Data for the analysis of interactive multibiomarker responses of a marine crustacean to long-term exposure to aquatic contaminants.

The data presented herein relates to the article entitled "Multibiomarker interactions to diagnose and follow-up chronic exposure of a marine crustacean to hazardous and noxious substances (HNS)" (Abreu et al., 2018). Multibiomarker approaches, including molecular, biochemical, physiological and behaviour parameters, are recognised as valuable and cost-effective to employ in integrated chemical and biological effects monitoring of aquatic contamination. Many biomarkers assessed in such programmes share common physiological pathways, showing concomitant or interdependent responses, which can reflect in increased energy costs related to physiological acclimation. Though, routine single biomarker data analysis, and exploratory principal component analysis, limit information obtained from the data collected and their functional interpretation. Ultimately, this influences the type of management actions taken to protect an affected ecosystem. This article presents data employed to develop an analytical approach accounting for multibiomarker interactions. The method was useful to diagnose and follow-up long-term exposure of the marine green crab (Carcinus maenas) to Hazardous and Noxious Substances (HNS).

The last frontier: Coupling technological developments with scientific challenges to improve hazard assessment of deep-sea mining.

The growing economic interest in the exploitation of mineral resources on deep-ocean beds, including those in the vicinity of sensitive-rich habitats such as hydrothermal vents, raise a mounting concern about the damage that such actions might originate to these poorly-know ecosystems, which represent millions of years of evolution and adaptations to extreme environmental conditions. It has been suggested that mining may cause a major impact on vent ecosystems and other deep-sea areas. Yet, the scale and the nature of such impacts are unknown at present. Hence, building upon currently available scientific information it is crucial to develop new cost-effective technologies embedded into rigorous operating frameworks. The forward-thinking provided here will assist in the development of new technologies and tools to address the major challenges associated with deep sea-mining; technologies for in situ and ex situ observation and data acquisition, biogeochemical processes, hazard assessment of deep-sea mining to marine organisms and development of modeling tools in support of risk assessment scenarios. These technological developments are vital to validate a responsible and sustainable exploitation of the deep-sea mineral resources, based on the precautionary principle.

The use of biomarkers as integrative tools for transitional water bodies monitoring in the Water Framework Directive context - A holistic approach in Minho river transitional waters.

The Water Framework Directive (WFD) provides an important legislative opportunity to promote and implement an integrated approach for the protection of inland surface waters, transitional waters, coastal waters and groundwaters. The transitional waters constitute a central piece as they are usually under high environmental pressure and by their inherent characteristics present monitoring challenges. Integrating water quality monitoring with biological monitoring can increase the cost-effectiveness of monitoring efforts. One way of doing this is with biomarkers, which effectively integrate physical-chemical status and biological quality elements, dealing holistically with adverse consequences on the health of water bodies. The new Marine Strategy Framework Directive (MSFD) already incorporates the biomarker approach. Given the recent activities of OSPAR and HELCOM to harmonize existing monitoring guidelines between MSFD and WFD the use of similar methodologies should be fostered. To illustrate the potential of the biomarker approach, juveniles of flounder (Platichthys flesus) were used to evaluate the quality of the Minho river-estuary water bodies. The use of juveniles instead of adults eliminates several confounding factors such changes on the biological responses associated with reproduction. Here, a panel of well-established biomarkers, EROD, AChE, SOD, CAT, GST, LPO, ENA and FACs (1-Hydroxyrene) were selected and measured along with a gradient of different physical conditions, and integrated with trace elements characterization on both biota and sediments. In general, a clear profile along the water bodies was found, with low seasonal and spatial variation, consistent with a low impacted area. Overall, the results support the use of both the battery of biomarkers and the use of juvenile flounders in the monitoring of the water quality status within the WFD.

Toxicity effects of hazardous and noxious substances (HNS) to marine organisms: acute and chronic toxicity of p-xylene to the amphipod Gammarus locusta.

Despite the recent focus on hazardous and noxious substances (HNS) spills preparedness and responses, much remains to be done regarding the threat posed by HNS spills on marine biota. Among the identified priority HNS, p-xylene was selected to conduct ecotoxicological assays. The aim of this study was to assess the performance of the amphipod Gammarus locusta under acute and chronic exposure to p-xylene simulating conditions of a spill incident. In the acute exposure (96 h) the p-xylene LC50 was estimated. In the chronic bioassay (36 d), an integration of organism-level endpoints (survival, growth rate, and sex ratio) with biochemical markers indicative of oxidative stress including catalase (CAT), glutathione S-transferase (GST), and superoxide dismutase (SOD) activities and lipid peroxidation (LPO) levels was determined. The aim was to increase the xylene ecotoxicological database and better predict its impact in aquatic environments. p-Xylene induced several chronic toxicity effects in G. locusta. Significant alterations in antioxidant enzymes and lipid peroxidation levels as well as growth rate and biased sex-ratio were observed. p-Xylene significantly affected the activities of CAT, SOD, and GST in G. locusta and produced oxidative damage by increasing levels of LPO in males. Further, impacts in key ecological endpoints, that is, growth and sex ratio, were noted that might be indicative of potential effects at the population level in a spill scenario. The present data may be useful to assist relevant bodies in preparedness and response to HNS spills.

Simulation of a Hazardous and Noxious Substances (HNS) spill in the marine environment: lethal and sublethal effects of acrylonitrile to the European seabass.

Despite the extensive maritime transportation of Hazardous and Noxious Substances (HNS), there is a current lack of knowledge on the effects posed by HNS spills on the marine biota. Among the HNS identified as priority, acrylonitrile was selected to conduct ecotoxicological assays. We assessed the acute and subletal effects of acrylonitrile in seabass, followed by a recovery phase to simulate the conditions of a spill incident. The work aimed at testing a broad range of biological responses induced by acrylonitrile. Sublethal exposure to the highest two doses increased the fish mortality rate (8.3% and 25% mortality in 0.75 and 2 mg L(-1) acrylonitrile concentrations), whereas no mortality were observed in control and 0.15 mg L(-1) treatments. Additionally, important alterations at sub-individual level were observed. Acrylonitrile significantly induced the activities of Catalase- CAT and Glutathione S-Transferase - GST; and the levels of DNA damage were significantly increased. Conversely, Superoxide Dismutase- SOD - activity was found to be significantly inhibited and no effects were found on Lipid Peroxidation- LPO and ethoxyresorufin O-deethylase - EROD - activity. Following a 7d recovery period, the levels of CAT, GST and EROD fell to levels at or below those in the control. In the 2 mg L(-1) group, SOD remained at the levels found during exposure phase. This study has gathered essential information on the acute and subletal toxicity of acrylonitrile to seabass. It also demonstrated that 7d recovery allowed a return of most endpoints to background levels. These data will be useful to assist relevant bodies in preparedness and response to HNS spills.

A real-time PCR assay for differential expression of vitellogenin I and II genes in the liver of the sentinel fish species Lipophrys pholis.

Abstract The recent advances in molecular biology techniques have prompted the use of vitellogenin (VTG) gene expression as a sensitive and reliable indicator of estrogenic chemicals (EC) exposure. However, data on the dynamic response of the different VTGs genes upon EC exposure is still poorly understood, particularly in sentinel fish species used in field monitoring studies. Hence, the present study aimed at developing a sensitive real-time PCR assay for determining the response of VTG I and II in the recently proposed marine sentinel species Lipophrys pholis upon exposure to the model EC 17α-ethinylestradiol (EE2). The findings of the laboratory study indicate that L. pholis VTG I proved to be not only more inducible but also more sensitive to EE2 exposure than VTG II, for the same range of concentrations. In fact, VTG I gene induction was 475-fold higher than VTG II at 15 ng/L EE2, and 13-fold at 5 ng/L EE2. Overall, the findings of the present study indicate that in the field, expression of VTG I in L. pholis should be preferentially used in the screening of EC exposure because of its higher sensitivity. Furthermore, the present study favors L. pholis integration in monitoring programs associated with EC's pollution within the European water policy legislation.

Zebrafish (Danio rerio) life-cycle exposure to chronic low doses of ethinylestradiol modulates p53 gene transcription within the gonads, but not NER pathways.

Parental full life-cycle exposure to ethinylestradiol (EE2) significantly affects embryo development and survival. One of the possible mechanisms of action of EE2 may involve the impairment of an organism's ability to repair DNA damage. DNA repair mechanisms have sophistically evolved to overcome DNA damaging hazards that threaten the integrity of the genome. In the present study, changes in the transcription levels of key genes involved in two of the most thoroughly studied DNA repair systems in mammals were evaluated in adult zebrafish (Danio rerio) gonad upon full life-cycle exposure to chronic environmentally low levels of EE2 (i.e., 0.5, 1 and 2 ng/L EE2). Real time PCR was used to analyse the expression levels of nucleotide excision repair genes (NER) as well as the tumor suppressor p53 and downstream selected effectors, i.e., p21 (cyclin-dependent kinase inhibitor), GADD45α (growth arrest and DNA damage induced 45, alpha), bax (bcl2-associated X protein) and p53 key regulator MDM2 (murine double minute 2 protein). NER genes transcription levels in gonads did not differ significantly among treatments. In contrast, the number of transcripts of p53 gene was significantly increased in male gonads at all EE2 exposure concentrations and in females at 1 ng/L EE2. Despite the increase in p53 transcripts, transcription levels of p21, GADD45α and bax genes were not affected upon EE2 treatment, whereas MDM2 gene expression significantly increased in females at the intermediate EE2 dose (1 ng/L). Overall, the present study indicate that chronic low levels of EE2 significantly modulates the transcription of p53, a key gene involved in DNA repair, particularly in male zebrafish gonads, which supports the hypothesis of an impact of EE2 in male gonad DNA repair pathways.

Review of oil and HNS accidental spills in Europe: identifying major environmental monitoring gaps and drawing priorities.

The European Atlantic area has been the scene of a number of extensive shipping incidents with immediate and potential long-term impacts to marine ecosystems. The occurrence of accidental spills at sea requires an effective response that must include a well executed monitoring programme to assess the environmental contamination and damage of the affected marine habitats. Despite a number of conventions and protocols developed by international and national authorities that focused on the preparedness and response to oil and HNS spills, much remains to be done, particularly in relation to the effectiveness of the environmental monitoring programmes implemented after oil and HNS spills. Hence, the present study reviews the status of the environmental monitoring programmes established following the major spill incidents over the last years in European waters, aiming at identifying the key monitoring gaps and drawing priorities for an effective environmental monitoring of accidental spills.

Drifting towards the surface: a shift in newborn pipefish's vertical distribution when exposed to the synthetic steroid ethinylestradiol.

Endocrine disrupting chemical (EDC) effects during early life have the potential to modulate population structure, either directly through increased mortality or by causing inappropriate aggregation events, thus affecting the number of young that will reach adulthood. An alteration in the dispersal and recruitment patterns can also impair the connectivity among geographically distant populations. However, the detection of EDC-induced effects occurring after egg hatch, when newborns increase their chances of contacting with environmentally dispersed contaminants, is not a simple process as effects might be masked by the large natural mortality rates that usually occur during fish early life. Since there is a lack of information regarding the impact of EDCs on fish early life dispersal patterns, particularly on vertical migrations patterns, the effects of environmentally relevant concentrations of EE(2) on the vertical distribution of newborn fish was assessed through an ex situ exposure experiment. Syngnathus abaster newborns were exposed to EE(2) (nominal concentrations of 8, 12 and 36 ng L(-1)) and the dynamics of their vertical distribution was monitored for up to 40 d. No significant differences in overall mortality were observed between treatments or in the dynamics of the registered death curves. Nevertheless, an alteration in the distribution patterns was observed. The commonly benthic newborn tended to shift their vertical distribution towards the surface, in a dose-dependent manner. Curiously, a follow up of the exposed pipefish confirmed that EE(2) effects were also noticeable upon sexual maturity, namely by the alteration of several primary and secondary sexual characters. The observation that vertical distributional patterns, at least in pipefish, are clearly altered at environmentally relevant EE(2) concentrations indicates that EDC's impact in fish larvae behaviour should be considered when addressing the effects of contaminants, given the obvious implications on population connectivity, stability and persistence.

The unpredictable effects of mixtures of androgenic and estrogenic chemicals on fish early life.

Estuarine shallow areas and coastal lagoons are known to receive and concentrate multiple inputs, either from land, rivers or coastal areas, being intensively impacted by chemical contamination, namely endocrine disrupting chemicals (EDCs). Despite the ubiquitous coexistence of several classes of EDCs in most of these aquatic ecosystems, there is still limited information regarding their combined effects. Furthermore, given the immediate implications for population dynamics, the available laboratory studies almost invariably focus on very specific life history stages, such as embryonic development or reproduction, thus creating a gap on our knowledge of what happens in between. During this 'intermediate phase', the newborn larvae and juveniles face numerous challenges whose outcome may impair reproduction or even survival. The black-striped pipefish, Syngnathus abaster, member of the Syngnathidae family (comprising pipefish, seahorses and seadragons), usually breeds in coastal areas such as estuaries, where its newborns are immediately exposed to EDCs. Given the ongoing decline of pipefish populations, together with the observed shrinkage and fragmentation of seagrass meadows, known to be impacted by EDCs, a first reasonable question to address is if pipefish newborns respond to environmentally relevant concentrations of ubiquitous EDCs, either single or in combination. Hence, a seven days exposure experiment to the estrogenic chemical ethinylestradiol (EE(2)) and the androgenic chemical tributyltin (TBT), single and in binary mixtures, was conducted. Selected behavioural (e.g. predator avoidance) and developmental variables (e.g. growth) were monitored in pipefish juveniles after EDCs insult. The obtained results indicate that EE(2), TBT, or their combined exposure, do impact pipefish early life. However, the pattern of results emerging from the measured variables clearly indicates that mixtures significantly modulate newborn responses in distinct ways when compared to individual chemical's exposure. These findings further demonstrate the importance of addressing the issue of chemical mixtures of pollutants acting through dissimilar mode of action. Independently of all the observed response variations, an ultimate conclusion seems certain: EE(2) and TBT, single or in combination, induce disruption patterns able to imbalance pipefish survival. Since these (as well as other) contaminants are present in estuarine areas, profound implications in population structure could be expected, ranging from a decrease in recruitment to a disruption of sexual selection. Inexorably, these stressors simultaneously operate in already declining populations.

Tributyltin-induced imposex in marine gastropods involves tissue-specific modulation of the retinoid X receptor.

Despite the large number of studies on the phenomenon of imposex, the mechanism underlying the abnormal growth of male sexual characters onto females in numerous gastropod species is yet to be fully elucidated. Although several hypotheses have been raised over the years, a convincing body of evidence indicates that tributyltin-induced imposex involves the abnormal modulation of the retinoid X receptor (RXR). Here, we investigate the RXR gene transcription at different timings and tissues upon exposure to environmentally relevant concentrations of tributyltin (TBT) (100 ng Sn/L TBT) in both genders of the imposex susceptible gastropod Nucella lapillus. RXR gene transcription was determined at two time-points (i.e., before and after imposex initiation) by quantitative Real Time PCR in potential target tissues: the central nervous system (CNS), penis/penis forming area (PFA), gonads and digestive gland. TBT-exposure altered transcription of RXR gene in a tissue and sex specific manner. In the CNS, a significant down-regulation was observed in females both before and after imposex initiation (P≤0.01 and P≤0.05, respectively). A similar trend was observed in male CNS at the first time-point, although differences between control and the TBT-exposed group were just above significance (P=0.059). The penis/PFA showed no differences in transcription of RXR gene between control and TBT exposed female snails before imposex induction, or before and after imposex initiation for males. However, male penis showed higher transcription of RXR gene in comparison to the PFA of females. After imposex has been induced, a significant (P≤0.001) increase in transcription of RXR gene was observed in penis of females with vas deference sequence index (VDS) levels of 3-4 in comparison with the PFA of both control and imposex females with VDS 1-2. At advanced stages of imposex, females displayed RXR transcription patterns in penis identical to those of males, which points to a functional role of RXR in the penis of both genders. In the other tissues, gonads and digestive gland, RXR gene transcription was not affected by TBT, at any of the analysed time-points. These patterns of RXR gene transcription upon TBT exposure highlight the pivotal involvement of the CNS in the mechanism of imposex induction. We integrate the results in a conceptual model, and discuss the central role of RXR and the retinoic acid signalling pathways in imposex and male genitalia formation in gastropods.

Hazardous and Noxious Substances (HNS) in the marine environment: prioritizing HNS that pose major risk in a European context.

Increases in the maritime transportation of Hazardous and Noxious Substances (HNS), alongside the need for an effective response to HNS spills have led environmental managers and the scientific community to focus attention on HNS spill preparedness and responsiveness. In the context of the ARCOPOL project, a weight-of-evidence approach was developed aimed at prioritizing HNS that pose major environmental risks to European waters. This approach takes into consideration the occurrence probability of HNS spills in European Atlantic waters and the severity of exposure associated with their physico-chemical properties and toxicity to marine organisms. Additionally, a screening analysis of the toxicological information available for the prioritization of HNS was performed. Here we discuss the need for a prioritization methodology to select HNS that are likely to cause severe marine environmental effects as an essential step towards the establishment of a more effective preparedness and response to HNS incidents.

Sexing blennies using genital papilla morphology or ano-genital distance.

Information on the genital morphology of male and female Lipophrys pholis is provided, as well as for two other sympatric blenniid species, Coryphoblennius galerita and Lipophrys trigloides. The use of non-invasive sex determination procedures described may be extremely useful not only for ecological studies but also as a proxy for the detection of environmental exposure to endocrine disruptive chemicals, given that blennies have been proposed as potential sentinel species for chemical contamination.

Validating a multi-biomarker approach with the shanny Lipophrys pholis to monitor oil spills in European marine ecosystems.

Oil spills are an importance source of polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. Intertidal communities are particularly sensitive since most organisms from these ecosystems are sessile or present reduced mobility. Hence, it is important to validate the use of resident species as sentinels to characterize the impact of oil spills on the rocky shores and the improvement during the restoration process. Recently the advantages of using the shanny Lipophrys pholis in pollution monitoring within the northwestern Atlantic coast has been pointed out. Therefore, with the aim of further validating the use of L. pholis in pollution monitoring associated with petrogenic hydrocarbon contamination, a multi-biomarker approach study was carried out 1 week after a moderate oil spill from the waste treatment plant (WTP) of the major Portuguese refinery in the north of Portugal (Petrogal). Fish collected at 2 km from the accident displayed a significant induction of ethoxyresorufin-O-deethylase activity (EROD) and fluorescent aromatic compounds (FACs) in bile (up to a 5-fold induction) in comparison with the pre-spill scenario, and a 15% induction in erythrocytic nuclear abnormalities (ENA), a biomarker of genotoxicity. In contrast, no significant differences were recorded in the reference site. In order to better characterize the time-course accumulation of FACs in bile after a PAH insult, laboratory exposure of L. pholis to benzo[a]pyrene (B[a]P) was performed. A clear dose-response accumulation of B[a]P metabolites was observed that closely reflected nominal exposure concentrations already after 3d. Overall, the findings of the present study highlight the potential of L. pholis in pollution monitoring dealing not only with chronic contamination, but also with oil spill accidents of a moderate scale. Taking into consideration that EROD and FACs determinations in L. pholis are cost effective, rapid and easy to use, they offer a great potential to be incorporated into risk assessment of PAHs in the scope of national monitoring programs and the European Water Policy legislation.

Disruption of zebrafish (Danio rerio) embryonic development after full life-cycle parental exposure to low levels of ethinylestradiol.

Exposure of fish to the synthetic estrogen ethinylestradiol (EE2) has been shown to induce a large set of deleterious effects. In addition to the negative impact of EE2 in reproductive endpoints, concern has recently increased on the potential effects of EE2 in fish embryonic development. Therefore, the present study aimed at examining the effects of EE2 on the full embryonic development of zebrafish in order to identify the actual phases where EE2 disrupts this process. Hence, zebrafish were exposed to environmentally relevant low levels of EE2, 0.5, 1 and 2ng/L (actual concentrations of 0.19, 0.24 and 1ng/L, respectively) from egg up to eight months of age (F(1)), and the survival as well as the occurrence of abnormalities in their offsprings (F(2)), per stage of embryonic development, was investigated. A thorough evaluation of reproductive endpoints and transcription of vtg1 gene in the parental generation (F(1)) at adulthood, was performed. No significant differences could be observed for the two lowest EE2 treatments, in comparison with controls, whereas vtg1 transcripts were significantly elevated (40-fold) in the 2ng/L EE2 treatment. In contrast to the findings in the F(1) generation,a significant concentration-dependent increase in egg mortality between 8 and 24hours post-fertilization (hpf) was observed for all EE2 treatments, when compared with controls. The screening of egg and embryo development showed a significant increase in the percentage of abnormalities at 8 hpf for the highest EE2 concentration, a fact that might explain the increased embryo mortality at the 24 hpf time-point observation. Taken together, these findings indicate that the two lowest tested EE2 concentations impact late gastrulation and/or early organogenesis, whereas exposure to 2ng/L EE2 also disrupts development in the blastula phase. After early organogenesis has been completed (24 hpf), no further mortality was observed. These results show that increased embryo mortality occurs at EE2 levels below those inducing reproductive impairment and vtg1 gene induction in the male parental generation, thus suggesting that EE2 may impact some fish populations at levels below those inducing an increase in vtg1 transcripts. Hence, these findings have important implications for environmental risk assessment, strongly supporting the inclusion of embryonic development studies in the screening of endocrine disruption in wild fish populations.

Organotin levels in seafood from Portuguese markets and the risk for consumers.

Because of their ubiquity in the aquatic environment, the antifouling agent tributyltin (TBT) and other organotins (OTs) accumulate through the food chain, resulting in the occurrence of OTs in seafood products. Despite a high number of studies on the negative impact of TBT in female prosobranch gastropods, few works exist in Europe reporting the levels of these compounds in edible parts of marine organisms used in Human diet. Therefore, within the scope of an EU project OT-SAFE the levels of several OTs were evaluated in the most relevant seafood products for Portuguese consumers. Butyltins (BTs) have been detected in all analysed groups (fish, crustaceans, bivalves, cephalopods), whereas triphenyltin, tricyclohexyltin, monooctyltin and dioctyltin could not be detected and tetrabutyltin was present above detection limits in a single sample. In general, levels of BTs in edible parts of fish, crustaceans and cephalopods collected in Portuguese markets during this study are in the lower range of that reported for these animal groups from other locations (i.e. below 30ngg(-1) wet weight). In contrast, moderate to high concentrations have been observed in bivalves (up to 275ng TBTg(-1) wet weight). While most samples showed TBT plus DBT levels below the tolerable average residue levels (TARL), which may indicate low risk for consumer, four bivalve samples displayed BT levels above TARL, thus indicating that higher bivalve consumer groups may be at risk. The results found are discussed in relation to the potential risk for consumers and integrated with recent finds on the molecular targets of OTs in mammals.

Vitellogenin gene expression in the intertidal blenny Lipophrys pholis: a new sentinel species for estrogenic chemical pollution monitoring in the European Atlantic coast?

The presence of estrogenic chemicals (ECs) in the aquatic environment is a growing problem. While most attention was initially given to fresh water and estuarine ecosystems, it is now evident that coastal marine areas are also vulnerable to these pollutants. The use of vitellogenin induction in male fish, a specific biomarker of EC exposure, has been the most widely applied methodology. However, in some occasions, the high mobility and migratory behaviour of common sentinel fish species makes data interpretation difficult. Hence, there is the need to validate new sentinel marine fish species which should display, among other features, a strong homing behaviour. The shanny, Lipophrys pholis, is an intertidal fish that combines many of the required characteristics for a sentinel species: abundance and easy of catch, wide geographical distribution and restricted home range. Thus, in order to evaluate, in the field, the species sensitivity to ECs, L. pholis males were collected at two sites reflecting different degrees of anthropogenic contamination. The vitellogenin II gene (VTGII) was isolated and its liver expression evaluated by RT-PCR in the field samples. A significant induction of gene expression was observed in the specimens collected in the urban area, if compared to the reference site, which suggests exposure to ECs. Moreover, a 21-days laboratory exposure to environmental relevant concentrations of ethinylestradiol (EE2) was also performed. A significant induction of L. pholis VTGII gene in EE2 exposed males was observed suggesting similar sensitivity to that of other marine/estuarine fishes. Even though further validation is currently in progress, the available data indicates that L. pholis is responsive to ECs, thus favouring its future integration in monitoring programmes designed to evaluate the presence of ECs in European marine ecosystems.

Organ-specific accumulation and elimination patterns of PCBs in adult seabass (Dicentrarchus labrax).

The present study quantifies and explains the differences between measured organ-specific polychlorinated biphenyls (PCBs) accumulation and elimination kinetics in seabass (Dicentrarchus labrax), comparing these results with calculations using the generic model OMEGA. Specimens of approximately 250 g were selected and fed with contaminated food with PCBs 18, 44, 49, 52, 101, 105, 118, 138, 180 and 187. The OMEGA model was applied using a measured biomass (re)production coefficient and the food ingestion coefficient. It produced estimations of total body concentrations within a factor between 1.5 and 7.0 times of the measured and it also reproduced the general trend of the PCB concentrations during the experiment. This study allows us to confirm that internal distribution of PCBs is slow, producing significant differences in the tissues elimination rate constants and consequently in tissues bioaccumulation factors (BAF). The distribution of PCBs within the organism was slower than reported before, due to the bigger body size and the high lipid content of these fish.

Anti-androgenic effects of sewage treatment plant effluents in the prosobranch gastropod Nucella lapillus.

Recent studies performed under laboratory conditions have shown that single exposure to high levels of several xenoestrogens is able to induce imposex in at least two neogastropod species. In an attempt to evaluate if xenoestrogens, at environmentally relevant conditions, do contribute to imposex induction, we have tested the effects of a mixture containing xenoestrogens (municipal sewage effluents) on imposex development in the dogwhelk Nucella lapillus. Exposure for 3 months to the raw (0.25% and 1%) and the final sewage effluent (12.5% and 50%) rendered no increase in the severity of imposex. Conversely, as exposure to high concentrations of natural steroids, estradiol and estrone, had previously been shown to partially rescue imposex development under laboratory conditions, we have also tested if exposure to the final sewage effluent could ameliorated the severity of imposex induction by tributyltin (TBT). The results demonstrated that co-exposure to the final sewage effluent leads to a decrease trend in the severity of imposex in the presence of TBT. Within the studied imposex parameters, the Relative Penis Size index (RPSI) was the most affected with a 50% decrease in the effluent 12.5% plus TBT exposed group and 25% decrease in the effluent 50% plus TBT, if compared with the TBT alone. Overall, our results give further support to the use of imposex in N. lapillus as a specific biomarker of TBT contamination. However, in areas of high inputs of sewage effluents, the assessment of TBT contamination by the use of the imposex phenomenon should ideally also include data on the tissue levels of butyltins.