Effects of polystyrene nano- and microplastics and of microplastics with sorbed polycyclic aromatic hydrocarbons in adult zebrafish.

The presence of nanoplastics (NPs) and microplastics (MPs) in the environment is recognised as a global-scale problem. Due to their hydrophobic nature and large specific surface, NPs and MPs can adsorb other contaminants, as polycyclic aromatic hydrocarbons (PAHs), and modulate their bioavailability and hazard. Adult zebrafish were exposed for 3 and 21 days to: (1) 0.07 mg/L NPs (50 nm), (2) 0.05 mg/L MPs (4.5 µm), (3) MPs with sorbed oil compounds of the water accommodated fraction (WAF) of a naphthenic crude oil (MPs-WAF), (4) MPs with sorbed benzo(a)pyrene (MPs-B(a)P), (5) 5 % WAF and (6) 21 µg/L B(a)P. Electrodense particles resembling NPs were seen in the intestine lumen close to microvilli. MPs were abundantly found in the intestine lumen, but not internalised into the tissues. After 21 days, NPs caused a significant downregulation of cat, and upregulation of gpx1a and sod1, while MPs upregulated cyp1a and increased the prevalence of liver vacuolisation. No histopathological alteration was observed in gills. In this study, contaminated MPs did not increase PAH levels in zebrafish but results highlight the potential differential impact of plastic particles depending on their size, making it necessary to urgently address the ecotoxicological impact of real environmental NPs and MPs.

Sustainable PHBH-Alumina Nanowire Nanocomposites: Properties and Life Cycle Assessment.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) is a bio-based polyester with the potential to replace some common polymers of fossil origin. However, PHBH presents serious limitations, such as low stiffness, tendency to undergo crystallization over long time periods and low resistance to thermal degradation during processing. In this work, we studied the use of alumina nanowires to generate PHBH-alumina nanocomposites, modifying the properties of PHBH to improve its usability. Solvent casting and melt blending were used to produce the nanocomposites. Then, their physicochemical properties and aquatic toxicity were measured. Finally, LCA was used to evaluate and compare the environmental impacts of several scenarios relevant to the processing and end of life (EoL) conditions of PHBHs. It was observed that, at low concentrations (3 wt.%), the alumina nanowires have a small positive impact on the stiffness and thermal degradation for the samples. However, for higher concentrations, the observed effects differed for each of the applied processing techniques (solvent casting or melt blending). The toxicity measurements showed that PHBH alone and in combination with alumina nanowires (10 wt.%) did not produce any impact on the survival of brine shrimp larvae after 24 and 48 h of exposure. The 18 impact categories evaluated by LCA allowed defining the most environmentally friendly conditions for the processing and EoL of PHBHs, and comparing the PHBH-related impacts to those of some of the most common fossil-based plastics. It was concluded that the preferable processing technique for PHBH is melt blending and that PHBH is unquestionably more environmentally friendly than every other analyzed plastic.

Screening of the Toxicity of Polystyrene Nano- and Microplastics Alone and in Combination with Benzo(a)pyrene in Brine Shrimp Larvae and Zebrafish Embryos.

The occurrence of nanoplastics (NPs) and microplastics (MPs) in aquatic ecosystems and their capacity to sorb hydrophobic pollutants is nowadays an issue of great concern. This study aimed to assess the potential bioavailability and acute toxicity of polystyrene (PS) NPs (50 and 500 nm) and of MPs (4.5 µm), alone and with sorbed benzo(a)pyrene (B(a)P), in the embryo/larval stages of brine shrimps and zebrafish. Exposure to pristine plastics up to 50.1 mg PS/L did not cause significant impact on brine shrimp survival, while some treatments of plastics-B(a)P and all concentrations of B(a)P (0.1-10 mg/L) resulted acutely toxic. In zebrafish, only the highest concentrations of MPs-B(a)P and B(a)P caused a significant increase of malformation prevalence. Ingestion of NPs was observed by 24-48 h of exposure in the two organisms (from 0.069 to 6.87 mg PS/L). In brine shrimps, NPs were observed over the body surface and within the digestive tract, associated with feces. In zebrafish, NPs were localized in the eyes, yolk sac, and tail at 72 h, showing their capacity to translocate and spread into the embryo. MP ingestion was only demonstrated for brine shrimps. In zebrafish embryos exposed to plastics-B(a)P, B(a)P appeared in the yolk sac of the embryos. The presence of B(a)P was also noticeable in brine shrimps exposed to 500 nm NPs-B(a)P. In conclusion, NPs entered and spread into the zebrafish embryo and PS NPs, and MPs were successful vectors of B(a)P to brine shrimp and zebrafish embryos. Particle size played a significant role in explaining the toxicity of plastics-B(a)P. Our study provides support for the idea that plastics may pose a risk to aquatic organisms when combined with persistent organic pollutants such as B(a)P.

Accumulation, Depuration, and Biological Effects of Polystyrene Microplastic Spheres and Adsorbed Cadmium and Benzo(a)pyrene on the Mussel Mytilus galloprovincialis.

Filter feeders are target species for microplastic (MP) pollution, as particles can accumulate in the digestive system, disturbing feeding processes and becoming internalized in tissues. MPs may also carry pathogens or pollutants present in the environment. This work assessed the influence of polystyrene (PS) MP size and concentration on accumulation and depuration time and the role of MPs as vectors for metallic (Cd) and organic (benzo(a)pyrene, BaP) pollutants. One-day exposure to pristine MPs induced a concentration-dependent accumulation in the digestive gland (in the stomach and duct lumen), and after 3-day depuration, 45 µm MPs appeared between gill filaments, while 4.5 µm MPs also occurred within gill filaments. After 3-day exposure to contaminated 4.5 µm MPs, mussels showed increased BaP levels whilst Cd accumulation did not occur. Here, PS showed higher affinity to BaP than to Cd. Three-day exposure to pristine or contaminated MPs did not provoke significant alterations in antioxidant and peroxisomal enzyme activities in the gills and digestive gland nor in lysosomal membrane stability. Exposure to dissolved contaminants and to MP-BaP caused histological alterations in the digestive gland. In conclusion, these short-term studies suggest that MPs are ingested and internalized in a size-dependent manner and act as carriers of the persistent organic pollutant BaP.

Uptake and effects of graphene oxide nanomaterials alone and in combination with polycyclic aromatic hydrocarbons in zebrafish.

Because of its surface characteristics, once in the aquatic environment, graphene could act as a carrier of pollutants, such as polycyclic aromatic hydrocarbons (PAHs), to aquatic organisms. In this study we aimed to (1) assess the capacity of graphene oxide (GO) to sorb PAHs and (2) to evaluate the toxicity of GO alone and in combination with PAHs on zebrafish embryos and adults. GO showed a high sorption capacity for benzo(a)pyrene (B(a)P) (98% of B(a)P sorbed from a nominal concentration of 100 µg/L) and for other PAHs of the water accommodated fraction (WAF) of a naphthenic North Sea crude oil, depending on their log Kow (95.7% of phenanthrene, 84.4% of fluorene and 51.5% of acenaphthene). In embryos exposed to different GO nanomaterials alone and with PAHs, no significant mortality was recorded for any treatment. Nevertheless, malformation rate increased significantly in embryos exposed to the highest concentrations (5 or 10 mg/L) of GO and reduced GO (rGO) alone and with sorbed B(a)P (GO-B(a)P). On the other hand, adults were exposed for 21 days to 2 mg/L of GO, GO-B(a)P and GO co-exposed with WAF (GO + WAF) and to 100 µg/L B(a)P. Fish exposed to GO presented GO in the intestine lumen and liver vacuolisation. Transcription level of genes related to cell cycle regulation and oxidative stress was not altered, but the slight up-regulation of cyp1a measured in fish exposed to B(a)P for 3 days resulted in a significantly increased EROD activity. Fish exposed to GO-B(a)P and to B(a)P for 3 days and to GO + WAF for 21 days showed significantly higher catalase activity in the gills than control fish. Significantly lower acetylcholinesterase activity, indicating neurotoxic effects, was also observed in all fish treated for 21 days. Results demonstrated the capacity of GO to carry PAHs and to exert sublethal effects in zebrafish.

Bioaccumulation, cellular and molecular effects in adult zebrafish after exposure to cadmium sulphide nanoparticles and to ionic cadmium.

Few works have addressed the effects provoked by the exposure to cadmium containing nanoparticles (NPs) on adult zebrafish (Danio rerio). We studied the effects of CdS NPs (5 nm) or ionic cadmium (10 µg Cd/L) after 3 and 21 d of exposure and at 6 months post-exposure (mpe). Acute toxicity was recorded after exposure to both forms of cadmium. Significant cadmium accumulation was measured in the whole fish after both treatments and autometallography showed a higher accumulation of metal in the intestine than that in the liver. Histopathological alterations, such as inflammation in gills and vacuolization in the liver, were detected after the exposure to both cadmium forms and, in a lower extent, at 6 mpe. X-ray analysis proved the presence of CdS NPs in these organs. The hepatic transcriptome analysis revealed that gene ontology terms such as "immune response" or "actin binding" were over-represented after 21 d of exposure to ionic cadmium respect to CdS NPs treatment. Exposure to CdS NPs caused a significant effect on pathways involved in the immune response and oxidative stress, while the exposure to ionic cadmium affected significantly pathways involved in DNA damage and repair and in the energetic metabolism. Oxidative damage to liver proteins was detected after the exposure to ionic cadmium, while a stronger destabilization of the hepatocyte lysosomal membrane was recorded under exposure to CdS NPs. In summary, although ionic cadmium provoked stronger effects than CdS NPs, both cadmium forms exerted an array of lethal and sublethal effects to zebrafish.

Impacts of dietary exposure to different sized polystyrene microplastics alone and with sorbed benzo[a]pyrene on biomarkers and whole organism responses in mussels Mytilus galloprovincialis.

Due to their hydrophobicity and relatively large surface area, microplastics (MPs) can act as carriers of hydrophobic pollutants in the ocean and may facilitate their transfer to organisms. This study examined effects of dietary exposure to polystyrene MPs of 0.5 and 4.5 µm alone and with sorbed benzo[a]pyrene (BaP) on mussels Mytilus galloprovincialis in order to elucidate the effects of MP size and the presence of sorbed BaP on the organism. MPs were provided daily, mixed with algae, during 26 days at equivalent mass (0.058 mg/L), corresponding to 1000 particles/mL for 4.5 µm MPs and to 7.44 × 105 particles/mL for 0.5 µm MPs. Effects were determined on early cellular biomarkers in hemocytes, structure and cell type composition of digestive tubules (DTs), histopathology and whole organism responses (condition index (CI), clearance rate (CR), food absorption efficiency (AE), respiration rate (RR) and scope for growth (SFG)). BaP concentrations in mussels increased with time, in particular when sorbed to smaller MPs. Large MPs were abundant in the lumen of stomach and DTs, but were also occasionally found within epithelial cells. Effects in all treatments increased with exposure time. MPs with sorbed BaP were more toxic than MPs alone according to hemocyte viability and catalase activity and to the quantitative structure of DT epithelium. Higher toxicity of small MPs compared to larger ones was recorded for DNA damage and cell composition of DTs. At tissue level a slight increase in prevalence of inflammatory responses occurred in all exposed groups. At whole organism level a compensatory effect was observed on absorption efficiency across MP treatments at day 26, resulting in increased SFG in mussels exposed to small MPs with sorbed BaP. This could be related to an increased energy need to deal with stress observed in biomarkers. Further work is required to understand the Trojan horse effect of a variety of plastic type, size, shape combinations together with a wide variety of pollutants.

Waterborne exposure of adult zebrafish to silver nanoparticles and to ionic silver results in differential silver accumulation and effects at cellular and molecular levels.

Effects of silver nanoparticles (Ag NPs) on freshwater species have been reported in several studies, but there is not information on the potential long-term consequences of a previous exposure. In this work, we investigated the long-term effects of maltose-coated Ag NPs (20 nm) and of ionic silver (10 µg/L) after 21 days of exposure and at 6 months post-exposure (mpe) in adult zebrafish. Exposure resulted in significant silver accumulation in the whole body of fish exposed to ionic silver, but not in those exposed to Ag NPs. However, autometallography revealed metal accumulation in the liver and intestine of fish treated with the two silver forms and especially in the intestine of fish exposed to Ag NPs. X-ray microanalysis showed the presence of silver in gills, liver and intestine and of Ag NPs in gill and liver cells. Inflammation and hyperplasia were evident in the gills after both treatments and these histopathological conditions remained at 6 mpe. According to the hepatic transcriptome analysis, at 3 days ionic silver regulated a larger number of transcripts (410) than Ag NPs (129), while at 21 days Ag NPs provoked a stronger effect (799 vs 165 regulated sequences). Gene ontology terms such as "metabolic processes" and "response to stimulus" appeared enriched after all treatments, while "immune system" or "reproductive processes" were specifically enriched after the exposure to Ag NPs. This suggests that the toxicity of Ag NPs may not be solely related to the release of Ag ions, but also to the NP form. No evident effects were found on protein oxidation or on hepatocyte lysosomal membrane stability during exposure, but effects recorded on liver lysosomes and persistent damage on gill tissue at 6 mpe could indicate potential for long-term effects in exposed fish.

Cellular and molecular responses of adult zebrafish after exposure to CuO nanoparticles or ionic copper.

Due to their antimicrobial, electrical and magnetic properties, copper nanoparticles (NPs) are suitable for a vast array of applications. Copper can be toxic to biota, making it necessary to assess the potential hazard of copper nanomaterials. Zebrafish (Danio rerio) were exposed to 10 µg Cu/L of CuO NPs of ≈100 nm (CuO-poly) or ionic copper to compare the effects provoked after 3 and 21 days of exposure and at 6 months post-exposure (mpe). At 21 days, significant copper accumulation was only detected in fish exposed to ionic copper. Exposure to both copper forms caused histopathological alterations that could reduce gill functionality, more markedly in the case of ionic copper. Nevertheless, at 6 mpe higher prevalences of gill lesions were detected in fish previously exposed to CuO-poly NPs. No relevant histological alterations were detected in liver, but the lysosomal membrane stability test showed significantly impaired general health status after exposure to both metal forms that lasted up to 6 mpe. 69 transcripts appeared regulated after 3 days of exposure to CuO-poly NPs, suggesting that NPs could produce oxidative stress and reduce metabolism and transport processes. Thirty transcripts were regulated after 21 days of exposure to ionic copper, indicating possible DNA damage. Genes of the circadian clock were identified as the key genes involved in time-dependent differences between the two copper forms. In conclusion, each copper form showed a distinct pattern of liver transcriptome regulation, but both caused gill histopathological alterations and long lasting impaired health status in adult zebrafish.

Developmental and reproductive toxicity of PVP/PEI-coated silver nanoparticles to zebrafish.

Cellular and molecular mechanisms of toxicity of silver nanoparticles (NPs) and their toxicity to fish embryos after waterborne exposure have been widely investigated, but much less information is available regarding the effect of Ag NPs on physiological functions such as growth or reproduction. In this work, the effects of waterborne exposure of adult zebrafish (Danio rerio) to PVP/PEI coated Ag NPs (~5nm) on reproduction (fecundity) were investigated. Moreover, the development of the embryos after parental exposure was compared with the development of embryos after direct waterborne exposure to the NPs. For this, two experiments were run: 1) embryos from unexposed parents were treated for 5days with Ag NPs (10µgAgL-1-10mgAgL-1) and development was monitored, and 2) selected breeding zebrafish were exposed for 3weeks to 100ngAgL-1 (environmentally relevant concentration) or to 10µgAgL-1 of Ag NPs, fecundity was scored and development of resulting embryos was monitored up to 5days. Waterborne exposure of embryos to Ag NPs resulted in being highly toxic (LC50 at 120h=50µgAgL-1), causing 100% mortality during the first 24h of exposure at 0.1mgAgL-1. Exposure of adults, even at the environmentally relevant silver concentration, caused a significant reduction of fecundity by the second week of treatment and resulting embryos showed a higher prevalence of malformations than control embryos. Exposed adult females presented higher prevalence of vacuolization in the liver. These results show that Ag NPs at an environmentally relevant concentration are able to affect population level parameters in zebrafish.

Acute toxicity, bioaccumulation and effects of dietary transfer of silver from brine shrimp exposed to PVP/PEI-coated silver nanoparticles to zebrafish.

The extensive use and release to the aquatic environment of silver nanoparticles (NPs) could lead to their incorporation into the food web. Brine shrimp larvae of 24h showed low sensitivity to the exposure to PVP/PEI-coated Ag NPs (5nm), with EC50 values at 24h of 19.63mgAgL-1, but they significantly accumulated silver after 24h of exposure to 100µgL-1 of Ag NPs. Thus, to assess bioaccumulation and effects of silver transferred by the diet in zebrafish, brine shrimp larvae were exposed to 100ngL-1 of Ag NPs as an environmentally relevant concentration or to 100µgL-1 as a potentially effective concentration and used to feed zebrafish for 21days. Autometallography revealed a dose- and time-dependent metal accumulation in the intestine and in the liver of zebrafish. Three-day feeding with brine shrimps exposed to 100ngL-1 of Ag NPs was enough to impair fish health as reflected by the significant reduction of lysosomal membrane stability and the presence of vacuolization and necrosis in the liver. However, dietary exposure to 100µgL-1 of Ag NPs for 3days did not significantly alter gene transcription levels, neither in the liver nor in the intestine. After 21days, biological processes such as lipid transport and localization, cellular response to chemical stimulus and response to xenobiotic stimulus were significantly altered in the liver. Overall, these results indicate an effective dietary transfer of silver and point out to liver as the main target organ for Ag NP toxicity in zebrafish after dietary exposure.

Effects of metal-bearing nanoparticles (Ag, Au, CdS, ZnO, SiO2) on developing zebrafish embryos.

Due to the increasing commercialization of consumer and industrial products containing nanoparticles (NPs), an increase in the introduction of these materials into the environment is expected. NP toxicity to aquatic organisms depends on multiple biotic and abiotic factors, resulting in an unlimited number of combinations impossible to test in practice. The zebrafish embryo model offers a useful screening tool to test and rank the toxicity of nanomaterials according to those diverse factors. This work aims to study the acute and sublethal toxicity of a set of metal-bearing NPs displaying different properties, in comparison to that of the ionic and bulk forms of the metals, in order to establish a toxicity ranking. Soluble NPs (Ag, CdS and ZnO) showed the highest acute and sublethal toxicity, with LC50 values as low as 0.529 mg Ag l(-1) for Ag NPs of 20 nm, and a significant increase in the malformation prevalence in embryos exposed to 0.1 mg Cd l(-1) of CdS NPs of ∼4 nm. For insoluble NPs, like SiO2 NPs, acute effects were not observed during early embryo development due to the protective effect of the chorion. But effects on larvae could be expected, since deposition of fluorescent SiO2 NPs over the gill lamella and excretion through the intestine were observed after hatching. In other cases, such as for gold NPs, the toxicity could be attributed to the presence of additives (sodium citrate) in the NP suspension, as they displayed a similar toxicity when tested separately. Overall, the results indicated that toxicity to zebrafish embryos depends primarily on the chemical composition and, thus, the solubility of the NPs. Other characteristics, such as size, played a secondary role. This was supported by the observation that ionic forms of the metals were always more toxic than the nano forms, and bulk forms were the least toxic to the developing zebrafish embryos.

Short-term effects on antioxidant enzymes and long-term genotoxic and carcinogenic potential of CuO nanoparticles compared to bulk CuO and ionic copper in mussels Mytilus galloprovincialis.

The aim of this work was to study short-term effects on antioxidant enzyme activities and long-term genotoxic and carcinogenic potential of CuO nanoparticles (NPs) in comparison to bulk CuO and ionic copper in mussels Mytilus galloprovincialis after 21 days exposure to 10 µg Cu L(-1). Then, mussels were kept for up to 122 days in clean water. Cu accumulation depended on the form of the metal and on the exposure time. CuO NPs were localized in lysosomes of digestive cells, as confirmed by TEM and X ray microanalysis. CuO NPs, bulk CuO and ionic copper produced different effects on antioxidant enzyme activities in digestive glands, overall increasing antioxidant activities. CuO NPs significantly induced catalase and superoxide dismutase activities. Fewer effects were observed in gills. Micronuclei frequency increased significantly in mussels exposed to CuO NPs and one organism treated with CuO NPs showed disseminated neoplasia. However, transcription levels of cancer-related genes did not vary significantly. Thus, short-term exposure to CuO NPs provoked oxidative stress and genotoxicity, but further studies are needed to determine whether these early events can lead to cancer development in mussels.

Short- and long-term responses and recovery of mussels Mytilus edulis exposed to heavy fuel oil no. 6 and styrene.

Biomarkers have the potential to be used to assess the impact of anthropogenic discharges in marine waters. We have used a suite of biomarkers spanning from enzymatic to histopathological alterations and general stress responses to assess the short- and long-term impact on mussels Mytilus edulis of heavy fuel oil no. 6 and styrene. Mussels were exposed for 5 months, with a refilling of the exposure system, to a water soluble fraction of heavy fuel and, then, kept for a month in clean water for recovery. In a second experiment, mussels were exposed to styrene for 19 days and maintained in clean water for up to 4 months. Chemical body tissue levels reflected the weathering processes of these compounds. Acyl-CoA oxidase activity was induced in oil-exposed mussels after refilling, whereas styrene inhibited it after 19 days of exposure and after 2 weeks in clean water. Gamete development and alkali-labile phosphate levels suggest that neither oil nor styrene behaved as endocrine disruptors. Neutral red retention time was lower in treated groups than in controls. Lysosomal membrane stability was significantly reduced in exposed groups and recovered after withdrawal of oil but not after removal of styrene. Neither oil nor styrene exposure affected the condition index except for the reduction seen in mussels exposed to oil for 1 month. Biomarker response index discriminated exposed mussels, which showed higher values, and returned to control levels after recovery. Results obtained from these pilot experiments can help to identify relevant monitoring tools to assess the impact of oil and chemicals in marine spill scenarios.

Biomarkers and transcription levels of cancer-related genes in cockles Cerastoderma edule from Galicia (NW Spain) with disseminated neoplasia.

Disseminated neoplasia (DN) is a pathological condition reported for several species of marine bivalves throughout the world, but its aetiology has not yet been satisfactorily explained. It has been suggested that chemical contamination could be a factor contributing to neoplasia. The aim of the present study was to compare cell and tissue biomarkers and the transcription level of cancer-related genes in cockles (Cerastoderma edule) affected by DN with those of healthy cockles in relation to chemical contaminant burdens. For this, cockles were collected from a natural bed in Cambados (Ria de Arousa, Galicia) in May 2009. The prevalence of DN was 12.36% and 3 degrees of DN severity were distinguished. No significant differences in metal accumulation, non-specific inflammatory responses and parasites were observed between healthy and DN-affected cockles. Lysosomal membrane stability was significantly reduced in cockles affected by DN, which indicates a poorer health condition. Very low frequencies of micronuclei were recorded and no significant differences were detected between DN severity groups. Haemolymph analyses showed a higher frequency of mitotic figures and binucleated cells in cockles affected by moderate and heavy DN than in healthy ones. Neoplastic animals showed significantly higher transcription levels of p53 and ras than healthy cockles and mutational alterations in ras gene sequence were detected. Low concentrations of metals, polycyclic aromatic hydrocarbons, polychlorinated biphenyls and phthalate esters were measured in cockles from Cambados. In conclusion, cockles affected by DN suffer a general stress situation and have altered patterns of cancer-related gene transcription. Further studies are in progress to elucidate mechanisms of carcinogenesis in this species.

Combined use of native and caged mussels to assess biological effects of pollution through the integrative biomarker approach.

Native and caged mussels were used in combination for the monitoring of pollution biological effects through an integrative biomarker approach. Mussels (Mytilus galloprovincialis) were deployed in cages in two well-known model localities with different pollution levels in the Basque coast. After 3 weeks caged and native mussels were collected from each site and a suite of effect and exposure biomarkers (from molecular/cellular to organism level) was applied and chemical contaminants (metals, PAHs, PCBs, phthalates and nonylphenol ethoxylates) were analytically determined. Integrative biomarker indices and pollutant indices of tissues were calculated. Several biomarkers used herein responded similarly in native and caged mussels, whereas others exhibited significant differences. Overall, biomarkers in-a-suite depicted site-specific profiles useful for the diagnostic of mussel health status and therefore for ecosystem health assessment in marine pollution biomonitoring. On the other hand, biomarkers and bioaccumulation exhibited different response times, which was especially evident when comparing biomarker and pollutant indices of tissues. The suite of biomarkers was more sensitive after caging (short-term response), whereas tissue pollutant concentrations were more sensitive in native mussels (long-term response). Thus, the combination of native and caged mussels is highly recommended to monitor biological effects of pollution in mussels through the integrative biomarker approach, especially in chronically polluted sites.

Marine ecosystem health status assessment through integrative biomarker indices: a comparative study after the Prestige oil spill "Mussel Watch".

Five integrative biomarker indices are compared: Bioeffects Assessment Index (BAI), Health Status Index (HSI), integrated biological response (IBR), ecosystem health condition chart (EHCC) and Integrative Biomarker Index (IBI). They were calculated on the basis of selected biomarker data collected in the framework of the Prestige oil spill (POS) Mussel Watch monitoring (2003-2006) carried out in Galicia and the Bay of Biscay. According to the BAI, the health status of mussels was severely affected by POS and signals of recovery were evidenced in Galicia after April-04 and in Biscay Bay after April-05. The HSI (computed by an expert system) revealed high levels of environmental stress in 2003 and a recovery trend from April-04 to April-05. In July-05, the health status of mussels worsened but in October-05 and April-06 healthy condition was again recorded in almost all localities. IBR/n and IBI indicated that mussel health was severely affected in 2003 and improved from 2004 onwards. EHCC reflected a deleterious environmental condition in 2003 and a recovery trend after April-04, although a healthy ecosystem condition was not achieved in April-06 yet. Whereas BAI and HSI provide a basic indication of the ecosystem health status, star plots accompanying IBR/n and IBI provide complementary information concerning the mechanisms of biological response to environmental insult. Overall, although the integrative indices based on biomarkers show different sensitivity, resolution and informative output, all of them provide coherent information, useful to simplify the interpretation of biological effects of pollution in marine pollution monitoring. Each others' advantages, disadvantages and applicability for ecosystem health assessment are discussed.

Responses of conventional and molecular biomarkers in turbot Scophthalmus maximus exposed to heavy fuel oil no. 6 and styrene.

Several accidental spills in European coastal areas have resulted in the release of different toxic compounds into the marine environment, such as heavy fuel oil type no. 6 in the "Erika" and "Prestige" oil spills and the highly toxic styrene after the loss of the "Ievoli Sun". There is a clear need to develop tools that might allow assessing the biological impact of these accidental spills on aquatic organisms. The aim of the present study was to determine the short-term effects and recovery after exposure of juvenile fish (Scophthalmus maximus) to heavy fuel oil no. 6 and styrene by using a battery of molecular, cell and tissue level biomarkers. Turbots were exposed to styrene for 7 days and to the diluted soluble fraction of the oil (10%) for 14 days, and then allowed to recover in clean seawater for the same time periods. cyp1a1 transcript was overexpressed in turbots after 3 and 14 days of exposure to heavy fuel oil, whereas ahr transcription was not modulated after heavy fuel oil and styrene exposure. pparα transcription level was significantly up-regulated after 3 days of treatment with styrene. Liver activity of peroxisomal acyl-CoA oxidase (AOX) was significantly induced after 14 days of oil exposure, but it was not affected by styrene. Hepatocyte lysosomal membrane stability (LMS) was significantly reduced after exposure to both treatments, indicating that the tested compounds significantly impaired fish health. Both AOX and LMS values returned to control levels after the recovery period. No differences in gamete development were observed between fuel- or styrene- exposed fish and control fish, and vitellogenin plasma levels were low, suggesting no xenoestrogenic effects of fuel oil or styrene. While styrene did not cause any increase in the prevalence of liver histopathological alterations, prevalence of extensive cell vacuolization increased after exposure to heavy fuel oil for 14 days. In conclusion, the suite of selected biomarkers proved to be useful to determine the early impact of and recovery from exposure to tested compounds in turbot.

Transcriptional responses of cancer-related genes in turbot Scophthalmus maximus and mussels Mytilus edulis exposed to heavy fuel oil no. 6 and styrene.

Recent spills in European waters have released polycyclic aromatic hydrocarbons, important components of heavy fuel oil, and the hydrocarbon styrene. Heavy fuel oil and styrene are classified as potentially genotoxic and carcinogenic. Here we investigate transcription of genes involved in cancer development in the liver of juvenile turbots and in the digestive gland of mussels exposed to heavy fuel oil and to styrene and after a recovery period. In turbot, oil produced a significant up-regulation of p53 and gadd45α after 14 days exposure. cyclin G1 was up-regulated after 7 days treatment with styrene. In mussels, ras was down-regulated in both treatments after the recovery periods. No mutations in ras hotspots were detected in exposed mussels. gadd45α was up-regulated after the recovery period of the styrene experiment. Overall, transcriptional responses differed in mussels compared to turbot. Turbot responded to hydrocarbon exposure by triggering cell cycle arrest (p53) and DNA repair (gadd45α).

Application of a battery of biomarkers in mussel digestive gland to assess long-term effects of the Prestige oil spill in Galicia and Bay of Biscay: correlation and multivariate analysis.

According to published reports, selected cell and tissue-level biomarkers revealed alterations in the health status of mussels collected after the Prestige oil spill (POS) in Galicia and the Bay of Biscay (2003-2006). Presently, univariate and multivariate analysis provides a comprehensive view of the whole data set. Correlation and principal component analysis (PCA) were performed using biomarkers corresponding to or derived from those data, say: induction, AOX(exp), and inhibition, AOX(eff), of Acyl-CoA oxidase enzyme activity; membrane labilisation period, LP, volume density, Vv(LYS), and surface-to-volume ratio, S/V(LYS), of digestive cell lysosomes; volume density of neutral lipids, Vv(NL); volume density of basophilic cells, Vv(BAS), mean luminal radius to mean epithelial thickness in digestive gland epithelium, MLR/MET; gonad index, GI; cumulative intensity of inflammatory responses, CI(IR) and parasitic infestation, CI(PI). The PCA results were used to classify sampling times and localities by means of hierarchical cluster analysis. Biomarkers were more frequently correlated with naphthalene than with total PAH concentration in mussel tissues. Many biological parameters were correlated with each other in agreement with their involvement in toxic processes or their role in the mussels' response against environmental insult. Naphthalene appears to be the most effective toxic herein, which supports that the reported effects may be mainly due to POS. According to the PCA, 4 groups of biomarkers explained 67.37% variability and the factors that most strongly contributed were S/V(LYS), CI(IR), Vv(BAS) and MLR/MET. These factors were used in the hierarchical cluster analyses carried out. The cluster analysis performed to classify sampling times discriminated 3 significantly different periods after POS: (a) a high affection period (until April-04); (b) the beginning of the recovery; and (c) an advanced recovery status in which geographical variability increased (from April-05 onwards). As regards the clustering of localities, major geographical differences were not detected but three outliers were identified: (a) early and highly impacted localities in Galicia and Bay of Biscay; (b) Aguiño that was characterised by extremely high Vv(BAS) during 2003; and (c) localities that were subjected to persistent chronic pollution.