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Coelomocytes, immunocompetent cells of lumbricids, have received special attention for ecotoxicological studies due to their sensibility to pollutants. Their in vitro responses are commonly quantified after in vivo exposure to real or spiked soils. Alternatively, quantifications of in vitro responses after in vitro exposure are being studied. Within this framework, the present study aimed at optimizing the neutral red uptake (NRU) assay in primary culture of Eisenia fetida coelomocytes for its application in soil toxicity testing. Optimized assay conditions were: earthworm depuration for 24 h before retrieving coelomocytes by electric extrusion; 2 × 10(5) seeded cells/well (200 µl) for the NRU assay and incubation for 1 h with neutral red dye. Supplementation of the culture medium with serum was not compatible with the NRU assay, but coelomocytes could be maintained with high viability for 3 days in a serum-free medium without replenishment. Thus, primary cultures were used for 24 h in vitro toxicity testing after exposure to different concentrations of Cd, Cu, Ni and Pb (ranging from 0.1 to 100 μg/ml). Primary cultures were sensitive to metals, the viability declining in a dose-dependent manner. The toxicity rank was, from high to low, Pb > Ni > Cd > Cu. Therefore, it can be concluded that the NRU assay in coelomocytes in primary cultures provides a sensitive and prompt response after in vitro exposure to metals.

In estuaries and coastal areas, intertidal organisms may be subject to thermal stress resulting from global warming, together with pollution. In the present study, the combined effects of thermal stress and exposure to Cd were investigated in the endo-lysosomal system of digestive cells in mussels, Mytilus galloprovincialis. Mussels were maintained for 24h at 18°C and 26°C seawater temperature in absence and presence of 50 μg Cd/L seawater. Cadmium accumulation in digestive gland tissue, lysosomal structural changes and membrane stability were determined. Semi-quantitative PCR was applied to reveal the changes elicited by the different experimental conditions in hexosaminidase (hex), β-glucuronidase (gusb), cathepsin L (ctsl) and heat shock protein 70 (hsp70) gene transcription levels. Thermal stress provoked lysosomal enlargement whilst Cd-exposure led to fusion of lysosomes. Both thermal stress and Cd-exposure caused lysosomal membrane destabilisation. hex, gusb and ctsl genes but not hsp70 gene were transcriptionally up-regulated as a result of thermal stress. In contrast, all the studied genes were transcriptionally down-regulated in response to Cd-exposure. Cd bioaccumulation was comparable at 18°C and 26°C seawater temperatures but interactions between thermal stress and Cd-exposure were remarkable both in lysosomal biomarkers and in gene transcription. hex, gusb and ctsl genes, reacted to elevated temperature in absence of Cd but not in Cd-exposed mussels. Therefore, thermal stress resulting from global warming might influence the use and interpretation of lysosomal biomarkers in marine pollution monitoring programmes and, vice versa, the presence of pollutants may condition the capacity of mussels to respond against thermal stress in a climate change scenario., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A 3-4 wk cross-transplantation experiment was carried out in order to investigate the sensitivity, rapidity, durability and reversibility of lysosomal and tissue-level biomarkers in the digestive gland of mussels. Four localities in the Basque coast with different levels of chemical pollution and environmental stress were selected. Lysosomal membrane stability (LP) and lysosomal structural changes (VvL; S/VL; NvL) and changes in cell-type composition in digestive gland epithelium (VvBAS) were investigated to determine short (2d) and mid-term (3-4 wk) responses after cross-transplantation. Mussels from Txatxarramendi presented VvBAS<0.1 μm(3)/μm(3) (unstressed) whilst VvBAS>0.12 μm(3)/μm(3) was recorded in mussels from Plentzia (moderate stress) and VvBAS>0.2 μm(3)/μm(3) in Arriluze and Muskiz (high stress). Accordingly, LP<10 min (high stress) was recorded in mussels from Muskiz and Arriluze and LP~15 min (low-to-moderate stress) in those from Plentzia and Txatxarramendi. According to the VvL, S/VL and NvL data, a certain lysosomal enlargement was envisaged in mussels from Arriluze in comparison with those from Txatxarramendi and Plentzia. Mussels from Muskiz exhibited a peculiar endo-lysosomal system made of abundant tiny lysosomes (low VvL and high S/VL and NvL values). Lysosomal and tissue-level biomarkers were responsive after 2d cross-transplantation between the reference and the polluted localities, which indicated that these biomarkers were quickly induced and, to a large extent, reversible. Moreover, the tissue-level biomarker values were maintained during the entire period (3-4 wk) of cross-transplantation, which evidenced the durability of the responsiveness. In contrast, comparisons in the mid-term were unfeasible for lysosomal biomarkers as these exhibited a seasonal winter attenuation resulting from low food availability and low temperatures. In conclusion, lysosomal enlargement and membrane stability and changes in cell-type composition were sensitive, rapid and reversible responses to changes in environmental stress whilst durability of the response could not be demonstrated for lysosomal responses by interferences with the seasonal variability., (© 2013.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The social amoeba Dictyostelium discoideum has been proposed for assessing stress responses to pollutants in soil and it has already been successfully employed in the aquatic environment. Presently, we developed the DDDC assay (D. discoideum developmental cycle assay) for both soil toxicity assessment and soil health screening. The DDDC assay is primarily aimed at determining the capacity of D. discoideum to undergo its developmental programme forming a fruiting body, measured in terms of fruiting body formation inhibition and fruiting body size factor, which may be considered an indication of its ecological fitness (potential for spore dispersal). A second objective of the solid phase DDDC assay is to identify potential mechanisms of toxic action on the developmental cycle, for which three checkpoints are examined: (a) aggregation arrest, (b) migration arrest, and (c) culmination arrest. Presently, conditions for the DDDC assay such as soil texture, soil water content, soil pH, food availability and incubation time were investigated and optimized. In addition, both solid and liquid phase variants of the DDDC assay were applied to assess the toxicity of Hg, at regulatory concentrations. The developmental cycle and ecological fitness were affected from the exposure to 0.3 mg Hg/kg dry-wt soil onwards. The DDDC assay has been shown to be a high sensitivity test., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

This is a pilot study for assessing soil ecosystem health in chronically polluted sites on the basis of a 3-tier approach (screening+scoring+understanding) designed to be cost-effective and scientifically based, and to provide straightforward advice and support to managers and stakeholders involved in environmental protection. For the initial screening (Tier 1), the use of a highly sensitive, low-cost biomarker such as neutral red uptake (NRU) in earthworm coelomocytes is proposed. In sites where an alteration in NRU has been established, the stress level may be further assessed by utilising a suite of low-cost and rapid biomarkers of effect integrated in an integrative biological response (IBR) index to obtain an objective (scored) assessment of the induced stress syndrome (Tier 2). The IBR/n index is based on the integration of biomarkers at different levels of biological organisation. Acyl-CoA oxidase activity (AOX), catalase activity (CAT), lipofuscin optical density (LOD%), NRU and the mean epithelial thickness (MET) have been used to calculate the IBR/n index. Biomarkers are determined in earthworms, Eisenia fetida, exposed ex situ to real soils (three mining sites and a reference) for 3, 10 and 17d. The 3d NRU (Tier 1) provided signal of stress. After 3d, PCA, based on the suite of biomarkers (Tier 2), discriminated reference and polluted sites according to toxicity profiles and at 17d, the most polluted site is segregated from less polluted and reference sites. Soils were classified as harmful, unhealthy (not apparently toxic) or healthy. Soils were investigated by microarray transcriptomics (Tier 3), to understand the causes (aetiology) and consequences (prognosis) of health impairment. Tier 3 discriminates, according to stress syndrome traits, soils that did not fall into the category of highly stressed and revealed the main agent causing toxicity at each site by identifying the toxicity mechanisms and biological responses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The biological effects of a coastal process water (PW) discharge on native and caged mussels (Mytilus edulis) were assessed. Chemical analyses of mussel tissues and semi permeable membrane devices, along with a suite of biomarkers of different levels of biological complexity were measured. These were lysosomal membrane stability in haemocytes and digestive cells; micronuclei formation in haemocytes; changes in cell-type composition in the digestive gland epithelium; integrity of digestive gland tissue; peroxisome proliferation; and oxidative stress. Additionally the Integrative Biological Response (IBR/n) index was calculated. This integrative biomarker approach distinguished mussels, both native and caged, exhibiting different stress conditions not identified from the contaminant exposure. Mussels exhibiting higher stress responses were found with increased proximity to the PW discharge outlet. However, the biological effects reported could not be entirely attributed to the PW discharge based on the chemicals measured, but were likely due to either other chemicals in the discharge that were not measured, the general impact of the processing plant and or other activities in the local vicinity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

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.

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.

The biological impact of a treated produced water (PW) was investigated under controlled laboratory conditions in the blue mussel, Mytilus edulis. Mussel health status was assessed using an integrated biomarker approach in combination with chemical analysis of both water (with SPMDs), and mussel tissues. Acyl-CoA oxidase activity, neutral lipid accumulation, catalase activity, micronuclei formation, lysosomal membrane stability in digestive cells and haemocytes, cell-type composition in digestive gland epithelium, and the integrity of the digestive gland tissue were measured after 5 week exposure to 0%, 0.01%, 0.1%, 0.5% and 1% PW. The suite of biomarkers employed were sensitive to treated PW exposure with significant sublethal responses found at 0.01-0.5% PW, even though individual chemical compounds of PW were at extremely low concentrations in both water and mussel tissues. The study highlights the benefits of an integrated biomarker approach for determining the potential effects of exposure to complex mixtures at low concentrations. Biomarkers were integrated in the Integrative Biological Response (IBR/n) index., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

Lysosomal biomarkers are early warning signals of the biological effects caused by environmental pollutants but the promptness of lysosomal responses to pollutants has not been investigated yet. This work is aimed to determine the response-time of digestive cell lysosomes in mussels exposed to metals and hydrocarbons. Mussels, Mytilus galloprovincialis, were exposed, under laboratory conditions to Cd and to the water-accommodated fraction of a lubricant oil. One mussel per experimental group was sacrificed and processed every hour from 0 h to 30 h. Changes in AcP activity, immunoreactivity and LMS test based on AcP histochemistry, discriminates significantly control and exposed mussels within 5 h exposure. The present results suggested that after 15-20 h exposure digestive cell loss might be accompanied by increased AcP activity (extralysosomal) without a parallel increase in the levels of immunoreactive AcP protein, especially after Cd-exposure. The reduced labilisation period of lysosomal membrane constitute a cost effective early warning signal that, however, is not necessarily correlated with the exposure time. The routine application of immunochemical techniques deserves more research efforts before its implementation although, these techniques are very valuable to understand and interpret correctly lysosomal responses to pollutants.

Lysosomal responses (enlargement and membrane destabilisation) in mussel digestive cells are well-known environmental stress biomarkers in pollution effects monitoring in marine ecosystems. Presently, in laboratory and field studies, both responses were measured separately (in terms of lysosomal volume density--Vv--and labilisation period--LP) and combined (lysosomal response index--LRI) in order to contribute to their understanding and to develop an index useful for decisions makers. LRI integrates Vv and LP, which are not necessarily dependent lysosomal responses. It is unbiased and more sensitive than Vv and LP alone and diminishes background due to confounding factors. LRI provides a simple numerical index (consensus reference=0; critical threshold=1) directly related to the pollution impact degree. Moreover, LRI can be represented in a way that allows the interpretation of lysosomal responses, which is useful for environmental scientists.

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are beta-glucuronidase (beta-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that beta-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas beta-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, beta-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that beta-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed.

A biomonitoring program was carried out in spring and autumn in three pollution hot-spots and sensitive areas of the NW Mediterranean Sea using red mullets (Mullus barbatus) as sentinel organisms and a battery of biomarkers together with gonad histology. In fish from anthropogenic impacted areas (Fos-sur-mer, Cortiou, Arenzano, Delta of Ebro) lysosomal membrane destabilization occurred indicating disturbed health. There were no significant differences in metallothionein (MT) levels among stations. Peroxisomal acyl-CoA oxidase (AOX) activity was highest in fish from Cortiou. Both MT levels and AOX activities were significantly correlated with gamete development. Prevalence of melanomacrophage centers were high in Cortiou in all samplings and in Fos-sur-mer in September samplings. In conclusion, the application of a battery of biomarkers in red mullets provided relevant data for the assessment of environmental pollution in the NW Mediterranean Sea but also showed the difficulties of using native fish as sentinels. For future studies caging strategies are recommended.

The natural variability in cell proliferation activity in the epithelium of the digestive gland and stomach was investigated in mussels, Mytilus galloprovincialis (Lmk), of different age and tidal level at different seasons. After treating mussels with the thymidine analogue bromodeoxyuridine (BrdU) for 6 hours, BrdU immunohistochemistry was performed every 2 hours for the next 36. The relative proportion of BrdU positive cells was quantified as BrdU labelling (per thousand). Marked seasonal differences were recorded in BrdU labelling, with much higher proliferating activity in summer than in autumn and winter. Cell proliferation seemed not to be significantly dissimilar between mussels of different age (size). In contrast, the digestive gland epithelium of mussels from intertidal and subtidal populations differed not only in the levels but also in the pattern of variation of BrdU labelling, which in intertidal mussels appeared to be modulated by photoperiod and tide, unlike in subtidal mussels, in which variations followed a circatidal pattern.

With the aim of assessing the biological effects of pollution along three gradients of pollution in the NW Mediterranean Sea, a biomonitoring survey was implemented using a battery of biomarkers (lysosomal membrane stability, lysosomal structural changes, metallothionein (MT) induction and peroxisome proliferation) in mussels over a period of two years as part of the EU-funded BEEP project. Mussels from the most impacted zones (Fos, Genova and Barcelona harbours) showed enlarged lysosomes accompanied by reduced labilisation period of lysosomal membranes, indicating disturbed health. MT levels did not reveal significant differences between stations and were significantly correlated with gonad index, suggesting that they were influenced by gamete development. Peroxisomal acyl-CoA oxidase (AOX) activity was significantly inhibited in polluted stations possibly due to interactions among mixtures of pollutants. In conclusion, the application of a battery of effect and exposure biomarkers provided relevant data for the assessment of biological effects of environmental pollution along the NW Mediterranean Sea.

Metallothioneins (MT) were localized by immunochemistry in different organs and cell compartments of turbot exposed to sublethal concentrations (100 ppb) of Cd for 7 days. The polyclonal rabbit anti-cod MT antibody (NIVA, Norway) applied herein exhibited positive cross-reactivity with turbot MTs. Immunoreactive MTs were localized in the branchial epithelium, in the liver and in the kidney of turbot. In Cd exposed fishes MTs were demonstrated mainly in branchial chloride cells (CC) and to a lesser extend in the area where progenitor cells are located and in the cells of the respiratory epithelium (secondary lamellae). A higher staining intensity for MTs was observed in CC of the interlamellar space of the main branchial epithelium in comparison with control CC. MT-staining was also observed in the chondroblasts of the cartilage and in the erythrocytes within blood vessels both in control and Cd-exposed specimens. MT immunoreaction was high in the liver hepatocytes and weak in the epithelium of the proximal portion of the kidney in exposed turbot. The tegument, spleen and muscle were devoid of any immunolabelling in both treatments. Ultrastructural studies at the transmission electron microscope revealed that Cd-induced MTs were mainly located in the cytoplasm of gill CC, the lysosomes and the cytoplasm of hepatocytes and in the basal labyrinth of kidney proximal nephrocytes. The differential localization/induction of MTs in different cell types described hereby suggests that the quantification of the specific expression of MT may be used in biomonitoring programs as a biomarker of Cd exposure in aquatic environments.

Cellular biomarkers of exposure and biological effects were measured in digestive gland of snails (Physa acuta) sampled in sites with and without active volcanism in São Miguel Island (Azores). Metal content in digestive cell lysosomes was determined by image analysis after autometallography (AMG) as volume density of autometallographed black silver deposits (Vv(BSD)). Lysosomal structural changes (lysosomal volume, surface and numerical densities - Vv(LYS,) Sv(LYS) and Nv(LYS-), and surface-to-volume ratio - S/V(LYS)-) were quantified by image analysis, after demonstration of beta-glucuronidase activity, on digestive gland cryotome sections. Additional chemical analyses (atomic absorption spectrophotometry) were done in the digestive gland of snails. The highest metal concentrations were found in snails from the active volcanic site, which agreed with high intralysomal Vv(BSD). Digestive cell lysosomes in snails inhabiting sites with active volcanism resembled a typical stress situation (enlarged and less numerous lysosomes). In conclusion, the biomarkers used in this work can be applied to detect changes in metal bioavailability due to chronic exposure to metals (volcanism), in combination with chemical analyses.

Whether the radial thickness (RT) of the chloragogenous tissue and intestinal epithelium of earthworms (Lumbricus terrestris) reflects the bioavailability of metals in soils was investigated in two areas, one with active volcanism (Furnas) and another with no volcanic activity since 3 million years ago (Santa Maria), in the Azores. Metal contents in soil samples and earthworms from the two areas were analyzed. Autometallography and measurements of the RT were performed in the chloragogenous tissue and intestinal epithelium. Earthworms from the active volcanic area demonstrated lower RT of chloragogenous tissue and intestinal epithelium as well as higher levels of bioavailable metals, especially Zn and Cd. Comparison of bioavailable metal contents between both areas suggests a higher risk for uptake of potentially toxic metals in the active volcanic area than in the non-active volcanic area, which is reflected by the lower RT of the chloragogenous tissue and intestinal epithelium in the former.

To assess the biological effects of the Prestige oil spill (November 2002) on coastal ecosystems, mussels (Mytilus galloprovincialis) were sampled in 22 locations along the coast of Galicia and the Bay of Biscay in April, July and September 2003 and in February, April, July and October 2004. Several cell and tissue-level biomarkers were measured in digestive gland. Flesh condition index and gamete development stages were assessed as supporting parameters. AOX activity, a marker of exposure to peroxisome proliferating compounds including PAHs, was particularly low in Galicia in 2003 and further was markedly increased in several localities in 2004. Values of the labilization period (LP) of the lysosomal membrane were low in all the studied localities, especially in Galicia in 2003. In 2004, LP values raised, evidencing a certain recovery in mussel's health. In agreement, the volume density of basophilic cells was markedly high in 2003 and showed a decreasing trend throughout 2004. Parameters defining the structure of digestive alveoli showed few variations between 2003 and 2004. Significant correlations between several biomarkers and total polycyclic aromatic hydrocarbons (PAHs) were found. In conclusion, employed biomarkers detected highest degree of disturbance in areas most impacted by the oil spill (Galicia) and were able to evidence a recovery trend during 2004, related to a decrease in total PAH concentrations in mussels.

Turbot (Scophthalmus maximus) were exposed to two sublethal concentrations (1 and 10 mg metal/l) of cadmium (8.9 and 89 microM Cd), copper (15.26 and 152.6 microM Cu) and zinc (15.3 and 153 microM Zn) for 7 days, and afterwards were maintained depurating for 14 days. Immunoreactive metallothioneins (irMTs) and metal ions were localized in the branchial epithelium by immunohistochemistry (using an anti-Cod MT antibody) and autometallography (AMG), respectively. Metal ions were demonstrated by AMG as black silver deposits (BSD), mainly in mucocytes (MC) and to a lesser extent in the other branchial cell-types (respiratory cells (RC), chloride cells (CC) and basal layer cells (BLC)). Irrespective of the metal supplied, BSD were rapidly visualized in MC after 1 h of exposure. This accumulation did not increase with increasing exposure time and concentration. Metallothionein expression was mainly observed in mature CC in the interlamellar space for all exposure conditions and it was shown that all mature cells express the same amount of irMT. The number of CC exhibiting irMT in metal-exposed turbots increased following short exposure times (1 h-1 day) in the filament epithelium and following longer exposure times (1-7 days) in the secondary lamellae. Total levels of irMT in the gills (quantified by image analysis and densitometry) increased significantly in metal-exposed turbot and were related to increased exposure times. It can be concluded that the total content of irMT in the gills of metal-exposed turbot is governed by changes in the number of mature CC expressing the protein. The quantification of total irMT in branchial CC can be considered as a reliable biomarker of metal exposure since reflects changes in metal bioavailability. This approach based on cell-selective immunohistochemistry can be simplified by only quantifying the number of mature CC. In addition, the dramatic increase of CC in the gills that produces epithelial thickening of the FE enhances migration of CC up to the edge of the SL and provokes the hypertrophy and fusion of secondary lamellae can be considered as unspecific biomarkers of effect indicating disturbed health in turbot.

In order to assess the biological effects of the Prestige oil spill (POS), mussels (Mytilus galloprovincialis), European hake (Merluccius merluccius) and European anchovy (Engraulis encrasicolus) were sampled between April and September 2003 in various geographical areas of Bay of Biscay: Galicia, Central Cantabrian and East Cantabrian. In mussels, several cell and tissue biomarkers were measured: peroxisome proliferation as induction of acyl-CoA oxidase (AOX) activity, lysosomal responses as changes in the structure (lysosomal volume density, V(V(L)), surface-to-volume ratio, S/V(L), and numerical density, N(V(L))) and in membrane stability (labilization period, LP), cell type replacement as relative proportion of basophilic cells (volume density of basophilic cells, V(V(BAS))) in digestive gland epithelium, and changes in the morphology of digestive alveoli as mean luminal radius to mean epithelial thickness (MLR/MET). Additionally, flesh condition index (FCI) and gonad index (GI) were measured as supporting parameters. In hake and anchovy, liver histopathology was examined to determine the prevalence of parasites, melanomacrophage centers, non-specific lesions (inflammatory changes, atrophy, necrosis, apoptosis), early non-neoplastic toxicopathic lesions (i.e., hepatocellular nuclear polymorphism), foci of cellular alteration, benign and malignant neoplasms. In mussels, AOX induction was noticeable in April except in Galicia. LP values were low in all the geographical areas studied, indicating disturbed health, especially in Galicia. Alike, lysosomal enlargement was observed in most stations as shown by the extremely low S/V(L) values. V(V(BAS)) and MLR/MET values were markedly high. Overall, employed biomarkers detected exposure to toxic chemicals and disturbed health in mussels from Bay of Biscay, with impact decreasing from April to September. Although hepatocellular nuclear polymorphism and nematode parasitization in fish liver were remarkably prominent in some areas, they cannot be hitherto related to POS, since we lack historical data to determine whether the prevalences found were normal or significantly raised after POS.

Cellular biomarkers of exposure and biological effects were measured in hepatocytes of turbot exposed to either Cd, Cu or Zn at concentrations of 1 and 10 mg/l seawater for 7 days and after depuration for 14 days. Metal content in hepatocyte lysosomes was determined by image analysis after autometallography (AMG) as volume density of autometallographed black silver deposits (Vv(BSD)). Metallothionein (MT) levels were quantified on liver sections by microdensitometry after immunohistochemical staining with a polyclonal anti cod-MT antibody (MT-OD), and in the cytosolic fraction of hepatocytes by difference pulse polarography (MT-DPP). Lysosomal structural changes (lysosomal volume, surface and numerical densities--Vv(LYS), Sv(LYS) and Nv(LYS-), and surface-to-volume ratio S/V(Lys)) were quantified by image analysis after demonstration of beta-glucuronidase activity on liver cryotome sections. Vacuolisation produced by metal-exposure in hepatocytes was quantified by stereology as volume density of vacuoles (Vv(VAC)). Exposure time and metal concentrations significantly affected Vv(BSD) in lysosomes, MT levels and the degree of vacuolisation after 1 h and 1 day exposure to the three metals. The highest Vv(BSD), MT and Vv(VAC) values were recorded after 7 days exposure in all cases. MT-OD and MT-DPP were significantly correlated with Vv(BSD). Vv(LYS) in hepatocytes increased significantly after exposure to the metals. Exposure biomarkers returned to control values after depuration with the exception of those turbots that had been exposed to 10 mg Cd/l. Alike, Vv(LYS) and Sv(lys) (Cu exposure) and Nv(LYS) (Cd and Zn exposures) returned to control values after depuration. It has been therefore demonstrated that the biomarkers used are reversible and return towards control levels once metal exposure ceases. Overall, it is concluded that Vv(BSD), MT-levels and lysosomal responses are valuable biomarkers to assess metal exposure and its effects in turbot, although in quantitative terms the biomarker response varied between metals.

The present study was carried out to elucidate whether lysosomal size reduction in digestive cells of mussels Mytilus galloprovincialis constitutes a selective response against a particular group of organic chemical compounds, in contrast to the lysosomal enlargement characteristic of general stress response. Mussels were treated with di(2-ethylhexyl) phthalate (DEHP), benzo(a)pyrene (B[a]P), and the water accommodated fraction (WAF) of a lubricant oil, which were daily applied by either injection through the adductor muscle for 7 days or water-exposure for 21 days. Control mussels were either kept untreated in clean sea water, or treated with acetone (injection and water-exposure), vehicle used for DEHP and B[a]P. A third set of controls consisted of mussels with pierced shell kept in clean seawater. Digestive glands were excised at various treatment days and beta-glucuronidase activity was demonstrated in 8-microm cryotome sections. Lysosomal volume, surface and numerical densities, and surface-to-volume ratio were quantified by image analysis. Other sections were stained with oil red 0 to demonstrate neutral lipids and changes in lipid levels were quantified by image analysis. Neutral lipid accumulation in digestive cells was used as a complementary indication of exposure to organic chemicals. It resulted to be a very prompt and all-or-nothing response, which reached a plateau before 1 day of treatment with WAF, DEHP and B[a]P after both injection and water-exposure. DEHP-treatment induced a general stress response characterised by lysosomal enlargement in digestive cells, which was already induced after 1 day. Treatment with either WAF or B[a]P elicited a comparable biphasic response. A transient lysosomal enlargement, shorter with WAF than with B[a]P treatment, was evidenced after both injection and water-exposure. Further, under water-exposure conditions, WAF reduced the endo-lysosomal system in size more markedly than B[a]P. Such lysosomal size reduction constitutes a transient response after exposure to diverse organic xenobiotics (acetone, WAF and B[a]P). In addition, this lysosomal size reduction might be followed by a further lysosomal enlargement, which later might yet again give rise to an apparent lysosomal size reduction under chronic exposure conditions. As a whole, the lysosomal response is intricate and cannot be simply interpreted in environmental pollution monitoring programmes. Nevertheless, it still constitutes a powerful and sensitive biomarker extremely useful as early-warning signal.

The cellular processes involved in metal metabolism in molluscs are reviewed, with emphasis on the contribution of microscopy (AMG, ARG, EPMA, and SIMS) to both basic research of metal cell biology and applied environmental research. In molluscs, metal uptake may occur by facilitated diffusion, active transport, or endocytosis, and can be enhanced by MT synthesis or formation of mineralized granules. In aquatic molluscs, gills constitute a key interface for dissolved metal uptake, where metals are bound to MT, incorporated into lysosomes, and released basally towards the blood plasma and circulating hemocytes. However, particulate metal uptake is mainly achieved via the digestive tract by endocytosis; further metals are transferred first to lysosomes and then to residual bodies, especially in the digestive cells of the digestive gland. Additionally, metals can be accumulated selectively in specific cell types. As ligands pools differ from cell to cell, different metals may be retained in different cell types. Class "a" metals are localized in cells with granules composed of carbonate, oxalate, phosphate, and sulfate (oxygen donors), whereas "b" metals are associated with those cell types rich in sulfur and nitrogen ligands (sulfur donors). In molluscs, oxygen donors occur in connective tissue calcium cells and basophilic cells, whereas sulfur donors are present in digestive cells, podocytes, nephrocytes, and rhogocytes. Hemocytes, which constitute the most relevant system for metal transport between tissues, move around the body and may penetrate tissues and remove metals from the inner medium to be accumulated in lysosomes as nondigested products. Rhogocytes also participate in metal mobilization, accumulation, and release. The assessment of metal levels in target cells of sentinel molluscs by microscopic techniques provides an early-warning measure, with promising applications as an exposure biomarker for environmental monitoring programs., (Copyright 2002 Wiley-Liss, Inc.)

The occurrence of changes in flesh and shell weights and in other biometric parameters of mussels, Mytilus galloprovincialis, has been related to different metal levels found in their soft tissues. The effects of clean and Zn-polluted environments and laboratory experiments where Zn-polluted mussels were exposed to sublethal concentrations of Zn, Cu and Cd were investigated. Zinc-polluted mussel shell weights increased significantly after a 51-day depuration period. Exposure of Zn-polluted mussels to Zn or Cd, however, caused a slightly reduced shell growth in comparison with depurating mussel Cu-exposures not causing any reduction in growth. Apart from metal concentrations, metal/shell weight indices have been used to assess metal bioavailability. Metal concentrations recorded in the soft tissues of depurating mussels increased without a source of 'extra' metals, while the Zn/shell-weight index was reduced, as expected from a depuration process, Cu and Cd/shell-weight indices remaining constant. Experimental exposure to Zn, Cu and Cd caused augmented values of Zn, Cu and Cd/shell-weight indices, respectively. These different findings were attributed to changes in flesh weight (related with gamete spawning) that would produce inconsistent estimates of whole metal concentration in soft tissues. Since changes in the tissue composition and in growth rates do not affect Zn/Cu ratios and metal/shell-weight indices, these parameters are proposed as reliable indices of metal bioavailability for 'Mussel-Watch' monitoring programmes. The most sensitive parameter is the metal/shell-weight index, which is, therefore, highly recommended to be used in 'Mussel-Watch' monitoring programmes in order to determine metal bioavailability in seawaters.

Lysosomal responses are widely accepted cellular effect biomarkers of general stress. Up to now, these biomarkers have been analysed by means of conventional techniques based on enzyme histochemical methods, where lysosomal enzymes such as acid phosphatase and beta-glucuronidase (beta-GUS) have been employed as markers of lysosomes. The aim of the present work was to develop more advanced and sensitive methods based on the use of polyclonal antibodies to measure lysosomal enzymes in different sentinel organisms. For this purpose, we have studied the cross-reactivity of two commercial polyclonal antibodies against the lysosomal enzymes acid phosphatase and beta-GUS with molluscan digestive gland by means of immunoblotting and immunohistochemistry. The antibody against acid phosphatase cross-reacted specifically with the lysosomal fraction of the digestive gland, while unspecific immunoreaction occurred with digestive gland whole homogenates and tissue sections. The antibody against beta-GUS cross-reacted specifically with digestive gland whole homogenates and tissue sections. The cross-reactivity of this antibody was tested also in crab hepatopancreas and mullet liver where the same successful results were obtained. The second aim of the present study was to test if the immuno-based approach was sensitive enough to detect lysosomal alterations provoked by contaminants. For this purpose two experiments were carried out with mussels treated with cadmium in two ways: in vivo treatment by injection and in vitro treatment using digestive gland explants. Afterwards immunoblotting studies with the antibody against beta-GUS were applied and immunoreactive bands were quantified by means of a gel analysis programme. We found that beta-GUS protein levels were higher in treated mussels when compared with controls in either in vivo or in vitro treatments. All these data suggest that the polyclonal antibody against beta-GUS is adequate to be used in immuno-based approaches to detect contaminant-induced lysosomal alterations.

This investigation attempts to determine the usefulness of autometallography to localise particular metals in certain key tissues of molluscs exposed to metal mixtures. For this purpose, winkles (Littorina littorea) removed from shell were exposed to very high concentrations of either copper (Cu), zinc (Zn) or a mixture of both metals (Cu&Zn) dissolved in sea-water for short periods of time. Protein-bound metals were detected by autometallography as black silver deposits (BSD) on histological sections of gills, foot, mantle, digestive gland/gonad complex, stomach and kidney. Copper was localised within cytoplasmic granules of gill ciliated cells, nephrocytes and stomach epithelial cells as well as within digestive cell lysosomes. Zinc was essentially found in the basal lamina (histological sense) of gill, stomach, kidney and digestive gland epithelia. BSD were also evidenced in cytoplasmic granules of pore cells present in parenchymal connective tissue of mantle, foot, gill, digestive gland and stomach. Copper and zinc concentrations were additionally calculated for the whole soft body as well as for certain organs by atomic absorption spectrophotometry (AAS). According to AAS, a synergistic phenomenon would contribute to increase the rate of Cu and Zn accumulation in presence of each other. However, after exposure to Cu&Zn autometallography did not evidence any synergistic phenomenon, and Cu and Zn were localised in their respective accumulation sites. In conclusion, autometallography might indicate the presence of certain metals in the environment irrespective of factors, such as "metal-metal interaction-like" phenomena, affecting metal concentrations in soft tissues.

The extent of autometallographical black silver deposits (BSD) has been semiquantified at the light microscope in the gills and digestive gland of either control mussels or Zn-polluted mussels after depuration and on exposure to sublethal concentrations of Cu, Zn, and Cd. The BSD extent in the gills and digestive gland of control mussels was much reduced compared to that in other experimental mussels. The extent of BSD in the gills of depurating mussels was reduced at short depuration times due to decreased levels in the abfrontal cells while in the digestive gland it did not change with the depuration period. The extent of BSD in digestive lysosomes of Cu- and Zn-exposed mussels followed a logarithmic pattern in relation to metal concentration increasing with metal concentrations in the digestive gland. However, a reduced extent of BSD was related to the presence of high metal concentrations under Cd-exposure conditions. This is because the great extent of BSD present in the lumen of the digestive tubules was not taken into account to carry out semiquantification, but, however, the chemical analysis measured the Cd content of these BSD. As such, the extent of BSD in digestive lysosomes followed a logarithmic pattern with total metal concentrations in the digestive gland of Cd-exposed mussels. Therefore, the semiquantitative estimation of BSD in the digestive lysosomes could be considered a reliable index to reflect changes in metal bioavailability in sea water.

Copper (Cu), zinc (Zn) and calcium (Ca) were demonstrated histochemically by means of conventional stains (rubeanic acid for copper, dithizone for zinc, and cobalt nitrate for calcium) and by autometallography in various tissues of winkles (Littorina littorea) sublethally exposed to either copper or zinc dissolved in sea water. Rubeanic acid and dithizone procedures exhibited poor sensitivity: there was no positive reaction after fixation tissues with Bouin's fixative, and only a weak reaction after ethanol fixation. Autometallography, however, produced a positive reaction with both fixatives in the form of black silver deposits in some key cell types. In winkles not exposed to either copper nor zinc, autometallographically demonstrated metals were found in the connective tissue pore cells, the lysosomes of digestive cells, the basal lamina of the digestive tubule epithelium, and cytoplasmic granules in the epithelial cells of the stomach wall. In addition, in winkles exposed to copper, metal deposits were present in some apical cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, and the circulating haemocytes. In winkles exposed to zinc, metal deposits were found in the basal cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, the apex and basal lamina of the nephrocytes in the kidney, and the connective tissue layer surrounding the blood vessels. Additionally, calcium was demonstrated histochemically in the cytoplasm of digestive cells, the cytoplasm of the epithelial cells of the stomach wall, the mucocytes of gills, the basal lamina of the kidneys, the haemocytes, the calcium and pore cells of connective tissue, and the oocyte cytoplasm. Metals were not detected by any procedure in sperm cells, in the cytoplasmic granules of oocytes, or in the basophilic cells in the digestive tubules. In conclusion, autometallography is a highly sensitive method and provides an excellent tool to localize protein-bound copper and zinc in molluscan tissues, and its use in combination with conventional histochemical or chemical methods is highly recommended.

The present investigation was to determine the extent to which mercury (Hg) provokes measurable effects on the structure of the digestive gland of slugs as well as to relate the extent of these effects to the cell and tissue distribution of Hg. For this purpose, slugs (Arion ater) received various dietary concentrations of Hg (from 0 to 1,000 microg Hg/g food) as chloride for 30 days and were histologically examined every third day. Autometallography was used to demonstrate Hg as black silver deposits (BSD) in paraffin sections. The lysosomes and residual bodies of digestive cells resulted to be the major accumulation sites. In addition, Hg was also evidenced in lipofuscine granules of vacuoles in excretory cells but, however, it was rarely observed within calcium cells. Generally, the extent of BSD increased with dietary Hg concentration and exposure time but, however, it became significant lowered after exposure to 1,000 microg Hg/g food for 30 when the digestive epithelium appeared almost devoid of digestive cells. On the other hand, significant changes were recorded in the quantitative structure of digestive tubules. Mean Epithelial Thickness (MET), Mean Luminal Radius (MLR) and Mean Diverticular Radius (MDR) were recorded as measures of the sublethal biological effect of Hg. MET, MLR/MET and MET/MDR were affected by Hg concentration (C), exposure time (T) and CxT interaction, changes in MET, MLR, MLR/MET and MET/MDR being explained by regression models after logarithmic transformation of the data. In order to explain the nature of the changes in the quantitative structure of the digestive tubules this investigation was complemented with qualitative histological observations. According to them, the excretory activity in digestive cells was initially enhanced. Afterwards, the relative numbers of digestive cells declined until the extreme cases of exposure to 1, 000 microg Hg/g for 27 to 30 days in which the digestive epithelium was mostly comprised of calcium and excretory cells. Concomitantly, some changes took place in blood vessels where Leydig cells became disrupted and the connective tissue layers thickened. Finally, it is suggested to use slugs in soil quality assessment as sentinel organisms ("Slug Watch") in which biomarkers of exposure to metallic pollutants and of biological effect are recorded.