Lipidomic analysis of mussel hemocytes exposed to polystyrene nanoplastics.

Plastics production and usage has exponentially increased in the last decades around the world. Due to the insufficient waste management, a significant amount of plastic ends up in the environment, where they tend to fragment into micro- and nano-plastics (NPs), and accumulate in aquatic organisms with still unknown effects. Although studies have indicated that lipid metabolism is a main target of NPs, this mechanism has not been extensively explored. In this study, we evaluated changes in the lipidome of mussel hemocytes after exposure to polystyrene (PS) NPs of 50 and 500 nm, at two different concentrations (106 and 109 particles/mL) for 24 h. The lipidome of hemocytes, analyzed by FIA-ESI (±) Orbitrap, was characterized by a relatively high abundance of cholesteryl esters (CEs) and phosphatidylcholine-plasmalogens (PC-Os/PC-Ps), involved in cell's defense against oxidative stress and membrane reorganization. In hemocytes exposed to PS NPs, a number of highly unsaturated membrane lipids were down-regulated, indicating a reorganization of the cell membranes after exposure to the particles and an oxidation of lipids with a high number of double bonds. This reduction was more evident after exposure to 50 nm NPs -both concentrations- and 500 nm NPs -high concentration-. The analysis of culture medium suggested increased release of vesicles enriched in triglycerides (TGs). The relevance of these responses to NP exposure on the immune function of hemocytes remains to be investigated.

Functionalization of Photosensitized Silica Nanoparticles for Advanced Photodynamic Therapy of Cancer.

BODIPY dyes have recently attracted attention as potential photosensitizers. In this work, commercial and novel photosensitizers (PSs) based on BODIPY chromophores (haloBODIPYs and orthogonal dimers strategically designed with intense bands in the blue, green or red region of the visible spectra and high singlet oxygen production) were covalently linked to mesoporous silica nanoparticles (MSNs) further functionalized with PEG and folic acid (FA). MSNs approximately 50 nm in size with different functional groups were synthesized to allow multiple alternatives of PS-PEG-FA decoration of their external surface. Different combinations varying the type of PS (commercial Rose Bengal, Thionine and Chlorine e6 or custom-made BODIPY-based), the linkage design, and the length of PEG are detailed. All the nanosystems were physicochemically characterized (morphology, diameter, size distribution and PS loaded amount) and photophysically studied (absorption capacity, fluorescence efficiency, and singlet oxygen production) in suspension. For the most promising PS-PEG-FA silica nanoplatforms, the biocompatibility in dark conditions and the phototoxicity under suitable irradiation wavelengths (blue, green, or red) at regulated light doses (10-15 J/cm2) were compared with PSs free in solution in HeLa cells in vitro.

Bioaccumulation, tissue and cell distribution, biomarkers and toxicopathic effects of CdS quantum dots in mussels, Mytilus galloprovincialis.

The bioaccumulation, cell, tissue distribution, and biological effects of 5 nm glutathione-capped CdS quantum dots (CdS QDs) in mussels was compared to bulk and aqueous Cd forms through a two-tier experimental approach. In the 1st tier, mussels were exposed for 3 d to 0.05, 0.5 and 5 mg Cd/l (QDs, bulk, aqueous), bioaccumulation, distribution and lysosomal responses were investigated. In the 2nd tier, mussels were exposed for 21 d to the same forms at the lowest effective concentration selected after Tier 1 (0.05 mg Cd/l), biomarkers and toxicopathic effects were investigated. Accumulation was comparable in QDs and aqueous Cd exposed mussels after 3 d. After 21 d, QDs exposed mussels accumulated less than mussels exposed to aqueous Cd and localised in the endo-lysosomal system and released to the alveoli lumen (21 d) after exposure to QDs and aqueous Cd. Intracellular levels of Cd increased on exposure to QDs and aqueous Cd, and to a lesser extent to bulk, and accompanied by the up-regulation of metallothionein 10 (1 d) and 20 (1, 21 d). Lysosomal membrane destabilisation depended on Cd2+ released by all forms but was marked after exposure to aqueous Cd (1 d). Toxicopathic effects (vacuolisation, loss of digestive cells and haemocytic infiltration) were evident after exposure to QDs (1 d) and aqueous Cd (21 d). Toxicity most likely depended on the ionic load resulting from Cd2+ release from the different forms of Cd; yet nanoparticle-specific effects of QDs cannot be disregarded.

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.

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.

Steroidogenesis and phase II conjugation during the gametogenesis of thicklip grey mullet (Chelon labrosus) from a population showing intersex condition.

Steroidogenesis, the process by which steroid hormones are synthesized, involves a vast number of enzymes and biochemical pathways that are susceptible to chemical modulation. Endocrine disrupting compounds (EDCs) are of special concern since they can alter hormone homeostasis by interfering with synthesis, transport and elimination of hormones. It is important to understand gender differences and the natural variation in steroid balance through gamete development in fish exposed to EDCs. The aim of this study was to determine mRNA levels of genes encoding for Steroidogenic Acute Regulatory (star) protein; the steroidogenic enzymes P450 11ß hydroxylase (cyp11b1) and P450 aromatase (cyp19a1a); as well as the phase II conjugation enzymes sulfotransferase (sult) and UDP-glucuronosyltransferase (ugt), together with the activity of P450 aromatase and plasma levels of 17ß-estradiol (E2) and 11-ketotestosterone (11-KT), at different gametogenic stages and in intersex individuals of the thicklip grey mullet Chelon labrosus. Results demonstrated that the transcription levels of star, sult and ugt and levels of E2 and 11-KT in plasma significantly changed with the interaction between gender and reproductive stage. Cyp11b1 and cyp19a1a transcription levels were significantly different between genders while the activity of P450 aromatase varied significantly between genders and reproductive stages. Results from a multivariate assessment demonstrated that measured endpoints distinguished male, female and intersex mullets at immature gametogenic stage. Intersex distinction was based on sult, ugt and cyp19a1a transcript levels and P450 aromatase activity. The present work provides data to be used in future experimental designs with C. labrosus species, and gives new clues about the molecular events that lead to intersex occurrence in mullets.

Mugilid fish are sentinels of exposure to endocrine disrupting compounds in coastal and estuarine environments.

Effects on fish reproduction can result from a variety of toxicity mechanisms first operating at the molecular level. Notably, the presence in the environment of some compounds termed endocrine disrupting chemicals (EDCs) can cause adverse effects on reproduction by interfering with the endocrine system. In some cases, exposure to EDCs leads to the animal feminization and male fish may develop oocytes in testis (intersex condition). Mugilid fish are well suited sentinel organisms to study the effects of reproductive EDCs in the monitoring of estuarine/marine environments. Up-regulation of aromatases and vitellogenins in males and juveniles and the presence of intersex individuals have been described in a wide array of mullet species worldwide. There is a need to develop new molecular markers to identify early feminization responses and intersex condition in fish populations, studying mechanisms that regulate gonad differentiation under exposure to xenoestrogens. Interestingly, an electrophoresis of gonad RNA, shows a strong expression of 5S rRNA in oocytes, indicating the potential of 5S rRNA and its regulating proteins to become useful molecular makers of oocyte presence in testis. Therefore, the use of these oocyte markers to sex and identify intersex mullets could constitute powerful molecular biomarkers to assess xenoestrogenicity in field conditions.

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.

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.

Retinoid X receptor (RXR), estrogen receptor (ER) and other nuclear receptors in tissues of the mussel Mytilus galloprovincialis: cloning and transcription pattern.

Bivalve molluscs accumulate chemical compounds from the environment that could cause alterations in lipid homeostasis and endocrine system. In vertebrates such cell processes are modulated by transcription factors belonging to the superfamily of nuclear receptors (NRs). The goal of this study was to clone fragments of mussel Mytilus galloprovincialis NR genes that could mediate cell responses such as peroxisome proliferation and endocrine disruption. PCR-based screening of mussel digestive gland cDNA using degenerate primers provided cDNA fragments or whole ORFs of retinoid X receptor (RXR), estrogen receptor (ER) and 5 proteins belonging to the NR1 subfamily highly similar to the arthropod ecdysone inducible protein E75. NR1G, whose whole ORF was cloned, is related to the nematode and trematode G group of NR1 receptors; NR1DEF is related to the D, E and F groups, and NR1Dv1, NR1Dv2 and NR1DΔ belong to the D group. mRNA transcripts for all these receptors were detected in gill, mantle and digestive gland. In all cases, except ER, transcript levels were lower in June than in January. NR1Dv1 and NR1DΔ did not show identical transcription levels, although both were at their lowest in digestive gland in June. On the contrary, NR1Dv2 and NR1DΔ transcription profiles were similar. Further studies are needed to determine the function(s) of mussel RXR, ER and novel NR1 subfamily receptors and their possible role in the regulation of physiological cell responses and/or adaptive response to xenobiotic exposures.

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.

Fate and effects of graphene oxide alone and with sorbed benzo(a)pyrene in mussels Mytilus galloprovincialis.

Graphene oxide (GO) has gained a great scientific and economic interest due to its unique properties. As incorporation of GO in consumer products is rising, it is expected that GO will end up in oceans. Due to its high surface to volume ratio, GO can adsorb persistent organic pollutants (POPs), such as benzo(a)pyrene (BaP), and act as carrier of POPs, increasing their bioavailability to marine organisms. Thus, uptake and effects of GO in marine biota represent a major concern. This work aimed to assess the potential hazards of GO, alone or with sorbed BaP (GO+BaP), and BaP alone in marine mussels after 7 days of exposure. GO was detected through Raman spectroscopy in the lumen of the digestive tract and in feces of mussels exposed to GO and GO+BaP while BaP was bioaccumulated in mussels exposed to GO+BaP, but especially in those exposed to BaP. Overall, GO acted as a carrier of BaP to mussels but GO appeared to protect mussels towards BaP accumulation. Some effects observed in mussels exposed to GO+BaP were due to BaP carried onto GO nanoplatelets. Enhanced toxicity of GO+BaP with respect to GO and/or BaP or to controls were identified for other biological responses, demonstrating the complexity of interactions between GO and BaP.

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).

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.

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.

Effects of microplastics alone or with sorbed oil compounds from the water accommodated fraction of a North Sea crude oil on marine mussels (Mytilus galloprovincialis).

Microplastics (MPs) can adsorb persistent organic pollutants such as oil hydrocarbons and may facilitate their transfer to organisms (Trojan horse effect). The aim of this study was to examine the effects of a 21 day dietary exposure to polystyrene MPs of 4.5 µm at 1000 particles/mL, alone and with sorbed oil compounds from the water accommodated fraction (WAF) of a naphthenic North Sea crude oil at two dilutions (25 % and 100 %), on marine mussels. An additional group of mussels was exposed to 25 % WAF for comparison. PAHs were accumulated in mussels exposed to WAF but not in those exposed to MPs with sorbed oil compounds from WAF (MPs-WAF), partly due to the low concentration of PAHs in the studied crude oil. Exposure to MPs or to WAF alone altered the activity of enzymes involved in aerobic (isocitrate dehydrogenase) and biotransformation metabolism (glutathione S-transferase). Prevalence of oocyte atresia and volume density of basophilic cells were higher and absorption efficiency lower in mussels exposed to MPs and to WAF than in controls. After 21 days MPs caused DNA damage (Comet assay) in mussel hemocytes. In conclusion, a Trojan horse effect was not observed but both MPs and oil WAF caused an array of deleterious effects on marine mussels at different levels of biological organization.

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

In order to assess the long-term lysosomal responses to the Prestige oil spill (POS), mussels, Mytilus galloprovincialis, were collected in 22 localities from Galicia and the Bay of Biscay (North Iberian peninsula) in July, and September 2003, April, July, and October 2004-2005 and April 2006. Lysosomal membrane stability (labilisation period, LP) and lysosomal structural changes (lysosomal volume density, Vv(L) and lysosomal surface-to-volume ratio, S/V(L)) were measured as general stress biomarkers. The most remarkable long-term effects after the POS were drastic changes in lysosomal size (lysosomal enlargement) and membrane stability (extremely low LP values) up to April-04. Later on, a recovery trend was envisaged all along the studied area after July-04, albeit membrane stability continued to be below 20 min throughout the studied period up to April-06, which indicates a "distress-to-moderate-stress" condition. Lysosomal Response Index (LRI) revealed that environmental stress was more marked in Galicia than in the Bay of Biscay, mainly in the first sampling year, although a "moderate-to-high-stress" condition persisted until July-05. Overall, although lysosomal size returned to reference values, membrane stability was not fully recovered indicating a stress situation throughout the studied period.

Effects of the fuel oil spilled by the Prestige tanker on reproduction parameters of wild mussel populations.

The aim of this work was to assess possible effects of the Prestige oil spill on reproduction parameters of mussels along the Galician and Bay of Biscay coast. Studied endpoints included sex ratio, gonad histology and vitellogenin-like proteins using the alkali-labile phosphate (ALP) method. A high prevalence of haemocytic infiltration of follicles and severe oocyte atresia was found in most localities in April 2003. Spawning gonads were observed in most impacted populations in the same sampling. In April 2004 mature small sized follicles were observed. No histopathological changes were observed in April 2005 and 2006, except a high prevalence of necrotic gametes in 6 out of 22 localities in April 2006. Female ALP levels showed high interindividual variability in April 2004, which was reduced in April 2005 and 2006. No xenoestrogenic effects were observed in male mussels. Overall, gamete alterations were detected during 2003-2004 and a recovery trend was observed afterwards.

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: tissue-level biomarkers and histopathology.

In order to assess the biological effects of the Prestige oil spill (POS), mussels, Mytilus galloprovincialis, were collected in 22 localities along the North coast of the Iberian Peninsula over 3 years (April 2003-April 2006). Different tissue-level biomarkers including cell type composition (volume density of basophilic cells, Vv(BAS)) in digestive gland epithelium, structural changes of digestive alveoli (mean luminal radius to mean epithelial thickness, MLR/MET) and histopathological alterations (prevalence and intensity) of the digestive gland were analysed. Severe alterations in the general condition of the digestive gland tissue were observed all over the study area up to 2004-2005. High Vv(BAS) values were recorded mainly in Galicia but also to a lesser extent in the Bay of Biscay in 2003-2004. Atrophy of the digestive alveoli, measured in terms of MLR/MET, was detected all along the studied area up to 2006. Inflammatory responses cannot be related to pollution due to the POS: (a) prevalence and intensity of focal hemocytic infiltration were higher in the Bay of Biscay than in Galicia but they did not show a clear temporal trend; (b) high intensities of brown cell aggregates were only sporadically recorded; and (c) granulocytomas were more frequently recorded in the Bay of Biscay than in Galicia and especially in localities (i.e. Arrigunaga) subjected to chronic pollution. Likewise, Marteilia, trematodes, intracellular ciliates, unidentified eosinophilic bodies, R/CLO and Mytilicola did not follow any recognisable pattern that could be associated to the POS. In contrast, high Nematopsis intensities recorded in several localities in 2003 might suggest some response of local interest after the POS (i.e., in combination with particular factors/conditions). More data at a regional scale are needed before histopathology may provide a reliable ecosystem health assessment but the present results suggest that the approach is worthwhile. Overall, although Vv(BAS) returned to reference values by 2004-2005, MLR/MET values indicated that the mussel health condition was affected during the whole study period up to April 2006.

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.