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The North and Baltic Seas are heavily trafficked marine areas with extensive anthropogenic activities, including cargo and fishing vessels, waste dumping, oil platforms, industrial activities and contamination from coastal runoff. In order to evaluate the environmental health of these regions, we used the demersal fish dab (Limanda limanda) as a sentinel species. The current study used well-established biomarkers for PAH exposure and oxidative stress, measuring EROD activity, the acute antioxidant response as well as oxidation of proteins detected as protein carbonyl levels. Results show the strongest biomarker results in an area with extensive oil drilling, where dab displayed high levels of EROD activities. This was also seen in dab captured in the Baltic Sea where elevated levels of oxidized glutathione and a trend towards higher EROD activity were observed. The obtained results did, however, not indicate a coherent biomarker response. The study was conducted off shore where many areas have presumably low levels of pollutants, and we could detect minor effects using the biomarker approach.

Livers from dab (Limanda limanda), plaice (Pleuronectes platessa) and flounder (Platichthys flesus) sampled from the Baltic Sea were used to determine the interaction of flatfish CYP1A enzymes with 2,4,6-trinitrotoluene (TNT) in vitro. Competitive inhibition of 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-deethylase (MROD) could be demonstrated for all three flatfish species. The highest inhibition of CYP1A activities was measured in liver samples of flounder resulting in a half maximal inhibitory concentration (IC50) of 28.1 μM TNT. Due to their lower inhibition (EROD IC50 65.2 μM TNT, MROD IC50 40.3 μM TNT), dab liver samples were used to conduct in vitro metabolization experiments with TNT. The metabolization of TNT in fish was investigated with post-mitochondrial fractions (PMF) of dab liver as a model system after adding different cofactors. Rapid and time-dependent enzymatic degradation of TNT was observed. The concentrations of 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene increased in the samples over time. Additionally, 2,2,6,6-tetranitro-4,4-azoxytoluene was detected in one sample. The results of this study indicate that in vitro experiments are useful to investigate the xenobiotic metabolism of fish under controlled conditions prior to field studies. The metabolites found can serve as target compounds for marine monitoring of TNT contamination in munition dumpsites.

Microplastics are present in marine habitats worldwide and may be ingested by low trophic organisms such as fish larvae, with uncertain physiological consequences. The present study aims at assessing the impact of polyethylene (PE 10-45 mu M) microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae. Fish were fed an inert diet including 0, 10(4) and 10(5) fluorescent microbeads per gram from 7 until 43 days post-hatching (dph). Microbeads were detected in the gastrointestinal tract in all fish fed diet incorporating PE. Our data revealed an efficient elimination of PE beads from the gut since no fluorescent was observed in the larvae after 48 h depuration. While the mortality rate increased significantly with the amount of microbeads scored per larvae at 14 and 20 dph, only ingestion of the highest concentration slightly impacted mortality rates. Larval growth and inflammatory response through Interleukine-1-beta (IL-1 beta) gene expression were not found to be affected while cytochrome-P450-1A1 (cyp1a1) expression level was significantly positively correlated with the number of microbeads scored per larva at 20 dph. Overall, these results suggest that ingestion of PE microbeads had limited impact on sea bass larvae possibly due to their high potential of egestion.

Accepted manuscript version, licensed CC BY-NC-ND 4.0. Published version available at https://doi.org/10.1016/j.marenvres.2018.09.005. The present study investigates the potential long-term physiological effects on maturing polar cod (Boreogadus saida), an Arctic key species, after an acute exposure (48 h) to environmentally realistic concentrations of either mechanically dispersed oil (MDO), chemically dispersed oil (CDO) or burned oil residues (BO) (N = 58–60 per treatment). Following exposure, fish were monitored in a common tank supplied with clean water for a seven-month period coinciding with the period of reproductive development. Females exposed to BO residues were more frequently found in an earlier phase of gonadal maturation compared to unexposed females while no effects of different oil spill response (OSR) actions were seen in the reproductive development of males. Mechanically and chemically dispersed oil induced a transient short-term reduction in growth in the first week post-exposure. Overall, no significant long-term effects of exposure were seen in growth or mortality. Ultimately, this study provides information for the assessment of population consequences of different OSR actions as part of a net environmental benefit analysis.

Following the 2010 Gulf of Mexico oil spill, concerns were raised regarding exposure of fish to crude oil components, particularly polycyclic aromatic hydrocarbons (PAHs). This three year study examined hepatic enzymes in post-mitochondrial supernatant fractions from red snapper (Lutjanus campechanus) and gray triggerfish (Balistes capriscus) collected in the north central Gulf of Mexico between 2011 and 2014. Biomarker activities evaluated included benzo(a)pyrene hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), glutathione transferase (GST), and glutathione peroxidase (GPx). Mean EROD activity was higher in gray triggerfish (12.97 ± 7.15 pmol/min/mg protein [mean ± SD], n = 115) than red snapper (2.75 ± 1.92 pmol/min/mg protein, n = 194), p

In this study, biological responses and contaminant levels in biological tissues were investigated in grass goby fish specimens (Zosterisessor ophiocephalus) collected from five stations in a moderately polluted ecosystem, namely the Bizerte lagoon on the north coast of Tunisia. The following biomarkers were measured: muscular acetylcholinesterase (AChE), hepatic ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), lipoperoxidation (TBARS), condition factor (CF), and hepatosomatic index (HSI). These measurements were taken in parallel with the content of Organochlorine pesticides (OCPs), Polychlorinated biphenyls (PCBs), Polycyclic aromatic hydrocarbons (PAHs) and trace metals (As, Cr, Cu, Mn, Pb, V, Zn, Ag, Cd, Co and Ni) in muscle tissue. Total PAH concentrations ranged from 20.09 ± 0.68 to 105.77 ± 42.58 ng g(-1) dw, PCB from 33.19 ± 6.25 to 126.28 ± 7.37 ng g(-1) dw, OCP from 11.26 ± 1.62 to 19.17 ± 2.06 ng g(-1) dw, and metals from 107.83 ± 1.83 to 187.21 ± 2.00 mg/kg dw. The highest levels of pollutants and biomarkers were observed at station S1, located in the Bizerte channel. Elevated EROD, GST and CAT activities, as well as TBARS levels in liver were positively correlated with tissue contaminant levels at station S1. Significant negative correlations were also found between hexachlorobenzene (HCB) and dichlorodiphenyltrichloroethane (DDTs) body burden with AChE activity in muscle at station S2. The integration of biological responses and contaminant tissue content indicated that certain areas of the Bizerte lagoon, notably station S1, are significantly impacted by various human activities, which likely represent a threat for aquatic wildlife. On the basis of these results, and due to its ecological characteristics, the grass goby appears a suitable indicator species for pollution biomonitoring in coastal marine areas along the Mediterranean Sea.

The aim of this study was to characterize the responsiveness of CYP1A and ABC transport proteins in European Sea bass (Dicentrarchus labrax) waterborne exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) (46 pg/L) for 24 h and 7 days. Genes modulation (abcb1, abcc1-2, cyp1a), EROD activity were investigated in liver and 2,3,7,8-TCDD bioconcentration in liver and muscle. TCDD induced significantly cyp1a gene expression and EROD activity at 24 h and 7 d. A significant up-regulation of abcb1 was also observed but only after 7 days. No modulation of abcc1 and abcc2 genes was observed. Waterborne TCDD exposure was able to induce CYP1A and abcb1 encoding for P-glycoprotein in juvenile of European sea bass.

Dioxin-like compounds (DLC) induce toxic responses in early life stages of fish through activation of the aryl hydrocarbon receptor (AhR) which is frequently assessed by ethoxyresorufin-O-deethylase (EROD) activity. A novel spectrofluorimetric method was developed to quantitatively assess EROD activity in individual living embryos and prolarvae of a marine model fish species, the mummichog Fundulus heteroclitus. This in vivo method is based on the measurement of the production of resorufin by single live embryos or prolarvae after 5 h incubation with ethoxyresorufin. Freshly fertilized eggs were treated topically from 2.5 to 50 pg egg⁻¹, with 3,3',4,4',5-pentachlorobiphenyl (PCB126), a prototypical DLC. EROD activity was assessed in embryos (7 days post-fertilization) and prolarvae (16 days post-fertilization). Resorufin was measured both in the culture medium (25‰ seawater) and in whole fish homogenates, to assess the percentage retained in the body. Approximately 95% and 17% of the resorufin produced in vivo was retained in embryos and prolarvae respectively. EROD activity in homogenates of embryos and in the culture medium of prolarvae increased linearly with dose. EROD activity measured by the in vivo method was highly correlated to that measured by a traditional in vitro technique using S9 fractions for both embryos and prolarvae. Both in vivo and in vitro EROD activity were higher in prolarvae than in embryos pretreated with PCB126. EROD induction measured in prolarvae by the in vivo and in vitro methods were similar whereas higher induction was measured in vivo than in vitro in embryos. The in vivo method was more sensitive and as reliable as the in vitro technique, and required a lower number of fish (4 compared to 3 pools of 5). This in vivo method is useful to link EROD induction in individual embryos or prolarvae to other organism-level responses. Further studies with other categories of xenobiotics should be performed to assess potential toxic effects on resorufin absorption/excretion processes which could affect in vivo measurements.

This study investigates the use of a weight of evidence (WOE) approach to evaluate fish health status and biological effects (BEs) of contaminants for assessment of ecosystem health and discusses its potential application in support of the Marine Strategy Framework Directive (MSFD). External fish disease, liver histopathology and several BEs of contaminant exposure including 7-ethoxy resorufin O-de-ethylase (EROD), acetylcholinesterase (AChE), bile metabolites, vitellogenin (VTG) and alkali labile phosphates (ALP) were measured in two flatfish species from four locations in Ireland. Contaminant levels in fish were generally low with PCBs in fish liver below OSPAR environmental assessment criteria (EAC). There were consistencies with low PCB levels, EROD and PAH bile metabolite levels detected in fish. Dab from Cork, Dublin and Shannon had the highest relative prevalence of liver lesions associated with the carcinogenic pathway. An integrated biomarker response (IBR) showed promise to be useful for evaluation of environmental risk, although more contaminant parameters in liver are required for a full assessment with the present study.

Oil and chemical spills in the marine environment, although sporadic, are highly dangerous to biota inhabiting coastal and estuarine areas. Effects of spilled compounds in exposed organisms occur at different biological organization levels: from molecular, cellular or tissue levels to the physiological one. The present study aims to determine the specific hepatic gene transcription profiles observed in turbot juveniles under exposure to fuel oil n °6 and styrene vs controls using an immune enriched turbot (Scophthalmus maximus) oligo-microarray containing 2716 specific gene probes. After 3 days of exposure, fuel oil specifically induced aryl hydrocarbon receptor mediated transcriptional response through up-regulation of genes, such as ahrr and cyp1a1. More gene transcripts were regulated after 14 days of exposure involved in ribosomal biosynthesis, immune modulation, and oxidative response among the most significantly regulated functional pathways. On the contrary, gene transcription alterations caused by styrene did not highlight any significantly regulated molecular or metabolic pathway. This was also previously reported at cell and tissue level where no apparent responses were distinguishable. For the fuel oil experiment, obtained specific gene profiles could be related to changes in cell-tissue organization in the same individuals, such as increased hepatocyte vacuolization, decrease in melano-macrophage centers and the regulation of leukocyte numbers. In conclusion, the mode of action reflected by gene transcription profiles analyzed hereby in turbot livers could be linked with the responses previously reported at higher biological organization levels. Molecular alterations described hereby could be preceding observed alterations at cell and tissue levels.

Karstic areas in Yucatan are very permeable, which allows contaminants to move rapidly into the aquifer. In the present study, we evaluated gene expression of vitellogenin (VTG) and cytochrome P-450 1A (CYP1A) in caged juvenile zebrafish deployed for 15 days in 13 different water bodies, cenotes and aguadas, throughout karstic region of the Yucatan peninsula. Gene expression was evaluated using qRT-PCR. Results indicated induction of VTG in 7 water bodies with respect to reference cage. The highest relative VTG expression, about 3000 times higher than reference cage, was found in an aguada close to a cattle farm. CYP1A induction with respect to reference cage was observed in 3 water bodies, all of them located near villages or used for tourist activities. Pollutants and biomarkers of effect should be monitored in these water bodies in order to have a better understanding of the actual levels of pollutants that are present at Yucatan’s aquifer and the potential risk to human and environmental health.

A suite of general physiological indicators and hepatic biomarker responses were determined in red mullet (Mullus barbatus) from priority pollution areas of W Mediterranean Sea, including the highly metal polluted area of Portmán (Cartagena, SE Spain). Concentrations of metals and persistent organic pollutants in fish muscle tissue and sediment samples were also analysed. Our results showed that fish from Portmán accumulated the highest concentrations of mercury, lead and arsenic and also of some polycyclic aromatic hydrocarbons and polychlorinated biphenyls congeners. In addition, they had significantly lower condition factor, muscle lipid content and gonadosomatic index, as well as the lowest levels of DNA integrity and the highest ethoxyresorufin-O-deethylase (EROD) activity in liver of the areas investigated. Contaminant body burden in fish only partly corresponded to chemical characteristics of the sediments in the areas in which they were collected. Our findings indicate that red mullets from Portmán had suboptimal health status that warrant further study., Sí

Aim of this study was to evaluate the responsiveness of red mullet (Mullus barbatus) liver detoxification enzymes to PAHs at transcriptional and post-transcriptional levels in the field. Fish were captured in the north-eastern Adriatic Sea, close to an oil refinery. Sixteen PAHs (EPA) were determined in sediments and fish fillets; transcription levels of cyp1a, cyp3a and abcc2 genes and EROD, BROD, B(a)PMO, BFCOD, GST and UDPGT enzymatic activities were measured. Levels of PAHs in sediments reflect the oil pollution gradient of the area, with weak correspondence in fish fillets. cyp1a gene transcription and EROD, B(a)PMO and BFCOD activities were significantly induced in the oil refinery site, and a slight up-regulation of cyp3a and abcc2 was also observed. GST and UDPGT remained unchanged. The present study provides the first data on detoxification responses at transcriptional levels in the liver of red mullet and confirms phase I enzymes as suitable biomarkers of exposure to PAHs in field studies.

The relationship between condition factor (CF), peroxisome proliferator-activated receptors (PPARs), phase 1 biotransformation (CYP1A isoforms) and contaminant burden has been studied in Sarotherodon melanotheron from a contaminated tropical freshwater dam (Awba Dam) and compared to a reference site (Modete Dam) in Southwest, Nigeria. A total of 89 fish (57 males and 32 females) was collected from Awba Dam and 95 fish (48 males and 47 females) from the reference site. In general, fish sampled from Awba Dam were bigger than reference site. Sediment samples were also collected from both sites for contaminant analysis. Expression of ppar and cyp1 isoforms was analyzed using validated real-time PCR, while CYP1A and PPAR protein levels were analyzed using immunochemical method with specific antibodies. CYP-mediated catalytic responses (EROD, MROD and BROD) were performed by biochemical methods. We observed significant increases in ppar and cyp1 isoforms mRNA in both male and female fish from Awba Dam, compared to the reference site. Catalytic activities of EROD, MROD and BROD paralleled cyp1 transcript levels. Sex-related differences in PPAR and CYP1A protein levels were also observed, showing higher CYP1A proteins in males, compared with females, and higher PPAR proteins in females compared with males. Principal component analysis (PCA) biplot showed positive relationships between biological responses (ppar isoforms), condition factor (CF) and sediment PCBs, PAHs, OCPs and heavy metal concentrations. The present study shows that S. melanotheron inhabiting Awba Dam are severely affected by different classes of environmental contaminants that target metabolic processes (PPAR) and biotransformation pathways (CYP1A) in male and female fish, compared to a reference site. Interestingly, fish from Awba Dam were exhibiting good growth (evidence by high CF values) that paralleled increases in the transcriptional activation of ppar and cyp1 isoforms, despite the high contaminant burdens, suggesting a possible contaminant-induced obesogenic effects.

Pollutants such polycyclic aromatic hydrocarbons (PAHs) are released into the environment by urban communities and industries and the enzymes that catalyse the biotransformation of pollutants play a key role regarding the accumulation of these compounds in fish species inhabiting these areas. In this study the relationship between phase I (EROD activity) and phase II (GST activity) and PAH metabolites was measured in grey mullet (Mugil cephalus) after capture in the Douro estuary, and after long-term depuration in an unpolluted laboratory environment. The results showed a significant decrease in EROD activity after 1 month and in bile metabolites after 4 months in captivity, with both maintaining reduced levels at 4 and 8 months depuration. Liver GST activity did not showed significant changes. This study provides evidence that Douro estuary waters contain bioavailable PAHs that can be associated with the induction of cytochrome P450, and that mullets have the ability to metabolise and eliminate PAHs.

Biomarkers of organochlorine exposure, such as the induction of cytochrome P450 1A (CYP1A), can be used to assess the impact of environmental contaminants on the health of free-ranging marine mammal populations. The objective of the present study was to measure CYP1A in skin and liver biopsies obtained from live harbour seals (Phoca vitulina). Twelve harbour seal pups, aged three to five weeks, were captured from the Fraser River estuary, British Columbia, Canada, and temporarily held in captivity. Skin ( approximately 60 mg) and liver ( approximately 40 mg) biopsies, obtained while seals were under general anaesthesia, yielded sufficient tissue for the measurement of CYP1A by immunoblot analysis and ethoxyresorufin O-deethylase activity. A short-term exposure experiment, in which harbour seals (n=3) were treated orally with beta-naphthoflavone (BNF), resulted in increased hepatic and cutaneous CYP1A protein levels, consistent with observations in other mammals. This study is the first to measure CYP1A in skin and liver biopsies from live harbour seals and to report in vivo BNF-associated CYP1A induction in a marine mammal. The results demonstrate that microsamples collected using minimally-invasive techniques can provide toxicologically-relevant information form marine mammals.

Our goal was to study the involvement of cytochrome P450 genes in estrogen metabolism and the extent to which the potentially carcinogenic 4-hydroxyestradiol metabolite is formed by channel catfish (Ictalurus punctatus; CC). Estradiol metabolism and ethoxyresorufin-O-deethylase (EROD) activity were assessed in several tissues from fish collected from three variably contaminated sites in the Mississippi River Delta, from laboratory control fish, and from fish exposed to 20 mg/kg benzo(a)pyrene (BaP) i.p. for 4 days. Liver EROD activity was induced by BaP, but Delta fish EROD activities were not statistically higher than activities in control fish. Gill microsomal EROD activity was also induced by BaP, but activities were 8- to 77-fold lower than those from liver. The predominant estrogen metabolites formed by CC liver, gill and gonad microsomes were 2-hydroxyestradiol and estrone as detected by GC/MS. Liver and gill 2-hydroxyestradiol formation was induced in BaP-dosed fish. The trends in hydroxyestradiol formation in field collected fish were more variable. In all fish liver microsomes there was more 2- compared to 4-hydroxyestradiol formed. However, BaP-treatment increased the 4:2 hydroxyestradiol ratio from 0.04 in control fish to 0.2 in BaP-exposed fish, suggesting that BaP induces the formation of the potentially genotoxic estrogen metabolite. No detectable 4-hydroxyestradiol was produced by gill and gonad microsomes. These results will ultimately help in determining which fish P450 genes are susceptible to environmental contamination and may be involved in estrogen genotoxicity.

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.

The goal of these experiments was to explore the relationship between cytochrome P4501A (CYP1A) induction and the teratogenicity of sediments from the Atlantic Wood Industries Superfund site (Elizabeth River, VA) in Fundulus heteroclitus embryos. In these experiments we used embryos spawned from reference site adults to assess CYP1A activity and teratogenicity induced by aqueous Elizabeth River sediment extracts (ERSE). Embryo exposures to ERSE induced CYP1A activity and caused deformities, including pericardial edema, heart elongation and tail shortening. Co-exposures with various CYP1A inhibitors significantly decreased CYP1A activity and increased the teratogenicity of the sediment extract. Potential mechanisms for this increased toxicity are discussed herein.

CYP1A-immunopositive protein can be elevated in response to planar PAHs and PCBs in Mytilus sp. digestive gland whilst CYP3A-immunopositive protein has been associated with testosterone 6β-hydroxylation in fish. Levels of CYP1A- and CYP3A-immunopositive protein were determined in Mytilus galloprovincialis digestive gland microsomes collected from 12 sites in the Mediterranean Sea during May and September 2001. CYP1A-immunopositive protein was significantly highest at contaminated sites whilst CYP3A-immunopositive protein was significantly lowest. A weak negative correlation (r2=0.21) was seen between CYP1A- and CYP3A-immunopositive protein. Little evidence of differences at the different sampling times was observed. These results confirm previous work indicating elevation of CYP1A-immunopositive protein in Mytilus sp. digestive gland at contaminated sites. Further study is required to characterise CYP3A-like expression in Mytilus and to elucidate the consequences of possible CYP3A-like down-regulation at contaminated sites.

European flounders from two UK estuaries with different contaminant profiles were sampled and analysed for impacts of PAHs, heavy metals and estrogenic compounds by currently adopted measurement procedures used in statutory monitoring programs. These showed that fish from the polluted Tyne estuary had responded to all three classes of pollutants with elevations in hepatic EROD, MT and plasma VTG and that there was considerable interanimal variation in the magnitude of the response. Analysis of PAH metabolites in bile showed recent uptake of low molecular weight PAHs. The pollutant response was also apparent when hepatic levels of cytochrome P450 1A (CYP1A), metallothionein (MT) and vitellogenin (VTG) mRNAs were quantified by real time PCR. Whilst there did not appear to be a direct correlation between levels of gene transcripts and their protein products on an individual basis, these results demonstrate that measurements of mRNA levels of specific genes, or their protein products both demonstrate induction in polluted environments and are thus valid measures in biomonitoring studies. Procedurally, the use of the single generic technology of Q-PCR was much simpler and is worth pursuing in future studies.

The induction of hepatic cytochrome P450 1A (CYP 1A) is an important step in the response to contaminants such as polycyclic aromatic hydrocarbons (PAHs), and has been used as a biomarker of exposure in fish. Several consensus response elements have been identified, including eight potential xenobiotic response elements (XREs) in the promoter region of the European flounder cytochrome P450 1A gene. However not all of these sequences are necessarily active. To help elucidate the molecular regulation of this important gene, site directed mutagenesis and dual-luciferase reporter gene assays were employed to characterize the consensus transcription factor binding sites of the CYP 1A 5′ flanking region. Mutation of response elements situated −1103, −859, −709 and −172 bases upstream of the transcription start site reduced the induction to 2.75, 1.51, 3.25 and 3.05 fold, respectively, compared with the full-length promoter (4.0-fold induction) on exposure to the PAH 3-methylcholanthrene (3MC) (1.0 μM). These results indicate that four out of eight different XREs are functional in the control of CYP 1A in the flounder. The activity of these response elements adds to the evidence for considerable diversity in vertebrate CYP 1A regulation.

Chronic exposure to organic contaminants such as polychlorinated biphenyls (PCBs) can lead to the development of resistance to these chemicals, a condition associated with reduced response of CYP1A1, a pollutant-inducible biomarker. We measured CYP1A activity (ethoxyresorufin o-deethylase, EROD) and PCB concentrations in feral fish from the Town Branch/Mud River system (Logan County, KY), a stream historically contaminated with PCBs and partially remediated. As a first step in evaluating the possible development of resistant populations in this system, we measured CYP1A expression and PCB body burdens in resident fish from sites we previously characterized as containing biologically significant levels of CYP1A inducing compounds. Mean PCB concentrations in edible flesh ranged from 75.2 to 16.7 μg/g in fish collected from Town Branch remediated sites and were relatively low (1.23 μg/g) in Town Branch reference site fish. However, hepatic CYP1A activity was similar among individuals of most species collected from reference and contaminated/remediated sites. The absence of elevated CYP1A levels in resident fish species despite the presence of significant PCB body burdens may indicate these fish have developed reduced sensitivity to CYP1A induction, a condition associated with acquired resistance to toxicants.

Hepatic levels of the pollutant inducible enzyme, CYP1A, are strongly suppressed in spawning female fish, a phenomenon attributed to high plasma levels of the female sex steroid hormone, estradiol. To evaluate the contribution of estrogen metabolites to estradiol-mediated CYP1A regulation, we treated primary hepatocytes isolated from juvenile rainbow trout ( Oncorhynchus mykiss ) with vehicle, 17β-estradiol, or the estrogen metabolite, estriol, alone and in combination with each other and with the potent CYP1A inducer, benzo[ a ]pyrene (B[ a ]P). We found dose-dependent suppression of B[ a ]P-induced CYP1A activity by both steroids relative to controls. At 10 −7 M doses, estradiol and estriol suppressed B[ a ]P-induced CYP1A activity by 3- and 2-fold, respectively. Although not statistically significant, mean basal CYP1A activity levels were 15- and 13-fold lower in estradiol and estriol treated hepatocytes, respectively, relative to vehicle treated controls. Combining doses of estradiol and estriol failed to produce synergistic suppression of either basal or B[ a ]P-induced CYP1A activity relative to treatment with either steroid alone. The observed suppression is well below the often strong suppression observed in spawning female fish. We conclude that factors in addition to estradiol and estriol are likely involved in producing sexual dimorphism in CYP1A expression observed in spawning fish.

Enzymes in the cytochrome P450 gene family 1 (CYP1) catalyze the metabolic activation of numerous hydrocarbon carcinogens and various natural compounds. CYP1 family members have been identified in several vertebrates, including fish, amphibians, birds, and mammals, and are inducible by aromatic hydrocarbons acting through the aryl hydrocarbon receptor (AHR). Together with its heterodimeric partner ARNT, the ligand-bound AHR binds conserved xenobiotic response elements (XREs) near the promoter of CYP1A and other genes. However, some populations of the Atlantic killifish Fundulus heteroclitus inhabiting highly contaminated sites are refractory to CYP1A induction by aromatic hydrocarbons. To better understand the mechanisms underlying this phenomenon, we are characterizing the AHR-CYP1A signaling pathway in this species. We report here the characterization of a genomic clone containing the 5(') end of the wild-type F. heteroclitus CYP1A gene. The 5(') coding sequence matches that of the F. heteroclitus CYP1A cDNA reported earlier [Comp. Biochem. Physiol. 121C (1998) 231]. Consistent with its inducibility by AHR agonists, the CYP1A gene contains three consensus XREs (5(')CACGC3(')) within 1.6 kb of the putative transcriptional start site. When oligonucleotides containing each of these sites were analyzed in an electrophoretic mobility shift assay, one of these showed a strong, TCDD-inducible mobility shift in the presence of in vitro expressed mouse AHR protein. These sequence data and initial functional characterization provide a valuable tool for the study of genetic variations in CYP1A expression and activity in sensitive and resistant populations. These studies may ultimately shed light on the importance of P4501A activity in xenobiotic toxicity.

The clam Scrobicularia plana and the polychaete worm Nereis diversicolor were collected in several sites from a littoral enclosure in SW Spain. The aim of our study was to relate various biomarker responses in these species to a pollution gradient caused by untreated domestic discharges and to verify the adequacy of the selected species as sentinels in this habitat. The biomarkers selected were the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPX) and DT-diaphorase (DT-D). In addition, the activities of cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity, the phase II detoxifying enzyme glutathione S-transferase (GST) and the neurotoxicity marker acetylcholinesterase (AChE) were measured. Metallothionein levels were selected as biomarkers of heavy metals exposure in both species. The results suggest a different response in the water filtering organism (clam) and the sediment eater (polychaete), probably as a consequent of different pollution exposure and that samples from the “Cano Sancti-Petri” were exposed to biologically active compounds that altered some of their biochemical responses. AChE was the most sensitive biomarker in both species and N. diversicolor proved to be a more robust sentinel in this ecosystem.

Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 micro(micro)M) with or without 0.2 micro(micro)g/l of the CYP1A inducer 3,3',4,4',5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.

Benzo( a )pyrene (BaP) and polychlorinated biphenyls (PCBs) often co-exist in contaminated environments. Polychlorobiphenylols (OH-PCBs), formed by CYP-dependent monooxygenation of PCBs, are potent inhibitors of the glucuronidation of hydroxylated BaP metabolites. We hypothesized that OH-PCBs could drive the biotransformation of (−)BaP-7,8-dihydrodiol (BaP-7, 8-D) away from detoxication and towards formation of the reactive metabolite. A mixture of five OH-PCBs with 4–6 Cl atoms was infused into isolated, perfused, biliary intact livers ( n =3 fish) removed from 3-methylcholanthrene-induced channel catfish. Controls ( n =3) were infused with vehicle. Subsequently, [ 3 H]-BaP-7, 8-D was infused into each liver and bile was collected for 1 h. The livers were taken for analysis of metabolites and DNA adducts. Induction status was confirmed by EROD assay. Bile was analyzed for metabolites. It was found that preinfusion of the mixture of OH-PCBs reduced the extent of glucuronidation of BaP-7, 8-D and increased the formation of DNA adducts 5-fold over controls. GSH conjugates, tetrols and triols were increased in the OH-PCB-infused fish, providing further support for our hypothesis that if the glucuronidation were inhibited, CYP-dependent activation would increase. These studies suggest a mechanism for synergy of toxicity of PAH and PCBs.

Although elasmobranchs are widely distributed species, commonly found on sandy, gravely or muddy bottoms, several biological aspects of their metabolism still remain poorly investigated. In this work the efficiency of antioxidant system and detoxification enzymes were investigated in the coastal spotted dogfish Scyliorhinus canicula and in the red mullet Mullus barbatus for comparison with a teleost species. Organisms were sampled during a bottom trawl survey and analyzed for the biotransformation activity (EROD), levels of metallothioneins, catalase, glutathione S-transferases and total oxyradical scavenging capacity (TOSC) toward peroxyl radicals and hydroxyl radicals. EROD activity in the elasmobranchs was more than one order of magnitude lower than in the red mullets, while similar levels of metallothioneins were measured in these species. S. canicula showed significantly lower antioxidant enzymes and a more reduced efficiency in neutralizing OH; on the other hand the scavenging capability toward ROO was comparable in S. canicula and M. barbatus.

Induction of cytochrome P4501A in fish is a well-known indicator of exposure to polycyclic aromatic hydrocarbons (PAHs) and determination of PAH metabolites in bile by fixed wavelength fluorescence (FF) or synchronous fluorescence spectroscopy (SFS), has become an useful method in monitoring programs. In this work the relationship between cytochrome P4501A (EROD activity) and levels of biliary PAH metabolites was measured in the European eel Anguilla anguilla, in both field and laboratory conditions: organisms were sampled on a seasonal basis from the Orbetello lagoon (Tuscany) to characterize the natural variability of these biological parameters, while in laboratory eels were intraperitoneally injected with benzo[a]pyrene to investigate temporal and dose-dependent induction patterns. Results showed that induction of cytochrome P450 and accumulation of PAHs metabolites in bile are not necessarily correlated either in field, or in laboratory investigations; different seasonal changes were measured in natural conditions and slight variations in dose and time response patterns were also obtained in laboratory exposures.

The purpose of the present study was to elucidate in vitro effects of Hg(2+), Zn(2+), Ni(2+) and Cd(2+) on cytochrome P4501A1 (CYP1A1) dependent EROD activities in leaping mullet liver microsomes. Fish captured from the most polluted part of Izmir Bay, had highly elevated EROD activities, and induced CYP1A1 protein levels as determined by Western blotting. Although all of the metal ions caused inhibition of the initial velocity of the reaction, Hg(2+) and Cd(2+) exhibited much higher inhibitory effect at lower concentrations and they were evidently more potent inhibitors than others. The inhibitor concentration giving 50% inhibition (IC(50) values) of Zn(2+), Ni(2+), Cd(2+) and Hg(2+) of initial EROD activity were 107, 16, 1.3 and 0.15 micromolar, respectively. Glutathione (GSH) at 0.5 mM final concentration, completely reversed Ni(2+) and Cd(2+) inhibition of EROD activity indicating the protective action of GSH.

The relative potency of polycyclic aromatic compounds as aryl hydrocarbon receptor (AhR) agonists in fish was determined using data on CYP1A induction or AhR binding for 74 polycyclic aromatic hydrocarbons (PAHs) and heterocycles in teleost, avian, or mammalian systems from 18 published papers. Each PAH was assigned a fish potency factor relative to the potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin as an AhR agonist. Two and three ring unsubstituted PAHs were generally inactive in fish, avian, and mammalian systems. Benzo[k]fluoranthene and indeno[1,2,3-cd]pyrene were consistently the most potent PAHs, with fish potency factors of 0.001-0.002. Common structural features associated with higher potency PAHs included 4-6 rings containing fluoranthene or phenanthrene structures with an exposed bay region. These results show that PAHs can have similar potency as many dioxin-like PCBs, and AhR mediated toxicity should be considered in assessing the risks of PAHs in fish.

To assess chemical contaminant stress in the marine environment, ethoxyresorufin-O-deethylase (EROD) activity and cytochrome P450 1A (CYP1A) expression were measured in 88 English Sole (Pleuronectes vetulus) collected during May and June 1999 from four sites in Vancouver Harbour and at an expected reference site outside the harbour. Hepatic microsomes were prepared from the fish and analyzed for total CYP content, EROD activity, and CYP1A protein levels. Hepatic EROD activity and CYP1A protein levels were elevated in fish from two sites in the inner harbour. A comparison with sediment chemistry data showed that fish with increased EROD activity and CYP1A levels came from sites containing relatively high levels of polycyclic aromatic hydrocarbons and polychlorinated biphenyls. Unexpectedly high levels of EROD activity and CYP1A protein were also found in fish from a reference site near Gibsons, in Howe Sound. The elevated EROD activity and CYP1A expression in fish from this site cannot be explained by the chemical analysis data collected.

Investigators from the six PICES member nations conducted a May, 1999 interdisciplinary research assessment of Vancouver Harbour, BC, Canada. Sediment contaminants were analyzed at several sites along an assumed pollution gradient extending eastward into the harbour from the relatively less-impacted Howe Sound area (located outside and seaward, to the west). Subsets of a few representative benthic biota (English sole for general contaminants; specific mollusks for TBT) at the sampling sites were concurrently assessed for specific tissue contaminants. Measurements of biotic response to contaminants—across a wide spectrum of biological and ecosystem organization, and ranging from selected biomarkers and tissue responses, to observations at the organism and community level—were likewise conducted. Parameters examined included: P-450 expression, EROD induction and histopathologic assessment of toxicopathic liver lesions in English sole; prevalence and frequency of imposex in neogastropods; perturbations in benthic community responses to pollutants; tissue assays of various bivalve shellfish species for PSP. Results affirmed a consistent east-to-west trend in declining contaminant-associated effects, across a range of biological assessments, thus providing strong evidence that these effects are pollution-related. Specific findings revealed a significant distortion of benthic community structure along the pollution gradient, as well as biomarker evidence of histological and biochemical stress in English sole from central and inner harbour sites. Although the study reaffirms prior assumptions that various species of neogastropods have been extirpated from the harbour, most likely from the antifoulant uses of tributyltin, some of the adverse impacts from TBT may be declining. Also noted were strong biochemical inferences that sediments in the relatively “uncontaminated” Howe Sound, may well contain significant P450-inducing pollutants which were not included as analytes in the design of this limited and preliminary assessment. During the brief sampling period, PSP contamination did not appear to be a significant issue for the harbour.

The initial sampling in the Marine Monitoring Program (MOMAM), coordinated by the Ministry of Marine Affairs (IEAPM), was performed along the southeast coast of Brazil. Orthopristis ruber samples were collected at Guanabara, Sepetiba and Ilha Grande Bays. Microsomal CYP1A levels and cytosolic cholinesterase (ChE), catalase (CAT) and glutathione S-transferase (GST) activities were measured in the liver of these fish according to established procedures. CAT activity and CYP1A content were significantly higher (P⩽0.05) in fish caught at Guanabara Bay, which might be due to higher levels of peroxisome proliferators and Ah receptor agonists, respectively, at this site compared to the other sites. Also, lower GST activity was observed in fish from this site, which may possibly be related to the presence of oxidative-stress inducing compounds.

Juvenile turbot (Scophthalmus maximus) were injected intraperitoneally with either corn oil or 5 mg/kg benzo[a]pyrene (BaP) dissolved in corn oil and sampled I and 3 days after injection. After 1 day, no elevation of 7-ethoxyresorufin O-deethylase (EROD) activity was observed, however bile metabolites (BaP-7,8 dihydrodiol representing 70% of the total metabolites) and a single hepatic DNA adduct spot (0.47 adducts/10(8) nucleotides) identified by 32P-postlabelling were formed. No BaP metabolites or DNA adducts were observed in either control or carrier control fish. Fish sampled after 3 days reported 5-fold higher (P0.05) levels of EROD activity, a shift in the bile metabolite profile towards BaP phenol formation (1OH and 30H BaP comprising up to 60% of total metabolites detected) and the formation of two adduct spots (0.86 and 0.71 adducts/10(8) nucleotides). These results show that BaP can be metabolised and form hydrophobic DNA adducts in turbot without EROD elevation. Following EROD elevation, a shift in the profile of both BaP metabolites and BaP metabolite-DNA interactions occurs indicative of other oxidative processes.

The short-term effects of the commercial PBDE flame retardant mixtures Penta-BDE and cta-BDE on the expression of cytochrome P450 1A (CYP1A), vitellogenin (Vtg) and zona radiata proteins (Zrp) were investigated in juvenile salmon (Salmo salar). For this purpose, groups of fish were dosed twice (oral intake at days I and 4) with 10 and 50 mg/kg body weight of both commercial mixtures. The fishes were sacrificed at day 7 (n = 5 for each group) and 14 (n = 6 for each group), and blood, liver, fillet, and brain were collected. Blanks and positive controls were also part of the experiment. The expressions of Vtg, Zrp, and CYPIA were measured with several techniques (EROD, ELISA, Western, Northern and Slot Blot). The values in the groups of fish treated with Penta-BDE or Octa-BDE did not significantly differ from the reference group for any of the parameters tested. In contrast, the positive control groups treated with estradiol-17beta for Vtg and Zrp expression, and beta-naphthoflavone for CYP1A expression did show a significant response, indicating the potential sensitivity of the fishes for the parameters measured. Since the results of the chemical analyses showed concentrations of a number of PBDE congeners in liver, fillet, and brain that were about three orders of magnitude above those of fish from the North Sea, it is concluded that the short-term toxicity of both commercial PBDE mixtures for these endpoints was low.

Killifish (Fundulus heteroclitus) from a highly contaminated site on the Elizabeth River are resistant to the acute toxicity and the cytochrome P4501A (CYP1A)-inducing activity of both the sediments from the site and chemically pure polycyclic aromatic hydrocarbons (PAHs). These effects are highly heritable for one generation, but heritable to a lesser degree by subsequent generations, in clean conditions in the laboratory. We show that offspring of this population of Elizabeth River killifish are also resistant to the teratogenicity and P4501A-inducing activity of PCB congener 126, a prototypical coplanar halogenated aromatic hydrocarbon (HAH). Furthermore, the pattern of greater resistance to acute toxicity and P4501A-inducing activity in the first generation and less in subsequent generations is also observed upon exposure to PCB-126.

Concentration dependent differences in acute and long-term effects of a 48 h exposure to mechanically or chemically dispersed crude oil were assessed on juvenile lumpsucker (Cyclopterus lumpus). Acute or post-exposure mortality was only observed at oil concentrations representing higher concentrations than reported after real oil spills. Acute mortality was more apparent in chemically than mechanically dispersed oil treatments whereas comparable EC50s were observed for narcosis. There was a positive correlation between EROD activity and muscle PAH concentration for the lower oil concentrations whereas higher concentrations inhibited the enzyme activity. The incidence of gill tissue lesions was low with no difference between dispersion methods or oil concentrations. A concentration dependent decrease in swimming- and feeding behavior and in SGR was observed at the start of the post-exposure period, but with no differences between corresponding oil treatments. Three weeks post-exposure, fish from all treatments showed as high SGR as the control fish.

Feral Atlantic cod (Gadus morhua) and corkwing wrasse (Symphodus melops) were investigated for polyaromatic hydrocarbon (PAH) contamination in the Karmsund strait, western Norway. This strait is highly contaminated with PAHs, and a main source is the chronic release of gas-scrubbing effluents from a local aluminium works. In both species, the level of biliary PAH metabolites and hepatic DNA adducts were higher in fish collected near the aluminium works. Interestingly, a significantly higher level of both biliary PAH metabolites and hepatic DNA adducts was found in corkwing wrasse as compared to cod, indicating a higher potential for genotoxic effects in this species. Hepatic cytochrome P4501A (CYP1A) in cod estimated by ethoxyresorufin-O-deethylase and an immunoassay technique (ELISA), seemed to be weakly induced at the contaminated sites. At the most contaminated site, skin ulcers and fin erosion were detected in about 70 and 45% of the cods, respectively. The data demonstrated that both cod and corkwing wrasse may be suitable target species for PAH pollution monitoring.

Medetomidine is being introduced as a new antifouling agent. As part of a large risk assessment campaign, we have studied responses to medetomidine in a number of fish species. The studied parameters include respiration, blood parameters, antioxidant enzymes, CYP1A, behaviour, pigmentation, reproduction and growth. The main observations from these studies are: 1. Body paleness was affected at water concentrations in the range of 0.5–50 nM, depending on species. In addition, impaired adaptation to the background colour was shown in fry from turbot and lumpfish. In rainbow trout, desensitization of melanophores (pigment cells) occurred in fish exposed to medetomidine for 21 days, but a prolonged study (54 days exposure time), showed that the melanophores were well functioning and in addition that no apoptosis had occurred. 2. CYP1A activities, measured as EROD activities, were increased at medetomidine water concentrations from 0.5 to 50 nM in rainbow trout, Atlantic salmon, turbot and three-spined stickleback. However, investigations in vitro showed medetomidine to be a potent inhibitor of EROD activity. 3. In lumpfish and turbot fry, a decreased oxygen consumption and respiration rate was observed from 2 nM medetomidine. This effect was reversible to a large extent.

The gulf killifish, Fundulus grandis, was used to determine the influence of biological rhythms on three biomarker responses. We first developed monoclonal antibodies against the model's immunoglobulins and vitellogenin in order to measure antibody responses and vitellogenesis, respectively. We then treated adults with 10, 1, 1, and 10 ppm of Aroclor 1254, tribuyltin, 3-methylcholanthrene, and nonyl-phenol, respectively, in mixtures over a 16-week period. The study followed Vibrio anguillarum-specific antibody responses, hepatic CYP1A, and plasma vitellogenin levels in the morning and again in the evening at 2-week intervals. The contaminated diet suppressed secondary antibody responses, but only in the morning. The contaminated diet also altered CYP1A, but not vitellogenesis. In addition, fish in the control group exhibited daily and seasonal differences in specific antibody levels and CYP1A induction. Moreover, circulating vitellogenin levels in control males sampled in the morning increased throughout the exposure, but remained below those of females. This study underscores the need to consider normal physiological rhythms when employing biomarkers in toxicology.

Recent studies demonstrating feminization of effluent-exposed wild-caught male fish in the UK have prompted much research regarding the estrogenic activity of effluent from municipal sewage treatment plants (MSTPs). To investigate the estrogenicity and cytochrome P450 1A (CYP1A) induction potency of MSTP effluent, two species of fish, adult male mummichogs, Fundulus heteroclitus, and juvenile sunshine bass, Morone saxatilis x Morone chrysops, were exposed to un-chlorinated effluent (75% effluent, 25% seawater) from a large MSTP in Yonkers, NY, USA. After a 21-day static-daily (75%) renewal exposure, significant elevations over controls were observed in levels of vitellogenin (VtG) in plasma (1730%) and liver (131%) in effluent-exposed sunshine bass. In contrast, hepatic VtG was not elevated in mummichogs; plasma VtG was not measured in this species. Effluent exposure elevated hepatic CYP1A protein (140-145%) and ethoxyresorufin-O-deethylase (EROD) activity (408-598%) in both species. These findings suggest ontogenetic and/or species differences in response to estrogenic compounds in MSTP effluent. Furthermore, the elevation of CYP1A in response to sewage effluent exposure indicates the presence of additional compounds that may alter xenobiotic and/or steroid biotransformation in fish.

The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goksøyr, A., Celander, M., Heida, H.,Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.

The effect of environmental pollutants present in sediments obtained from Bahía de Chetumal, a bay on the border between Mexico and Belize, was studied in nile tilapia (Oreochromis niloticus) intraperitoneally injected with sediment extracts from six different sites of the Bay. Sediment samples used for the study contained a variety of organic chemicals such as organochlorine pesticides, polychlorinated biphenyls (PCBs) and polynuclear aromatic hydrocarbons (PAHs). Total cytochrome P-450 and EROD activity were measured in fish liver. Haematological and histological analyses were also carried out. Hepatic P-450 content in treated fish increased from 43 to 240%, and EROD activity from 85 to 160% compared to controls. Extracts from two sampling sites inhibited EROD activity. There were positive significant correlations between P-450 content and the levels of PCBs 44 and 128. EROD activity correlated to HCB, op'-DDE, pp'-DDE, pp'-DDD, mirex and PCB 18 concentrations. Blood examination showed cell degeneration and binucleated leukocytes with abnormal chromatin. Extract treatment also resulted in foci of hyperplasia on the basement of gill lamellae, hypertrophy and oedema in gills and liver necrosis. Control fish showed no abnormalities. The results demonstrate that sediments from Bahía of Chetumal have the potential to cause histopathological, haematological and biochemical alterations in fish. The administration of sediment extracts to fish may serve as a useful test to screen the toxicity of sediments from different areas.