Enhanced discrimination of basophilic cells on mussel digestive gland tissue sections by means of toluidine-eosin staining.

Changes in the cell type composition of the digestive gland epithelium constitute a common and recognized biological response to stress in mussels. Usually, these changes are identified as alterations in the relative proportion of basophilic cells, determined in tissue sections stained with hematoxylin-eosin (H&E) and measured in terms of volume density of basophilic cells (VvBAS) after stereological quantification. However, the identification and discrimination of basophilic cells may be a difficult issue, even for a trained operator, especially when, in circumstances of environmental stress, basophilic cells lose their basophilia and the perinuclear area of digestive cells gains basophilia. Thus, the present study was aimed at exploring the best available practices (BAPs) to identify and discriminate basophilic cells on tissue sections of mussel digestive gland. In a first step, a thorough screening of potentially suitable staining methods was carried out; the final selection included several trichrome staining methods and some of their variants, as well as toluidine-based stains. Next, the sample processing (fixation/dehydration steps) was optimized. Toluidine-eosin (T&E) staining after fixation in 4% formaldehyde at 4 °C for 24 h was considered the BAP to identify and discriminate basophilic cells in the digestive gland of mussels. Using the mussel Mytilus galloprovincialis as a target organism, this approach was successfully applied to quantify VvBAS values after automated image analysis and compared with the conventional H&E staining in different field and laboratory tests. It is worth noting that VvBAS values were always higher after T&E staining than after H&E staining, apparently because discrimination of basophilic cells was enhanced. Thus, until more data are available, any comparison with VvBAS values obtained in previous studies using H&E staining must be done cautiously. Finally, the T&E staining was successfully used to discriminate basophilic cells in tissue sections of other marine molluscs of ecotoxicological interest, including Mytilus edulis, Mytilus trossulus, Crassostrea gigas and Littorina littorea.

Effects of soil organic matter content on cadmium toxicity in Eisenia fetida: implications for the use of biomarkers and standard toxicity tests.

Bioavailability is affected by soil physicochemical characteristics such as pH and organic matter (OM) content. In addition, OM constitutes the energy source of Eisenia fetida, a well established model species for soil toxicity assessment. The present work aimed at assessing the effects of changes in OM content on the toxicity of Cd in E. fetida through the measurement of neutral red uptake (NRU) and mortality, growth, and reproduction (Organisation for Economic Co-operation and Development [OECD] Nos. 207 and 222). Complementarily, metallothionein (MT) and catalase transcription levels were measured. To decrease variability inherent to natural soils, artificial soils (Organization for Economic Cooperation and Development 1984) with different OM content (6, 10, and 14%) and spiked with Cd solutions at increasing concentrations were used. Low OM in soil decreased soil ingestion and Cd bioaccumulation but also increased Cd toxicity causing lower NRU of coelomocytes, 100 % mortality, and stronger reproduction impairment, probably due to the lack of energy to maintain protection mechanisms (production of MT).Cd bioaccumulation did not reflect toxicity, and OM played a pivotal role in Cd toxicity. Thus, OM content should be taken into account when using E. fetida in in vivo exposures for soil health assessment.

Sex and sex-related differences in gamete development progression impinge on biomarker responsiveness in sentinel mussels.

In marine pollution monitoring, the biomarkers recorded in sentinel organisms are influenced by natural confounding factors that may jeopardise their interpretation. Among these confounding factors, little is known about the influence of sex along the annual reproductive cycle. The present investigation aims at contributing to understand how sex and sex-related differences in gamete development progression impinge on biomarker baseline values and on biomarker responsiveness to pollution in sentinel mussels. Mussels (Mytilus galloprovincialis) were collected from a relatively clean locality and from a chronically polluted site in the Basque Coast (Bay of Biscay) in January, April, August and November. Sex and gametogenesis stages were determined for each mussel. Tissue concentration of metals and PAHs was analysed. A battery of biomarkers was investigated: cytochrome c oxidase, pyruvate kinase and phosphoenolpyruvate carboxykinase enzyme activities; levels of protein carbonyls, malondialdehyde and 4-hydroxy-2-nonenal; lysosomal enlargement and membrane stability; intracellular neutral lipid accumulation; cell type composition and thinning of the digestive gland epithelium; and survival-in-air. Sex- and reproductive stage-related differences were found in bioaccumulation and in the values and responsiveness of most of the biomarkers. However, the patterns of sex-related differences were not consistent across all biomarkers. The differences in the biomarker responses between females and males also depended on the season, reflecting the progression of the gametogenesis cycle. Thus, selecting mussels of one specific sex does not seem to be a crucial requisite to carry out biomarker-based monitoring; yet, it is highly recommended to identify sex condition and gamete developmental stage of each mussel to test for the potentially confounding effects of sex, reproductive status and sex-related variability along the reproductive cycle.

Beta-glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells.

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.

Multi-annual survey of health status disturbance in the Bilbao estuary (Bay of Biscay) based on sediment chemistry and juvenile sole (Solea spp.) histopathology.

The Bilbao estuary (SE Bay of Biscay) is a recovering ecosystem whose sediments are still contaminated. They represent a potential risk for the biota including benthic and demersal species living in direct contact with the sediment. In this context, the present study aims to survey trends of the health status of the Bilbao estuary based on sediment chemistry and sole (Solea spp.) histopathology. Monitoring campaigns were carried out every autumn from 2011 to 2017 along the estuary. Contaminant levels were measured in sediments; liver, gills and gonads of juvenile fish were collected for histopathology. Overall, contaminant levels fluctuated throughout the years, with highest values recorded in the earlier years of the study period. Sole histopathology showed alterations of mild severity. Results permitted to assess the environmental health status of the Bilbao estuary during 7 years, although no clear temporal trend was detected. Longer-term monitoring programmes are necessary to confirm the ecosystem recovery.

The influence of short-term experimental fasting on biomarker responsiveness in oil WAF exposed mussels.

Mussels are widely used in toxicological experimentation; however, experimental setups are not standardized yet. Although there is evidence of changes in biomarker values during food digestion and depending on the mussel nutritive status, the mode of feeding differs among toxicological experiments. Typically, mussels are fed with different diets in different long-term experiments, while fasting is the most common approach for short-term studies. Consequently, comparisons among experiments and reliable interpretations of biomarker results are often unfeasible. The present investigation aimed at determining the influence of fasting (against feeding with Isochrysis galbana) on biomarkers and their responsiveness in mussels exposed for 96 h to the water accommodated fraction (WAF) of a heavy fuel oil (0%, 6.25%, 12.5% and 25% WAF in sea water). PAH tissue levels in digestive gland and a battery of biomarkers were compared. WAF exposure led to decrease of cytochrome-C-oxidase activity, modulated glutathione-S-transferase activity, augmented lipid peroxidation, inhibited acetyl cholinesterase (AChE) activity, and led to lysosomal enlargement (VvLYS and S/VLYS) and membrane destabilisation, lipofuscin accumulation, and histopathological alterations (VvBAS, MLR/MET and CTD ratio) in the digestive gland epithelium; and were integrated as IBR/n (biological response index). Overall, no significant changes were recorded in AChE activity, S/VLYS and CTD ratio in any experimental treatment, while all the other biomarkers showed significant changes depending on the fasting/feeding condition, the exposure to WAF and/or their interaction. As a result, the integrated biomarker index IBR/n was higher at increasing WAF exposure levels both in fasted and fed mussels albeit the response was more marked in the latter. The response profiles were qualitatively similar between fasted and fed mussels but quantitatively more pronounced in fed mussels, especially upon exposure to the highest concentration (25% WAF). Therefore, it is highly recommended that mussels are also supplied with food during short-term, like during long-term toxicological experiments. This practice would avoid the interference of fasting with biological responses elicited by the tested chemicals and allow for reliable comparison with data obtained in long-term experiments and monitoring programmes.

Assessment of ecosystem health disturbance in mangrove-lined Caribbean coastal systems using the oyster Crassostrea rhizophorae as sentinel species.

This investigation was aimed at contributing to develop a suitable multi-biomarker approach for pollution monitoring in mangrove-lined Caribbean coastal systems using as sentinel species, the mangrove cupped oyster, Crassostrea rhizophorae. A pilot field study was carried out in 8 localities (3 in Nicaragua; 5 in Colombia), characterized by different environmental conditions and subjected to different levels and types of pollution. Samples were collected in the rainy and dry seasons of 2012-2013. The biological effects at different levels of biological complexity (Stress-on-Stress response, reproduction, condition index, tissue-level biomarkers and histopathology) were determined as indicators of health disturbance, integrated as IBR/n index, and compared with tissue burdens of contaminants in order to achieve an integrative biomonitoring approach. Though modulated by natural variables and confounding factors, different indicators of oyster health, alone and in combination, were related to the presence of different profiles and levels of contaminants present at low-to-moderate levels. Different mixtures of persistent (As, Cd, PAHs) and emerging chemical pollutants (musk fragrances), in combination with different levels of organic and particulate matter resulting from seasonal oceanographic variability and sewage discharges, and environmental factors (salinity, temperature) elicited a different degree of disturbance in ecosystem health condition, as reflected in sentinel C. rhizophorae. As a result, IBR/n was correlated with pollution indices, even though the levels of biological indicators of health disturbance and pollutants were low-to-moderate, and seasonality and the incidence of confounding factors were remarkable. Our study supports the use of simple methodological approaches to diagnose anomalies in the health status of oysters from different localities and to identify potential causing agents and reflect disturbances in ecosystem health. Consequently, the easy methodological approach used herein is useful for the assessment of health disturbance in a variety of mangrove-lined Caribbean coastal systems using mangrove cupped oysters as sentinel species.

Reversible alterations in epithelial cell turnover in digestive gland of winkles (Littorina littorea) exposed to cadmium and their implications for biomarker measurements.

In marine molluscs, the epithelium of the digestive gland is composed of two cell types, namely, digestive and basophilic cells. Under normal physiological conditions digestive cells outnumber basophilic cells, but under different stress situations the composition of the epithelium changes, basophilic cells apparently replace digestive cell. Winkles, Littorina littorea, were exposed to 1.25mg/l Cd for 20 days to provoke cell type replacement. Then, animals were depurated in clean seawater for 10 days to determine whether cell type replacement was reversible. Digestive glands were fixed in Carnoy and paraffin embedded for histological analysis. The volume densities of basophilic cells (Vv(BAS)) and digestive cells (Vv(DIG)) were calculated by stereology on hematoxylin-eosin stained sections. Vv(BAS) increased and Vv(DIG) decreased in Cd-exposed animals. After estimation of cell size and absolute cell numbers, these changes were attributed to digestive cell loss and concomitant basophilic cell hypertrophy but not to increased numbers of basophilic cells. Cell type composition and cell size almost fully returned to normal values after 10-day depuration. Accordingly, PCNA immunohistochemistry demonstrated that proliferating digestive cells were more abundant in winkles exposed to Cd and after 10-day depuration than in control specimens, suggesting that net digestive cell loss was accompanied by increased digestive cell proliferation. Thus, Cd-exposure seems to provoke an enhanced digestive cell turnover in order to cope with Cd detoxification. Intralysosomal accumulation of metals (autometallographied black silver deposits; BSD) was used as a biomarker of exposure to Cd and lysosomal structural changes as an effect biomarker to see whether cell type composition might have any effect on these endpoints. BSD formed around Cd ions, in digestive cell lysosomes of Cd-exposed winkles whereas basophilic cells appeared devoid of them. After depuration, BSD were less conspicuous. Enlarged lysosomes were observed in Cd-exposed winkles, lysosome size returning to control levels after 10-day depuration. Changes in digestive cell proliferation, digestive cell loss and basophilic cell hypertrophy did not apparently affect the biomarkers investigated herein.

Digestive cell turnover in digestive gland epithelium of slugs experimentally exposed to a mixture of cadmium and kerosene.

Slugs, Arion ater (L), have been proposed as sentinel organisms to assess soil health. In slugs under the influence of pollutants, digestive cell loss and the concomitant increase of excretory cells of the digestive gland have been described. The aim of the present work was to determine up to what extent digestive cell loss affects biomarkers and whether the affectation is reversible after exposure to a mixture of metal and organic pollutants. Slugs were dosed with a mixture of cadmium and kerosene in the food for 27 days. Apart from chemical analyses, the volume density of black silver deposits (Vv(BSD)) after autometallography, and acyl-CoA oxidase (AOX) activity were used as biomarkers of exposure to metals and organic compounds, respectively. As effect biomarkers, changes in the volume density of the cell types that constitute the digestive gland epithelium were calculated. Proliferating cells were identified by means of bromodeoxyuridine (BrdU) immunohistochemistry. Results revealed that the mixture of pollutants provoked an increase in Vv(BSD) and AOX activity and a decrease in the number of digestive cells. These changes had no effect in the digestive gland accumulation capacity or in the effect and exposure biomarkers employed. BrdU-labelling showed that exposure to pollutants provoked an enhanced digestive cell proliferation.

Digestive cell lysosomes as main targets for Ag accumulation and toxicity in marine mussels, Mytilus galloprovincialis, exposed to maltose-stabilised Ag nanoparticles of different sizes.

Bioavailability and toxicity of maltose-stabilised AgNPs of different sizes (20, 40 and 100 nm) in mussels were compared with bulk and aqueous forms of the metal through a two-tier experimental approach. In the first tier, mussels were exposed for 3 d to a range of concentrations (0.75, 75, 750 µg Ag/l) in the form of Ag20-Mal, Ag40-Mal, Ag100-Mal, bulk Ag and aqueous Ag (as AgNO3), as well as to the concentrations of maltose used in the formulation of NPs. Mortality, bioaccumulation, tissue and cell distribution and lysosomal responses were investigated. In the second tier, mussels were exposed for 21 d to Ag20-Mal, Ag100-Mal, bulk Ag and aqueous Ag at the lowest effective concentration selected after Tier 1 (0.75 µg Ag/l), biomarkers and toxicopathic effects were investigated. Aqueous Ag was lethal within 3 d at 75 µg Ag/l; Ag NPs or bulk Ag did not produce significant mortality at 750 µg Ag/l. Ag accumulation was limited and metallothionein gene transcription was not regulated although metal accumulation occurred in digestive, brown and stomach epithelial cells and in gut lumen after exposure to AgNPs and aqueous Ag starting at low concentrations after 1 d. Electrondense particles (<10 nm) in lysosomes and residual bodies after exposure to AgNPs contained Ag and S (X-ray). Intralysosomal metal accumulation and lysosomal membrane destabilisation were enhanced after exposure to all the forms of Ag and more marked after exposure to Ag20-Mal than to larger NPs. 21 d exposure to AgNPs provoked digestive cell loss and loss of digestive gland integrity, resulting in atrophy-necrosis in digestive alveoli and oedema/hyperplasia in gills (Ag NP), vacuolisation in digestive cells (aqueous Ag) and haemocyte infiltration of connective tissue (all treatments). Intralysosomal metal accumulation, lysosomal responses and toxicopathic effects are enhanced at decreasing sizes and appear to be caused by Ag+ ions released from NPs, although the metal was not substantially accumulated.

Lysosomal responses to different gold forms (nanoparticles, aqueous, bulk) in mussel digestive cells: a trade-off between the toxicity of the capping agent and form, size and exposure concentration.

Gold nanoparticles (NPs) are increasingly used in technological materials and consumer products and may have toxicological characteristics distinct from bulk and aqueous gold. The aim of this work was to understand the effects of Au NPs especially, how the form, the size and the coating influence bioaccumulation/biodistribution and toxicity of NPs in mussels, Mytilus galloprovincialis. Mussels were exposed for 3 d to concentrations of Au (0.75, 75 and 750 µg Au/l) supplied as Au-Cit NPs (5 and 40 nm; Au5-Cit and Au40-Cit), bulk and aqueous Au (HAu(III)Cl4), and to the capping agent (Na-citrate) in doses used in the formulation of NPs (0.005, 0.5, 5 mg/l). Citrate-stabilised NPs formed stable suspensions of aggregates in seawater (SW) available for mussels. Au accumulation in soft tissues was similar in Au40-Cit and aqueous Au exposed mussels, lower in Au5-Cit and negligible after bulk exposure. Au NPs were identified (X-ray microanalysis) in different compartments of the endolysosomal system in digestive cells, and small size NPs (5 nm) were more accumulated than 40 nm NPs, aqueous and bulk. The degree of lysosomal membrane destabilisation was related with intralysosomal metal accumulation and depended on the form, NP size (Au5-Cit > Au40-Cit > aqueous > bulk) and concentration. Citrate alone provoked extreme reduction in lysosomal membrane stability. Toxicopathic alterations were recorded in digestive gland cells (vacuolisation, swollen RER, connective tissue disruption and cell death) especially in mussels exposed to 40 nm NPs. Deleterious effects resulted from digestive tract obliteration (agglomerates) and digestion malfunction. The toxic effect of Au-Cit NPs was influenced both by NP size, capping agent composition and the dose of capping agent carried by NPs, which was size dependent.

Nanoparticle size and combined toxicity of TiO2 and DSLS (surfactant) contribute to lysosomal responses in digestive cells of mussels exposed to TiO2 nanoparticles.

The aim of this investigation was to understand the bioaccumulation, cell and tissue distribution and biological effects of disodium laureth sulfosuccinate (DSLS)-stabilised TiO2 nanoparticles (NPs) in marine mussels, Mytilus galloprovincialis. Mussels were exposed in vivo to 0.1, 1 and 10 mg Ti/L either as TiO2 NPs (60 and 180 nm) or bulk TiO2, as well as to DSLS alone. A significant Ti accumulation was observed in mussels exposed to TiO2 NPs, which were localised in endosomes, lysosomes and residual bodies of digestive cells, and in the lumen of digestive tubules, as demonstrated by ultrastructural observations and electron probe X-ray microanalysis. TiO2 NPs of 60 nm were internalised within digestive cell lysosomes to a higher extent than TiO2 NPs of 180 nm, as confirmed by the quantification of black silver deposits after autometallography. The latter were localised mainly forming large aggregates in the lumen of the gut. Consequently, lysosomal membrane stability (LMS) was significantly reduced upon exposure to both TiO2 NPs although more markedly after exposure to TiO2-60 NPs. Exposure to bulk TiO2 and to DSLS also affected the stability of the lysosomal membrane. Thus, effects on the lysosomal membrane depended on the nanoparticle size and on the combined biological effects of TiO2 and DSLS.

Comparison of cytochemical procedures to estimate lysosomal biomarkers in mussel digestive cells.

Enlargement and membrane destabilisation in digestive cell lysosomes of mussels are biomarkers of pollution effect. Cytochemical methods are currently applied to determine lysosomal membrane stability (LMS) and lysosomal structural changes (LSC). LMS, determined after grading N-acetyl-beta-hexosaminidase activity on cryotome sections of digestive gland, is measured as labilisation period (LP). LSC, determined after image analysis of cryotome sections where beta-glucuronidase activity is revealed, are measured as lysosomal volume (Vv), surface (Sv), numerical (Nv) densities and surface-to-volume ratio (S/V). Both methods have now been compared in a field study. Mussels were collected from Biscay Bay (Plentzia, reference; Muskiz, moderately polluted) and North Aegean Sea (Olympiada, reference; Limani, heavily polluted). Higher Vv and Sv and lower S/V and LP were recorded in polluted sites than in reference sites. Significant correlations with LP were found for Vv and S/V. The cost/effectiveness and environmental significance of both methods are discussed.

Lysosomal responses to heat-shock of seasonal temperature extremes in Cd-exposed mussels.

The present study was aimed at determining the effect of temperature extremes on lysosomal biomarkers in mussels exposed to a model toxic pollutant (Cd) at different seasons. For this purpose, temperature was elevated 10°C (from 12°C to 22°C in winter and from 18°C to 28°C in summer) for a period of 6h (heat-shock) in control and Cd-exposed mussels, and then returned back to initial one. Lysosomal membrane stability and lysosomal structural changes in digestive gland were investigated. In winter, heat-shock reduced the labilisation period (LP) of the lysosomal membrane, especially in Cd-exposed mussels, and provoked transient lysosomal enlargement. LP values recovered after the heat-shock cessation but lysosomal enlargement prevailed in both experimental groups. In summer, heat-shock induced remarkable reduction in LP and lysosomal enlargement (more markedly in Cd-exposed mussels), which recovered within 3 days. Besides, whilst heat-shock effects on LP were practically identical for Cd-exposed mussels in winter and summer, the effects were longer-lasting in summer than in winter for control mussels. Thus, lysosomal responsiveness after heat-shock was higher in summer than in winter but recovery was faster as well, and therefore the consequences of the heat shock seem to be more decisive in winter. In contrast, inter-season differences were attenuated in the presence of Cd. Consequently, mussels seem to be better prepared in summer than in winter to stand short periods of abrupt temperature change; this is, however, compromised when mussels are exposed to pollutants such as Cd.

Season-dependent effects of elevated temperature on stress biomarkers, energy metabolism and gamete development in mussels.

In coastal areas, sessile species can be severely affected by thermal stress associated to climate change. Presently, the effect of elevated temperature on metabolic, cellular and tissue-level responses of mussels was determined to assess whether the responses vary seasonally with seawater temperature and reproductive stage. Mussels were collected in fall, winter and summer, and (a) maintained at 16, 12, and 20 °C respectively or (b) subject to gradual temperature elevation for 8 days (+1 °C per day; from 16 to 24 °C in fall, from 12 to 20 °C in winter and from 20 to 28 °C in summer) and further maintained at 24 °C (fall), 20 °C (winter) and 28 °C (summer) for the following 6 days. Temperature elevation induced membrane destabilization, lysosomal enlargement, and reduced the aerobic scope in fall and summer whereas in winter no significant changes were found. Changes at tissue-level were only evident at 28 °C. Gamete development was impaired irrespective of season. Since the threshold of negative effects of warming was close to ambient temperatures in summer (24 °C or above) studied mussel populations would be vulnerable to the global climate change.

Scenario-targeted toxicity assessment through multiple endpoint bioassays in a soil posing unacceptable environmental risk according to regulatory screening values.

Lanestosa is a chronically polluted site (derelict mine) where the soil (Lanestosa (LA) soil) exceeds screening values (SVs) of regulatory policies in force (Basque Country; Europe) for Zn, Pb and Cd. A scenario-targeted toxicity assessment was carried out on the basis of a multi-endpoint bioassay approach. Acute and chronic toxicity bioassays were conducted with selected test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates and with bioaccumulation studies in earthworms. Besides, the toxicity profile was compared with that of the mine runoff (RO) soil and of a fresh artificially polluted soil (LAAPS) resembling LA soil pollutant profile. Extractability studies in LA soil revealed that Pb, Zn and Cd were highly available for exchange and/or release into the environment. Indeed, Pb and Zn were accumulated in earthworms and LA soil resulted to be toxic. Soil respiration, V. fischeri, vegetative and developmental cycles of D. discoideum and survival and juvenile production of E. fetida were severely affected. These results confirmed that LA soil had unacceptable environmental risk and demanded intervention. In contrast, although Pb and Zn concentrations in RO soil revealed also unacceptable risk, both metal extractability and toxicity were much lower than in LA soil. Thus, within the polluted site, the need for intervention varied between areas that posed dissimilar risk. Besides, since LAAPS, with a high exchangeable metal fraction, was the most toxic, ageing under in situ natural conditions seemingly contributed to attenuate LA soil risk. As a whole, combining multi-endpoint bioassays with scenario-targeted analysis (including leaching and ageing) provides reliable risk assessment in soils posing unacceptable environmental risk according to SVs, which is useful to optimise the required intervention measures.

Tissue and cell distribution of copper, zinc and cadmium in the mussel, Mytilus galloprovincialis, determined by autometallography.

The localization of metals in selected tissues of metal-exposed mussels was investigated by means of autometallography. Mussels collected from a Zn-polluted site were (a) depurated or, alternatively, (b) exposed to either Cu, Zn or Cd for 41 d. Mussels collected from a clean site were used as experimental reference. Autometallographically demonstrated black silver deposits (BSD), indicating the presence of metals, were observed in gills, (a) in frontal cells and haemocytes of Cu-exposed mussels, (b) in secretory postlateral and abfrontal cells and in endothelial cells of Zn-exposed mussels, and (c) in frontal, postlateral and endothelial cells but mainly in abfrontal cells and haemocytes of Cd-exposed mussels. Autometallography also revealed the presence of BSD in connective tissue brown cells underlying the mantle. Additionally, adipogranular cells of the connective tissue surrounding the gonad follicles were positively stained but no BSD was found in gonad tissue. Scarce BSD were found in the cytoplasmic granules of the stomach wall of control mussels whilst depurating and metal-exposed mussels exhibited BSD lining the apex of the stomach epithelial cells. Contrary to the results previously obtained with marine gastropod molluscs, BSD were not found in the basal lamina of digestive tubules, stomach, and ducts of mussels. Highly conspicuous BSD were observed in digestive cell lysosomes of depurating and metal-exposed mussels. Basophilic cells were always devoid of BSD. Finally, BSD were also found in nephrocyte lysosomes and mineralized concretions.

Changes in mussel biometry on exposure to metals: implications in estimation of metal bioavailability in 'Mussel-Watch' programmes.

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.

Use of polyclonal antibodies for the detection of changes induced by cadmium in lysosomes of aquatic organisms.

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.

Autometallographical localisation of Cu and Zn within target cell compartments of winkles following exposure to Cu&Zn mixtures.

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.