We scored the aggressive behaviors of individual unexposed males and categorized these fish as either dominant or subordinate. We then exposed dominant males to EE 2 at doses of 0, 0.5, 5.0, and 50.0 ng/L for 48 h. Subsequent trials examined the agonistic behaviors of males in two testing scenarios: (1) a dyadic encounter with another male alone, and (2) a competitive spawning interaction with another male and three adult females. Competitive spawning tests were also used to assess the impacts of EE 2 exposure on courtship behavior and paternity using males that were homozygous for green fluorescent protein (GFP) expression under the control of the islet-1 promoter. We found that EE 2 at all exposure concentrations reduced male aggression during male-male dyadic encounters and caused a social dominance reversal in 50% of the fish at the highest exposure dose (50 ng/L EE 2 ). The frequency of courtship-specific behavior decreased in dominant males exposed to the steroid, though this effect was only significant for the lowest dose group (0.5 ng/L EE 2 ). In the highest exposure group (50 ng/L EE 2 ), 50% of dominant males relinquished paternal dominance. Our results show that short-term exposure to EE 2 at environmentally relevant levels can alter aggression, and shift individual social status and reproductive success in male zebrafish.

Aquatic toxicology has been defined as the qualitative and quantitative study of adverse or toxic effects of chemicals and other anthropogenic materials on aquatic organisms. The subject also includes the study of transport, distribution, transformation and ultimate fate of chemicals in the aquatic environment. Within this multidisciplinary field of science, studies of the biochemistry and function of biotransformation enzymes in aquatic organisms hold a central role. Metabolism or biotransformation through the phase 1 (cytochrome P-450 monooxygenase enzymes) and phase 11 (conjugating enzymes) pathway is a requisite for detoxification and excretion of lipophilic chemicals. In addition, such a transformation is also responsible for the activation of foreign chemicals to the intermediates that ultimately result in toxicity, carcinogenicity, and other adverse effects. The dual role of many of these enzyme systems, being involved in both xenobiotic and endogenous metabolism, furthermore makes interactions between foreign chemicals and physiological processes possible. Lastly, the response of some of these enzyme systems, in particular the cytochrome P-450 I Al subfamily, to organic xenobiotics such as oil hydrocarbons, PCBs, dioxins and dibenzofurens. makes analysis of enzyme levels by catalytic or immunochemical methods a potent way to monitor pollution effects at the molecular level. Several of these aspects will be discussed with special reference to fish.

The effects of exposure to metals under and laboratory conditions were investigated in the Mediterranean mussel Mytilus gdoprovinciulis. The examined biological responses included the concentrations of heavy metals, the level of glutathione, and the activity of several enzymes selected among glutathione-dependent oxidoreductases and hydrolases: glutathione reductase.

We have identified a novel vertebrate-specific gene by applying a Differential Display method on two distinct subtypes of pituitary melanotropes showing divergent secretory phenotypes of hypo-and hypersecretion. A paralogue of this gene was also identified. The existence of a long coiled-coil domain and a C-terminal transmembrane domain in the sequences, together with the Golgi distribution of the proteins in transfected cells, suggest that they can be considered as new members of the golgin family of proteins. Both genes were primarily expressed in (neuro)endocrine tissues in vertebrates thus supporting a role for these proteins in the regulated secretory pathway.

Two simple, accurate, precise, reproducible, requiring no prior separation and economical procedures for simultaneous estimation of Losartan Potassium and Amlodipine Besylate in tablet dosage form have been developed. First method employs formation and solving of simultaneous equation using 208 nm and 237.5 nm as two analytical wavelengths for both drugs in methanol. The second method is Q value analysis based on measurement of absorptivity at 242.5 nm (as an iso-absorptive point) and 237.5 nm. Losartan Potassium and Amlodipine Besylate at their respective max 208 nm and 237.5 nm and at isoabsorptive point 242.5 nm shows linearity in a concentration range of 2-20 g/mL. Recovery studies range from 99.95% for Losartan Potassium and 99.33% for Amlodipine Besylate in case of simultaneous equation method and 102.93% for Losartan Potassium and 101.02% for Amlodipine Besylate in case of Q -analysis method confirming the accuracy of the proposed method. The proposed method is recommended for routine analysis since it is rapid, simple, accurate and also sensitive and specific by no heating and no organic solvent extraction.

It is now well established that there is a diverse array of chemicals discharged into the environment that can mimic or antagonise the action of hormones. These endocrine-disrupting chemicals (EDCs) can thus interact with physiological systems and cause alterations in development, growth and reproduction in wildlife that are exposed to them. As yet, however, there is little information on the relative sensitivities of different wildlife groups to these chemicals and/or mixtures of them (e.g. estrogenic effluents) and hence, there are fundamental shortfalls in our knowledge of the ecological importance of endocrine disruption in wildlife. In this study, the effects of exposure to individual estrogenic chemicals (17a-ethinylestradiol; EE 2 , bisphenol-A, and 4-tert octylphenol) and a mixture containing these chemicals (treated sewage effluent) on embryo production in the prosobranch mollusc, Potamopyrgus antipodarum , were studied and compared with the effects of EE 2 and the same estrogenic effluent on vitellogenin induction and/or egg production in various species of freshwater fish (fathead minnow; Pimaphales promelas , rainbow trout (Oncorhynchus mykiss ); Cyprinus carpio , carp; Cyprinus carpio ). The lab-based studies demonstrated that all of the tested chemicals (known to be estrogenic and to cause reproductive effects in fish) also affected embryo production in P. antipodarum . Furthermore, exposure to EE 2 induced similar reproductive responses in the snails as in the fathead minnow (Pimephales promelas), stimulating egg/embryo production at low doses (up to 1 ng/l in the minnow and 25 ng/l in the snail) and causing inhibitory effects at higher doses. A similar pattern of embryo production occurred in P. antipodarum when it was exposed to a graded concentration of treated sewage effluent containing mixtures of estrogenic EDCs and hence, the total number of new embryos produced by the snails increased steadily over the 9 weeks exposure period in treated snails. Plasma vitellogenin concentrations in two species of male fish (the rainbow trout and the carp) also increased over the same time period. These data indicate that both the nature of the response and the relative sensitivities to environmental estrogens in P. antipodarum and three different fish species fish are comparable. P. antipodarum is thus, potentially a sensitive test organism for assessing estrogenicity of chemicals with a relevance to their activity in vertebrates. #

We investigated whether exposure to environmentally relevant concentrations of the bactericidal agent, triclosan, induces changes in the thyroid hormone-mediated process of metamorphosis of the North American bullfrog, Rana catesbeiana and alters the expression profile of thyroid hormone receptor (TR) ␣ and ␤, basic transcription element binding protein (BTEB) and proliferating nuclear cell antigen (PCNA) gene transcripts. Premetamorphic tadpoles were immersed in environmentally relevant concentrations of triclosan and injected with 1 × 10 −11 mol/g body weight 3,5,3 -triiodothyronine (T 3 ) or vehicle control. Morphometric measurements and steady-state mRNA levels obtained by quantitative polymerase chain reaction were determined. mRNA abundance was also examined in Xenopus laevis XTC-2 cells treated with triclosan and/or 10 nM T 3 . Tadpoles pretreated with triclosan concentrations as low as 0.15 ± 0.03 g/L for 4 days showed increased hindlimb development and a decrease in total body weight following T 3 administration. Triclosan exposure also resulted in decreased T 3 -mediated TR␤ mRNA expression in the tadpole tail fin and increased levels of PCNA transcript in the brain within 48 h of T 3 treatment whereas TR␣ and BTEB were unaffected. Triclosan alone altered thyroid hormone receptor ␣ transcript levels in the brain of premetamorphic tadpoles and induced a transient weight loss. In XTC-2 cells, exposure to T 3 plus nominal concentrations of triclosan as low as 0.03 g/L for 24 h resulted in altered thyroid hormone receptor mRNA expression. Exposure to low levels of triclosan disrupts thyroid hormone-associated gene expression and can alter the rate of thyroid hormone-mediated postembryonic anuran development.

The induction of hepatic CYP1A by selected polycyclic aromatic hydrocarbons (PAHs) was followed in Atlantic salmon (Salmo salar) by measurement of CYP1A messenger RNA (mRNA), CYP1A protein levels, and catalytically by the measurement of 7-ethoxyresorufin-O-deethylase activity. There was clear correspondence between all three methods of measurement both in terms of the specificity of response to five- and some four-ring PAHs and in terms of the dose-response relationship to methyl-cholanthrene. The level of induction was compared with that measured in salmon confined in sea pens around Shetland and exposed to crude oil spilled from the Braer in January 1993. This oil was rapidly dispersed by the extreme weather (turbulence) at the time of the spill. The time course of the hepatic CYP1A induction was followed and related to the levels of oil measured in water and the concentration of PAHs determined in the flesh of the fish. Again there was a good correspondence between the different methods of measuring CYP1A expression, and the results show a rapid induction response in fish at the most contaminated sites and small, insignificant changes occurring at the reference stations. There was a clear concentration response between CYP1A and catalytic activity and between the exposure observed.

Bioaccumulation potential in aquatic biota is typically expressed using ratios of chemical concentrations in organism tissue (typically whole body) relative to chemical exposure concentrations, such as bioconcentration factors (BCFs). Past reviews of metal BCFs for aquatic biota, which account for water-only exposures, have shown that BCFs are often highly variable between organisms and generally inversely related to exposure concentration. This paper further evaluates trends in metal bioaccumulation data by evaluating data for bioaccumulation factors (BAFs) and trophic transfer factors (TTFs). Bioaccumulation factor data were compiled from field studies that account for combined waterborne and dietary metal exposures. Trophic transfer factor data for metals were compiled from laboratory studies in which aquatic food chains were simulated. Natural aquatic food webs are rarely sufficiently understood to properly evaluate exact predator-prey relationships (i.e., TTFs). Results indicate that field BAFs, like laboratory BCFs, tend to be significantly (p ≤ 0.05) inversely related to exposure concentration. Bioaccumulation factors are frequently 100-1000 times larger than BCFs for the same metal and species. This difference is attributed to both lower exposure levels in the field and inclusion of the dietary exposure route. Trophic transfer factors for the metals reviewed, including selenium and methyl mercury were also observed to be inversely related to exposure concentration, particularly at lower exposure concentrations. These inverse relationships have important implications for environmental regulations (e.g., hazard classification and tissue residue-based water quality criteria) and for the use of metal bioaccumulation data in site-specific environmental evaluations, such as ecological and human health risk assessments. Data presented indicate that for metals and metalloids, unlike organic substances, no one BAF or TTF can be used to express bioaccumulation and/or trophic transfer without consideration of the exposure concentration.

Silver nanoparticles (AgNPs) are frequently used as antimicrobials. While the mechanism(s) by which AgNPs are toxic are unclear, their increasing use raises the concern that release into the environment could lead to environmental toxicity. We characterized the physicochemical behavior, uptake, toxicity (growth inhibition), and mechanism of toxicity of three AgNPs with different sizes and polyvinylpyrrolidone (PVP) or citrate coatings to the nematode Caenorhabditis elegans. We used wild-type (N2) C. elegans and strains expected to be sensitive to oxidative stress (nth-1, sod-2 and mev-1), genotoxins (xpa-1 and nth-1), and metals (mtl-2). Using traditional and novel analytical methods, we observed significant aggregation and extra-organismal dissolution of silver, organismal uptake and, in one case, transgenerational transfer of AgNPs. We also observed growth inhibition by all tested AgNPs at concentrations in the low mg/L levels. A metallothionein-deficient (mtl-2) strain was the only mutant tested that exhibited consistently greater AgNP sensitivity than wild-type. Although all tested AgNPs were internalized (passed cell membranes) in C. elegans, at least part of the toxicity observed was mediated by ionic silver. Finally, we describe a modified growth assay that permits differentiation between direct growth-inhibitory effects and indirect inhibition mediated by toxicity to the food source.

Zebrafish (Danio rerio) were exposed for 3 weeks to low concentrations of estradiol including environmentally relevant concentrations (5, 25 and 100 ng/l), encompassing either their embryo-larvae (from fertilization to 21 day post-fertilization (dpf)), juvenile (from 21 to 42 dpf) or adult life stages (>200 dpf) with a view to investigating the most sensitive life stage of the zebrafish to 17␤-estradiol (E2). At all sampling points, whole-body vitellogenin concentrations and gonadal development were analyzed in order to investigate the effects of estrogen exposure on these endpoint in the zebrafish. In the adult stage, additional endpoints were measured including secondary sexual characteristics (manifestation of the uro-genital papillae (UGP) in males), gonadal growth (the gonado-somatic index (GSI)) and sex ratio. For all the different life stage exposures, reproductive performance of the F0 generation was assessed (egg production) and survival and development of the F1 embryo-larvae. Exposure to low concentrations of E2 resulted in vitellogenin induction whatever the life stage exposed but these effects were reversible after depuration. The effective concentration for vitellogenin induction in zebrafish early life stages was 100 ng E2/l, and in adult male zebrafish the effective concentration for vitellogenin induction (between 5 and 25 ng/l) was lower than for the early life stage fish. Exposure to E2 prior to (from fertilization to 21 dpf) and during the time of sex differentiation (from 21 to 42 dpf) also caused disruptions in the process of sexual differentiation (resulting in formation of a retrogonadal cavity in presumptive male, germ cell development and leading to a significant change of the sex ratio towards the female sex at the dose of 100 ng E2/l for the fish exposure as embryo-larvae) and altered patterns of egg production in the subsequent adults. Exposure of adult fish to E2 resulted in a modification of the secondary sexual characteristic in males at 25 and 100 ng E2/l as well as a dose-dependent inhibition of egg production. The findings from this study show that the nature and intensity of the reproductive effects of E2 are dependent of the time and concentration of exposures of zebrafish to E2, some of these effects being permanent (effect on the sexual differentiation) while others being reversible (effect on the Vtg induction). This study demonstrated that early life stages of zebrafish are sensitive to low concentrations of E2 and provides relevant data that could be used for the adaptation of existing fish early life stage test for the in vivo testing of estrogenic compounds. The data presented raise further concerns about the effects of steroid estrogens in the environment on fish reproductive health.

Rapid progress in the field of genomics (the study of how an individual's entire genetic make-up, the genome, translates into biological functions) is beginning to provide tools that may assist our understanding of how chemicals can impact on human and ecosystem health. In many ways, if scientific and regulatory efforts in the 20th century have sought to establish which chemicals cause damage to ecosystems, then the challenge in ecotoxicology for the 21st century is to understand the mechanisms of toxicity to different wildlife species. In the human context, 'toxicogenomics' is the study of expression of genes important in adaptive responses to toxic exposures and a reflection of the toxic processes per se. Given the parallel implications for ecological (environmental) risk assessment, we propose the term 'ecotoxicogenomics' to describe the integration of genomics (transcriptomics, proteomics and metabolomics) into ecotoxicology. Ecotoxicogenomics is defined as the study of gene and protein expression in non-target organisms that is important in responses to environmental toxicant exposures. The potential of ecotoxicogenomic tools in ecological risk assessment seems great. Many of the standardized methods used to assess potential impact of chemicals on aquatic organisms rely on measuring whole-organism responses (e.g. mortality, growth, reproduction) of generally sensitive indicator species at maintained concentrations, and deriving 'endpoints' based on these phenomena (e.g. median lethal concentrations, no observed effect concentrations, etc.). Whilst such phenomenological approaches are useful for identifying chemicals of potential concern they provide little understanding of the mechanism of chemical toxicity. Without this understanding, it will be difficult to address some of the key challenges that currently face aquatic ecotoxicology, e.g. predicting toxicant responses across the very broad diversity of the phylogenetic groups present in aquatic ecosystems; estimating how changes at one ecological level or organisation will affect other levels (e.g. predicting population-level effects); predicting the influence of time-varying exposure on toxicant responses. Ecotoxicogenomic tools may provide us with a better mechanistic understanding of aquatic ecotoxicology. For ecotoxicogenomics to fulfil its potential, collaborative efforts are necessary through the parallel use of model microorganisms (e.g. Saccharomyces cerevisiae) together with aquatic (e.g. Danio rerio, Daphnia magna, Lemna minor and Xenopus tropicalis) and terrestrial (e.g. Arabidopsis thailiana, Caenorhabdites elegans and Eisenia foetida) plants, animals and microorganisms.

Animal alternatives research has historically focused on human safety assessments and has only recently been extended to environmental testing. This is particularly for those assays that involve the use of fish. A number of alternatives are being pursued by the scientific community including the fish embryo toxicity (FET) test, a proposed replacement alternative to the acute fish test. Discussion of the FET methodology and its application in environmental assessments on a global level was needed. With this emerging issue in mind, the ILSI Health and Environmental Sciences Institute (HESI) and the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) held an International Workshop on the Application of the Fish Embryo Test as an Animal Alternative Method in Hazard and Risk Assessment and Scientific Research in March, 2008. The workshop included approximately 40 scientists and regulators representing government, industry, academia, and non-governmental organizations from North America, Europe, and Asia. The goal was to review the state of the science regarding the investigation of fish embryonic tests, pain and distress in fish, emerging approaches utilizing fish embryos, and the use of fish embryo toxicity test data in various types of environmental assessments (e.g., hazard, risk, effluent, and classification and labeling of chemicals). Some specific key outcomes included agreement that risk assessors need fish data for decision-making, that extending the FET to include eluethereombryos was desirable, that relevant endpoints are being used, and that additional endpoints could facilitate additional uses beyond acute toxicity testing. The FET was, however, not yet considered validated sensu OECD. An important action step will be to provide guidance on how all fish tests can be used to assess chemical hazard and to harmonize the diverse terminology used in test guidelines adopted over the past decades. Use of the FET in context of effluent assessments was considered and it is not known if fish embryos are sufficiently sensitive for consideration as a surrogate to the sub-chronic 7-day larval fish growth and survival test used in the United States, for example. Addressing these needs by via workshops, research, and additional data reviews were identified for future action by scientists and regulators.

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kiobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic ,1and y-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle y-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic ,Iand y-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.

Pesticides (organochlorines-OC), polycyclic aromatic hydrocarbons (PAH) and heavy metals are toxic to fish and may be taken in through gills, skin and contaminated foods. Here we measure concentrations of OC, PAH and heavy metals, and their effects in the eel Anguilla anguilla from three locations in the Camargue Reserve in southern France. The Camargue Biosphere Reserve is the largest coastal wetland in Western Europe, and A. anguilla is a common predator at the top of the food chain. Livers and spleens were analyzed for histopathological, chemical and organo-somatic (HSI and SSI) effects. Gill, liver and spleen samples were collected for histopathological studies. Livers and muscles were sampled for metabolic parameters and persistent organic pollutant analysis. Total lipids were estimated by spectrophotometry and lipid-free residues were used in protein and glycogen analysis. OC pesticides were extracted from lipids of muscles and livers, analyzed by gas chromatography, and PAH from bile were analyzed by fixed wavelength fluorescence spectrofluorimetry. Heavy metals were measured by inductively coupled plasma with optical or with mass spectrometers. High concentrations of contaminants were found in eel tissues. La Capelière had the greatest OC and PAH concentrations; unexpected lesions in gills, livers and spleens were more common at the other sites. Liver and spleen tumors and lipidosis in livers were associated with chronic, and gill lesions with acute exposure. High pesticide and PAH concentrations and lesions in eels from the Camargue reserve demonstrate the contamination of the area. A more complete study in the Camargue reserve is necessary to better understand the impact on wildlife and humans. Also, this study suggests that eel biology must be better understood before continued use of this species as a biomonitor of polluted areas.

Environmental anti-androgens are increasingly being recognized as potential contributing factors in the chemically induced feminization of wild fish because, by blocking androgen action, they can produce phenotypic effects similar to environmental estrogens. The molecular mechanisms by which anti-androgens and estrogens exert feminizing effects, however, have not been systematically compared. Using a targeted approach, we profiled the expression responses of a suite of 22 genes involved in reproduction, growth and development (processes controlled by androgens and estrogens) in the liver and gonad in adult male and female fathead minnow (Pimephales promelas) exposed to the model anti-androgen flutamide and the model synthetic estrogen 17␣-ethinylestradiol (EE 2). Both flutamide (320 g/L) and EE 2 (10 ng/L) produced phenotypic effects indicative of feminization (induction of plasma vitellogenin, reduced gonadosomatic index, and reduced secondary sex characters), although for the chosen test concentrations EE 2 was the more potent. For the genes studied, flutamide and EE 2 produced distinct expression profiles, suggesting that they largely operate via distinct molecular mechanisms. As examples, in liver EE 2 (but not flutamide) exposure up-regulated estrogen receptor (ER) ␣ mRNA, whereas flutamide exposure increased ER␤ and ER␥ mRNAs in males and resulted in decreased androgen receptor (AR) mRNA in females. In the testis, flutamide up-regulated genes coding for enzymes involved in androgen biosynthesis (cytochrome P450 17 [CYP17] and 11␤-hydroxysteroid dehydrogenase [11␤-HSD]) implying an inhibitory action on androgen negative feedback pathways. EE 2 , in contrast, inhibited the expression of enzymes involved in androgen biosynthesis (CYP17, 11␤-HSD and 17␤-hydroxysteroid dehydrogenase [17␤-HSD]). There were also some commonalities in the molecular mechanisms of flutamide and EE 2 action, including the down-regulation of gonadal sex steroid receptor expression (gonadal AR and ovarian ER␣), increased expression of genes coding for estrogen-producing enzymes (cytochrome P450 19A and B [CYP19A and CYP19B]), decreased expression of genes involved in testis differentiation (anti-Mullerian hormone [AMH] and doublesex and mab-3 related transcription factor 1 [DMRT1]), and decreased expression of hepatic genes which mediate wider physiological processes such as somatic growth (growth hormone [GH], GH receptor [GHR], insulin-like growth factor-I [IGF-I], IGF-I receptor [IGF-IR], thyroid hormone receptor ␣ [TR␣] and ␤ [TR␤]).

With the aim of studying levels of antioxidant and peroxisomal enzymes and the structure of peroxisomes in relation to body burdens of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), mussels Mytilus gallopro6incialis, oysters Crassostrea sp., crabs Carcinus maenas and mullets Mugil cephalus were sampled in two Basque estuaries (Bay of Biscay): Urdaibai (Laida, Txatxarramendi, Arteaga, and downstream a sewage treatment plant-STP) and Plentzia. In general, animals showed higher concentrations of contaminants in winter than in summer and no relevant differences were detected among locations. Conversely, antioxidant enzyme activities were higher in summer. Enzyme expression was studied in mullets using immunochemical methods. By immunoblotting season-dependent differences were detected for Mn-superoxide dismutase (Mn-SOD). As for the immunohistochemical staining, mullets sampled in summer in Plentzia showed significantly higher optical densities for acyl-CoA oxidase and lower for both Cu,Zn-SOD and Mn-SOD than those collected downstream a STP as well as higher catalase immunostaining than those collected in winter. Peroxisomal volume density (V vp ) of mussels sampled in Laida and Txatxarramendi did not show seasonal variations, while for oysters collected in Laida and Arteaga V vp was higher in summer. Crab and mullet V vp were also higher in summer. In conclusion, the estuaries of Urdaibai and Plentzia can be considered as low to moderately polluted areas and levels of PAHs and PCBs do not show marked variations apart from seasonal variations. Animals can be adapted to low pollution conditions and, under these circumstances, seasonal factors might affect biomarker responses to a greater extent than pollution variations.

Five biomarkers (MT: metallothionein-like proteins, EROD: ethoxyresorufin ortho-dééthylase, DNA strand breaks, LPO: peroxidation of lipids, VG: vitellogenin-like protiens) were measured in the soft tissues of zebra mussels (Dreissena polymorpha) in order to assess the spatial variation of exposure to contaminants along the St Lawrence River (Canada). Fifteen mussels \25 mm shell length were analyzed from each of the 13 sampling sites. Significant differences between sites were noted for all biomarkers, but the general level of variability was low. Three biomarkers (DNA, LPO and VG) exhibited a similar pattern of spatial variation while MT and EROD had distinct and specific patterns. MT had the strongest discriminating power and EROD showed the largest range of variation among sites. Highest biomarker responses were measured in specimens from local contaminated sites such as harbors and industrial sectors. A positive relationship was found between MT and copper (Cu), but no significant correlation was observed between other biomarker responses and the levels of ten trace metals bioaccumulating in the zebra mussels tissues. Results indicate that the measurement of biomarker responses is technically feasible. The performance of each biomarker is assessed in the context of the role and advantages of selecting a battery of biomarkers for detecting contamination problems. The use of zebra mussels as a sentinel species for biomonitoring potential toxic effects in situ is discussed.

There is an increasing body of evidence to support the significant role of invertebrates in assessing impacts of environmental contaminants on marine ecosystems. Therefore, in recent years massive efforts have been directed to identify viable and ecologically relevant invertebrate toxicity testing models. Tigriopus, a harpacticoid copepod has a number of promising characteristics which make it a candidate worth consideration in such efforts. Tigriopus and other copepods are widely distributed and ecologically important organisms. Their position in marine food chains is very prominent, especially with regard to the transfer of energy. Copepods also play an important role in the transportation of aquatic pollutants across the food chains. In recent years there has been a phenomenal increase in the knowledge base of Tigriopus spp., particularly in the areas of their ecology, geophylogeny, genomics and their behavioural, biochemical and molecular responses following exposure to environmental stressors and chemicals. Sequences of a number of important marker genes have been studied in various Tigriopus spp., notably T. californicus and T. japonicus. These genes belong to normal biophysiological functions (e.g. electron transport system enzymes) as well as stress and toxic chemical exposure responses (heat shock protein 20, glutathione reductase, glutathione S-transferase). Recently, 40,740 expressed sequenced tags (ESTs) from T. japonicus, have been sequenced and of them, 5673 ESTs showed significant hits (E-value, >1.0E−05) to the red flour beetle Tribolium genome database. Metals and organic pollutants such as antifouling agents, pesticides, polycyclic aromatic hydrocarbons (PAH) and polychrlorinated biphenyls (PCB) have shown reproducible biological responses when tested in Tigriopus spp. Promising results have been obtained when Tigriopus was used for assessment of risk associated with exposure to endocrine-disrupting chemicals (EDCs). Application of environmental gene expression techniques has allowed evaluation of transcriptional changes in T. japonicus with the ultimate aim of understanding the mechanisms of action of environmental stressors. Through a better understanding of toxicological mechanisms, ecotoxicologists may use this ecologically relevant species in risk assessment studies in marine systems. The combination of uses as a whole-animal bioassay and gene expression studies indicate that Tigriopus may serve as an excellent tool to evaluate the impacts of marine pollution throughout the coastal region. The purpose of this review is to illustrate the potential of using Tigriopus to fulfill the niche as an important invertebrate marine model organism for ecotoxicology and environmental genomics. In addition, the knowledge gaps and areas for further studies have also been discussed.

In a partial life-cycle test, the impact of 17␣-ethinylestradiol (EE2) and 17␣-methyltestosterone (MT) on juvenile zebrafish was evaluated by use of vitellogenin measurements and gonadal development. Exposure to EE2 (1-25 ng/l) resulted in a dose-dependent increase in vitellogenin production starting at 2 ng/l. Significant changes in sex ratios in female direction were detected at 1 ng/l, with complete sex reversal taking place after exposure to 2 ng/l. No intersex fish were observed after exposure to EE2. Exposure to MT resulted in decreased vitellogenin concentrations. Complete sex reversal was detected in all MT concentrations used (26-1000 ng/l). A large proportion of intersex fish was observed after exposure to 1000 ng MT/l. The period of gonadal sex reversal in non-exposed zebrafish was also studied. The main morphological features of the transformation of ovaries into testis were observed 4-5 weeks after hatching.

Environmental toxicology has been expanding as growing concerns on the impact of produced and released chemical compounds over the environment and human health are being demonstrated. Among the toxic effects observed in organisms exposed to pollutants, those affecting skeletal tissues (osteotoxicity) have been somehow overlooked in comparison to hepato-, immune-, neuro- and/or reproductive toxicities. Nevertheless, sub-lethal effects of toxicants on skeletal development and/or bone maintenance may result in impaired growth, reduced survival rate, increased disease susceptibility and diminished welfare. Osteotoxicity may occur by acute or chronic exposure to different environmental insults. Because of biologically and technically advantagous features - easy to breed and inexpensive to maintain, external and rapid rate of development, translucent larvae and the availability of molecular and genetic tools - the zebrafish (Danio rerio) has emerged in the last decade as a vertebrate mode...

The induction of cytochrome P4501A1 (CYP1A1) in rat hepatoma cells has been used by some investigators to determine 'dioxin equivalents' in environmental samples, including extracts of fish tissues. However, the relative potency of inducing compounds may vary between species, suggesting the need for taxonspecific model systems. In this paper we present an initial characterization of CYP1A induction in one such system, a teleost liver cell line (PLHC-1) derived from a hepatocellular carcinoma of Poeciliopsis lucida (Hightower, L.E. and Renfro, J.L., 1988. J. Exp. Zool. 248, 290). Specific binding of the photoaffinity ligand 2-azido-3-[~zsI]iodo-7,8-dibromodibenzo-p-dioxin ([~25I]NaBr2DD) to proteins in PLHC-1 cytosol indicated the presence of the Ah receptor, which is known to control CYP1A induction in mammals. 3,3',4,4'-Tetrachlorobiphenyl (TCB) induced a microsomal protein in PLHC-1 cells that was recognized by monoclonal antibody (MAb) 1-12-3 to scup CYP1 A1 (P450E) on immunoblots. Immunohistochemical staining of whole cells with MAb 1-12 3 showed specific recognition of CYP1A induced by TCB. No staining was seen in untreated or vehicle-treated cells. There was an excellent quantitative correlation between amounts of CYPI A protein detected immunohistochemically and in immunoblots of cell homogenates. In a dose response experiment, maximal induction of ethoxyresorufin O-deethylase (EROD) activity occurred at 0.1 ¢tM TCB; at higher concentrations (1 and 10/tM), EROD activity was reduced as compared to the activity at 0.1 ¢tM TCB. In contrast, immunoreactive CYP1A protein increased with increasing TCB concentration. up to 10/.tM. The loss of EROD activity at high concentrations of TCB did not result from changes in cell number or viability. The apparent inhibition or inactivation of CYP1A catalytic activity by the higher Abbreviations used: AHH, aryl hydrocarbon hydroxylase; BCIP, 5-bromo-4-chloro-3-indolylphosphate; BNF, fl-naphthoflavone; ECs0, estimated concentration needed to produce 50% of the maximal response; EROD, ethoxyresorufin O-deethylase; HAH, halogenated aromatic hydrocarbons; MAb, monoclonal antibody; [~25I]N3Br2DD, 2-azido-3-[125I]iodo-7,8-dibromodibenzo_p_dioxin; NBT, nitro blue tetrazolium; P450, cytochrome P450; PAH, polynuclear aromatic hydrocarbons; PBS, phosphate-buffered saline; PCB, polychlorinated biphenyl; TEF, toxic equivalency factor; TCB, 3,3',4, TCDD, 2,3,7, TCDF, 2,3,7, concentrations of halogenated biphenyls has been seen, but not generally recognized, both in vivo and in cultured cells from diverse vertebrate species. PLHC-1 ceils may be a good model system for studying Ah receptor-mediated regulation of gene expression, for determining the fish-specific toxic or inducing potency of halogenated aromatic hydrocarbon congeners, and for investigating the mechanism of CYP1A inhibition or inactivation by environmental contaminants such as TCB.

In this study, the cladoceran Daphnia magna was exposed to two model organophosphorous and one carbamate pesticides including malathion, chlorpyrifos and carbofuran to assess acetylcholinesterase (AChE) and carboxylesterase (CbE) inhibition and recovery patterns and relate those responses with individual level effects. Our results revealed differences in enzyme inhibition and recovery patterns among the studied esterase enzymes and pesticides. CbE was more sensitive to organophosphorous than AChE, whereas both CbE and AChE showed equivalent sensitivities to the carbamate carbofuran. Recovery patterns of AChE and CbE activities following exposure to the studied pesticides were similar with 80-100% recoveries taking place 12 and 96 h after exposure to organophosphorous and carbamates pesticides, respectively. The physiological role of AChE and CbE inhibition patterns in Daphnia was examined by using organophosphorous and carbamate compounds alone and with specific inhibitors of CbE. Under exposure to organophosphorous pesticides, survival of Daphnia juveniles was impaired at AChE inhibition levels higher than 50% whereas under exposure to the carbamate carbofuran low levels of AChE inhibition affected mortality. Inhibition of CbE by 80-90% increased toxicity to organophosphorous and carbamate pesticides by up to two-and four-fold, respectively. Our results suggest that both AChE and CbE enzymes are involved in determining toxicity of Daphnia to the studied chemicals and that AChE inhibition levels higher than 50% can be considered of environmental concern to Daphnia species.

The estrogenic activity of compounds was evaluated in a comparative approach both with in vitro and in vivo assays. By comparing simultaneously obtained experimental data, we evaluated the differences in response sensitivity (by EC10) and concentration-response relationships (including EC50) in order to get an idea about the predictive value of in vitro assays for in vivo estrogenic potencies or effects in fish. Two human estrogen receptor-based assays, the MVLN-assay (transformed MCF-7 human breast cancer cell line) and the yeast estrogen screen (YES-screen) were used for the in vitro evaluation of the estrogenic potencies. An in vivo model with the female zebrafish (Danio rerio) with plasma vitellogenin (VTG) as a biomarker for exposure and the ovarian somatic index (OSI) as an effect endpoint was used for the in vivo work. Compounds tested were 17␤-estradiol (E2), estrone (E1), 17␣-ethynylestradiol (EE2) and the alkylphenolic compound nonylphenol (NP). All compounds were found to be estrogenic in both in vitro assays and were able to induce VTG and to reduce the ovarian somatic index in female zebrafish. The MVLN-assay appeared up to 15 times more sensitive than the YES-screen. Concentration-response relationships, determined by EC10 and EC50 (concentration of test compound causing 10% or 50% effect compared to control) for VTG and OSI were of the same order of magnitude, indicating that VTG induction as an exposure biomarker can be predictive for effects on ovaries in females. We further demonstrated that for E1 and NP, the in vitro observed estrogenic potencies, based on EC50 values, were of the same order of magnitude as the in vivo estrogenic potencies. For EE2, a difference between in vitro and in vivo relative estrogenic potency was observed, being about 25 times more potent in vivo than could be expected based on the in vitro results. These experimental results showed the suitability of in vitro assays for screening purposes with qualitative assessment of estrogenicity, but they meanwhile point to the need of in vivo tests for an accurate hazard assessment for wildlife.

Gold nanoparticles (AuNP) have potential applications in drug delivery, cancer diagnosis and therapy, food industry and environment remediation. However, little is known about their potential toxicity or fate in the environment. Mytilus edulis was exposed in tanks to750 ppb AuNP (average diameter 5.3 ± 1 nm) for 24 h to study in vivo biological effects of nanoparticles. Traditional biomarkers and an affinity procedure selective for thiol-containing proteins followed by two-dimensional electrophoresis (2DE) separations were used to study toxicity and oxidative stress responses. Results were compared to those obtained for treatment with cadmium chloride, a well known pro-oxidant. M. edulis mainly accumulated AuNP in digestive gland which also showed higher lipid peroxidation. One-dimensional SDS/PAGE (1DE) and 2DE analysis of digestive gland samples revealed decreased thiol-containing proteins for AuNP. Lysosomal membrane stability measured in haemolymph gave lower values for neutral red retention time (NRRT) in both treatments but was greater in AuNP. Oxidative stress occurred within 24 h of AuNP exposure in M. edulis. Previously we showed that larger diameter AuNP caused modest effects, indicating that nanoparticle size is a key factor in biological responses to nanoparticles. This study suggests that M. edulis is a suitable model animal for environmental toxicology studies of nanoparticles.

The use of aquatic plants as bio-indicators constitutes an irreplaceable tool for investigation in ecological research, applied to the conservation of littoral ecosystems. Today, studies in both the laboratory and the field have provided encouraging insights into the capacity of aquatic plants to act as biomonitors of environmental quality, through the use of biomarkers, and these are reviewed here. Photosynthetic activity, secondary metabolites, heat shock proteins, enzymes of detoxication, and oxidative stress biomarkers were measured in the case of various stressors, (e.g. light, thermal, hydric/haline stress, or herbicides, metals, organic contaminants). Most of them seem to be valuable and early markers of the environmental conditions, as demonstrated by experimentations carried out on Posidonia oceanica. Nevertheless, none can be in itself a valuable solution, and only a multiparametric approach, including both ‘physiological’ biomarkers, biomarkers of general stress and more specific biomarkers seems to be appreciable in an ecotoxicological diagnostic.

Quinolones are one of the most important group of synthetic antibiotics used in aquaculture. We studied the single substance and mixture toxicity of ten quinolones using a long term bioluminescence inhibition assay with the marine bacterium Vibrio fischeri as the test organism. All tested quinolones are highly toxic to the test organism with EC50 values ranging from 14 mg/l for ofloxacin to 1020 mg/l for pipemidic acid. Adapting the approach outlined in EEC directive 93/21/EEC to these results, all but one of the ten quinolones belong to the group classified as being 'very toxic to aquatic organisms' (EC50 below 1 mg/l). On the basis of the concentration -response relationships of the single compounds, the mixture toxicity of the ten compounds was estimated by the concepts of concentration addition and independent action. Complete concentration-response relationships were experimentally recorded for the quinolone mixture in three different mixture ratios, based on the relative toxicity of the components (EC50, EC1 and NOEC). The results show that the mixture toxicity of the quinolones is best predictable by concentration addition, whereas independent action underestimates the toxicity of the mixture. As the quinolones have an identical specific mechanism of action (the inhibition of bacterial gyrases), these results are in agreement with the pharmacological assumptions that form the basis of the concept of concentration addition. It is therefore concluded, that concentration addition can be useful for hazard assessment procedures of mixtures of similarly acting compounds. One important implication of this concept is that even mixture components that are present only at their individual no observed effect concentrations (NOECs) contribute to the overall toxicity of the mixture. Under these conditions more than 99% effect of the quinolone mixture are observed. This result emphasises the unsuitability of NOECs as an approximation of a 'safe' concentration.

Knowledge of possible toxic mechanisms (or modes) of action (MOA) of chemicals can provide valuable insights as to appropriate methods for assessing exposure and effects, thereby reducing uncertainties related to extrapolation across species, endpoints and chemical structure. However, MOA-based testing seldom has been used for assessing the ecological risk of chemicals. This is in part because past regulatory mandates have focused more on adverse effects of chemicals (reductions in survival, growth or reproduction) than the pathways through which these effects are elicited. A recent departure from this involves endocrine-disrupting chemicals (EDCs), where there is a need to understand both MOA and adverse outcomes. To achieve this understanding, advances in predictive approaches are required whereby mechanistic changes caused by chemicals at the molecular level can be translated into apical responses meaningful to ecological risk assessment. In this paper we provide an overview and illustrative results from a large, integrated project that assesses the effects of EDCs on two small fish models, the fathead minnow (Pimephales promelas) and zebrafish (Danio rerio). For this work a systems-based approach is being used to delineate toxicity pathways for 12 model EDCs with different known or hypothesized toxic MOA. The studies employ a combination of state-of-the-art genomic (transcriptomic, proteomic, metabolomic), bioinformatic and modeling approaches, in conjunction with whole animal testing, to develop response linkages across biological levels of organization. This understanding forms the basis for predictive approaches for species, endpoint and chemical extrapolation. Although our project is focused specifically on EDCs in fish, we believe that the basic conceptual approach has utility for systematically assessing exposure and effects of chemicals with other MOA across a variety of biological systems.

Mixtures of metals and polycyclic aromatic hydrocarbons (PAHs) occur ubiquitously in aquatic environments, yet relatively little is known regarding their combined toxicities. Emerging reports investigating the additive mortality in metal-PAH mixtures have indicated that more-than-additive effects are equally as common as strictly-additive effects, raising concern for ecological risk assessment typically based on the summation of individual toxicities. Moreover, the current separation of focus between in vivo and in vitro studies, and fine-and coarse-scale endpoints, creates uncertainty regarding the mechanisms of co-toxicity involved in more-than-additive effects on whole organisms. Drawing from literature on metal and PAH toxicity in bacteria, protozoa, invertebrates, fish, and mammalian models, this review outlines several key mechanistic interactions likely to promote more-than-additive toxicity in metal-PAH mixtures. Namely, the deleterious effects of PAHs on membrane integrity and permeability to metals, the potential for metal-PAH complexation, the inhibitory nature of metals to the detoxification of PAHs via the cytochrome P450 pathway, the inhibitory nature of PAHs towards the detoxification of metals via metallothionein, and the potentiated production of reactive oxygenated species (ROS) in certain metal (e.g. Cu) and PAH (e.g., phenanthrenequinone) mixtures. Moreover, the mutual inhibition of detoxification suggests the possibility of positive feedback among these mechanisms. The individual toxicities and interactive aspects of contaminant transport, detoxification, and the production of ROS are herein discussed.

Understanding how biomarkers relate to each other on exposure to particular contaminants in different species is key to their widespread application in environmental management. However, few studies have systematically used multiple biomarkers in more than a single species to determine the variability of sublethal effects of a particular contaminant. In this study, three marine invertebrates, the shore crab Carcinus maenas, the common limpet Patella vulgata and the blue mussel Mytilus edulis, were exposed over 7 days in the laboratory to environmentally realistic concentrations of the priority pollutant copper. A combination of molecular, cellular and physiological biomarkers was measured in each organism to detect the toxic effects of copper. Biomarkers included lysosomal stability (neutral red retention), neurotoxicity (acetylcholinesterase activity), metabolic impairment (total haemolymph protein), physiological status (heart rate) and induction of protective metallothionein proteins. P. vulgata was the most sensitive to copper with significant effects measured in all biomarkers at concentrations of 6.1 g Cu l −1 . In C. maenas, cellular and neurotoxic endpoints were affected significantly only at 68.1 g Cu l −1 . Exposure to copper also induced metallothionein production in crabs. Over a 7-day exposure period, M. edulis was the most tolerant species to copper with significant effects being observed at the cellular level only at 68.1 g Cu l −1 . In all three species, cellular and neurotoxic pathways were more sensitive to disruption than physiological processes (protein and heart rate). Results illustrate how a suite of biomarkers applied to different sentinel species can provide a 'diagnosis of stress', whereby, effects at the molecular level can be used to interpret the level of physiological impairment of the organism.

The purpose of this study was to examine the toxic effects of cadmium-telluride (CdTe) quantum dots on freshwater mussels. Elliption complanata mussels were exposed to increasing concentrations of CdTe (0, 1.6, 4 and 8 mg/L) and cadmium sulfate (CdSO 4 , 0.5 mg/L) for 24 h at 15 • C. After the exposure period, they were removed for assessments of immunocompetence, oxidative stress (lipid peroxidation) and genotoxicity (DNA strand breaks). Preliminary experiments revealed that CdTe dissolved in aquarium water tended to aggregate in the particulate phase (85%) while 15% of CdTe was found in the dissolved phase. Immunotoxicity was characterized by a significant decrease in the number of hemocytes capable of ingesting fluorescent beads, and hemocyte viability. The cytotoxic capacity of hemocytes to lyse mammalian K-562 cells was significantly increased, but the number of circulating hemocytes remained unchanged. Lipid peroxidation was significantly increased at a threshold concentration of 5.6 mg/L in gills and significantly reduced in digestive glands at a threshold concentration <1.6 mg/L CdTe. The levels of DNA strand breaks were significantly reduced in gills at <1.6 mg/L CdTe. In digestive glands, a transient but marginal increase in DNA strand breaks occurred at the lowest concentration and dropped significantly at the higher concentrations. A multivariate analysis revealed that the various response patterns differed based on the concentration of CdTe, thus permitting the identification of biomarkers associated with the form (colloidal vs. molecular) of cadmium. Crown

Dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP) is an organophosphorus (OP) insecticide and acaricide extensively used to treat external parasitic infections of farmed fish. In previous studies we have demonstrated the importance of the glutathione (GSH) metabolism in the resistance of the European eel (Anguilla anguilla L.) to thiocarbamate herbicides. The present work studied the effects of the antioxidant and glutathione pro-drug N-acetyl-l-cysteine (NAC) on the survival of a natural population of A. anguilla exposed to a lethal concentration of dichlorvos, focusing on the glutathione metabolism and the enzyme activities of acetylcholinesterase (AChE) and caspase-3 as biomarkers of neurotoxicity and induction of apoptosis, respectively. Fish pre-treated with NAC (1 mmol kg −1 , i.p.) and exposed to 1.5 mg l −1 (the 96-h LC 85 ) of dichlorvos for 96 h in a static-renewal system achieved an increase of the GSH content, GSH/GSSG ratio, hepatic glutathione reductase (GR), glutathione S-transferase (GST), glutamate:cysteine ligase (GCL), and ␥-glutamyl transferase (␥GT) activities, which ameliorated the glutathione loss and oxidation, and enzyme inactivation, caused by the OP pesticide. Although NAC-treated fish presented a higher survival and were two-fold less likely to die within the study period of 96 h, Cox proportional hazard models showed that hepatic GSH/GSSG ratio was the best explanatory variable related to survival. Hence, tolerance to a lethal concentration of dichlorvos can be explained by the individual capacity to maintain and improve the hepatic glutathione redox status. Impairment of the GSH/GSSG ratio can lead to excessive oxidative stress and inhibition of caspase-3-like activity, inducing cell death by necrosis, and, ultimately, resulting in the death of the organism. We therefore propose a reconsideration of the individual effective dose or individual tolerance concept postulated by Gaddum 50 years ago for the log-normal dose-response relationship. In addition, as NAC increased the tolerance to dichlorvos, it could be a potential antidote for OP poisoning, complementary to current treatments.

As a first stage in developing a DNA array-based approach to investigating the effects of pollutants on an environmentally relevant European fish species, we have constructed a 160-gene custom microarray for European flounder. Degenerate primers were used to amplify 110 different fragments of stress-related and other genes from European flounder cDNA and genomic DNA. Additionally, 22 fragments were obtained by suppressive subtractive hybridisation (SSH). These fragments were cloned and sequenced, then, with additional control genes, used to create a cDNA microarray for flounder. After optimisation of the arraying process, hepatic mRNA was isolated from flounder caught in the polluted Tyne and relatively unpolluted Alde estuaries. Fluorescent cDNA probes were synthesised from the mRNA and used in dual-colour hybridisations to the microarray. A number of transcripts were differentially expressed between Tyne and Alde female flounder but these changes were not significant, due to high inter-individual variation. However, in comparisons between Tyne and Alde male flounder, 11 transcripts were found to significantly differ in expression (P B/0.05). Seven transcripts were more highly expressed in the Tyne male fish (CYP1A, UDPGT, a-2HS-glycoprotein, dihydropyrimidine dehydrogenase, Cu/Zn SOD, aldehyde dehydrogenase and paraoxonase). Four transcripts (Elongation factor 1 (EF1), EF2, Int-6 and complement component C3) were found to be significantly less abundant in the Tyne male fish. Selected genes were assayed by real-time PCR, then normalised to a-tubulin. These assays confirmed the significance of the array results for CYP1A, UDPGT and EF1, but not for Cu/Zn SOD. This study provides a link between traditional single-gene biomarker studies and the emerging field of eco-toxicogenomics, demonstrating the utility of microarray studies on environmentally sampled, non-model organisms. #

The effects on the immune response of the mussel, Mytilus edulis, of short-term, in-vivo exposure to copper were investigated under laboratory-controlled conditions. Parameters measured concentrated on the ability of the blood cells to destroy invading pathogens and included changes in the number and character of the circulating haemocytes, peroxidase and phenoloxidase enzyme activity in the blood cells, intra-and extracellular superoxide radical production, phagocytosis and uptake of neutral red. Copper concentrations of 0.02 and 0.05 ppm were found to increase significantly the total number of circulating haemocytes, while 0.2 and 0.5 ppm decreased the proportion of eosinophilic to basophilic cells. Intracellular superoxide production significantly decreased on exposure to 0.5 ppm copper, whereas phagocytic activity was stimulated at 0.2 ppm but not at 0.5 ppm. Copper exposures of 0.2 and 0.5 ppm reduced the percentage of haemocytes showing binding of lectins from Galanthus ni6alis and Helix pomatia compared with haemocytes from mussels not dosed with copper. No significant alterations were found in peroxidase and phenoloxidase activity, binding of wheat germ agglutinin or uptake of neutral red. The results are discussed in the light of elucidating the possible relationship between environmental contaminants and increased disease susceptibility in aquatic organisms. The benefits of using a multi-assay approach to monitoring environmental pollution using such methods are highlighted.

In order to determine if cellular responses to contaminant exp+xure differed between ceII types, from within the same host, neutral red retention (NRR) was used as a determinant of lysosomal membrane damage in blood and digestive cells isolated from ~4yfi1u.s edrdis eqmsta3 in viva to the model hydrocarbon Ruoranthene. In addition, the Eansitivity of NRR as a Z%omarker of coc'aminant effect for isolated digestive cells, was compared to that of lysosom& latency, as an established biomarker of lysosomal damage, in tissue sections of digestive gkmd. The nsults of the NRR assay indicated a significant depression in the dye retention ime following exposure io i%oPaGhene, ad io its x&one vehide, which was indicative ofmcmbrane failure. There was, however. no significant difference lxiween the lysosomal reteztjon times for the two cell types under investigation indicating a contaminant ekt that is independent of the complement of the acid hydrolases within the lysosomes, and possibl:r t.heir rob within a particular cell type, and more toward a failure in a common controlling factor The comparison of NRR and latency for digestive cell lysosomes. following fluoranthene e.xposure, showed that there was good agreement between the tests in terms of their ability to demonstrate a detrimental contamimatrt effect. The latency test, ho;uever, failed in contrast to the NRR assay to detect any e!Tect resulting from exposure to the acetone vehicle alone.

This study investigated whether gonadal sex differentiation of zebrafish (Danio rerio) is susceptible to compounds that interfere with cytochrome P450 aromatase (P450arom). Treatment of zebrafish during the period of gonadal differentiation with either the non-steroidal aromatase inhibitor fadrozole or 17␣-methyltestosterone (MT) changed gonad morphological differentiation and altered the pattern of P450arom gene (CYP19) expression. Application of fadrozole (500 g/g of food) between days 35 and 71 post-fertilisation (pf) resulted in 100% masculinisation, i.e. the gonads of all individuals examined (n = 40) showed testicular morphology. At the same time, fadrozole treatment suppressed gonadal CYP19A (gonad-derived CYP19 gene) mRNA expression, as assessed by means of semi-quantitative RT-PCR. After termination of fadrozole treatment at day 71 pf and subsequent rearing of zebrafish under control conditions until 161 days pf, the gonads of all individuals still displayed testicular morphology. Gonadal CYP19A expression, however, showed a dimorphic pattern, with 14 out of 22 individuals having low CYP19A mRNA levels similar to those found in testes of control fish, while eight fish showed high, ovary-like levels of gonadal CYP19A mRNA. MT treatment (10 g/l) during the period of gonadal differentiation (days 35 to 71 pf) resulted in phenological feminisation, i.e., all fish examined (n = 28) showed an ovarian gonadal morphology. While gonadal CYP19A expression was suppressed compared to control fish, cerebral CYP19B (brain-derived CYP19 gene) mRNA expression was increased in 71-day-old MT-treated fish. The results from this study provide evidence that exogenous compounds interfering with the P450arom system are able to disrupt, even persistently, gonadal sex differentiation of the protogynic zebrafish.

The effects of microcystins from cyanobacterial cells on various oxidative stress biomarkers in liver, kidney and gill tissues in freshwater tilapia fish (Oreochromis sp.) were investigated under laboratory conditions. Microcystins are a family of cyclic peptide toxins produced by species of freshwater cyanobacteria (blue-green algae). Fish were exposed to the cyanobacterial cells in two ways: mixed with a commercial fish food or crushed into a commercial fish food so that the toxins were released. Two different exposure times were studied: 14 and 21 days. The oxidative status of fish was evaluated by analyzing the level of lipid peroxidation (LPO), as well as the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR). The findings of the present investigation show that microcystins induce oxidative stress in a time-dependent manner and that the type of administration of the cyanobacterial cells influences the extent of these effects. Thus, the crushed cyanobacterial cells (released toxins) induced the antioxidant defences studied and increased the LPO level to a greater extent than the non-crushed cells. The liver was the most affected organ followed by kidney and gills. These results together with reports that fish can accumulate microcystins mean that cyanobacterial blooms are an important health, environmental and economic problem.

In freshwater systems located in agricultural areas, organisms are exposed to a multitude of toxicologically and structurally different pesticides. For regulatory purposes it is of major importance whether the combined hazard of these substances can be predictively assessed from the single substance toxicity. For artificially designed multi-component mixtures, it has been shown that the mixture toxicity can be predicted by concentration addition (CA) in case of similarly acting substances and by independent action (IA), if mixtures are composed of dissimilarly acting substances. This study aimed to analyse whether these concepts may also be used to predictively assess the toxicity of environmentally realistic mixtures. For this purpose a mixture of 25 pesticides, which reflects a realistic exposure scenario in field run-off water, was studied for its effects on the reproduction of the freshwater alga Scenedesmus vacuolatus. The toxicity of the tested mixtures showed a good predictability by CA. This is consistent with the finding that the toxicity was dominated by a group of similarly acting photosystem II inhibitors, although the mixture included substances with diverse and partly unknown mechanisms of action.

A series of experiments was carried out to assess the effects of the toxic dinoflagellate Protogonyaulax tamarensis on shell-valve activity, rates of particle clearance, preingestive, and postingestive particle selection in seven species of bivalve molluscs from three geographic localities: Maine, Rhode Island, and Spain. The responses observed were species-specific and varied with collection locality. Responses included shell-valve closure and/or siphon retraction (Mya arenaria, Mytilus edulis, Geukensia demissa), reduced rates of particle clearance (M. arenaria, G. demissa), increased rates of particle clearance (Ostrea edulis), production of mucus (M. edulis from Spain and Rhode Island, Placopecten magellanicus, G. dernissa). Mortalities were noted in M. edulis from both Spain and Rhode Island. Two species (Modiolus modiolus and Spisula solidissma) exhibited no effects of the toxic dinoflagellates. Data are also presented for particle selection, clearance and filtration rates for the seven species. It is suggested that species which are periodically exposed to dinoflagellate blooms may have evolved mechanisms permitting them to exploit the toxic organisms as food with no ill effects.