Manu Soto

Chemical, biochemical and cellular responses in the digestive gland of the mussel Mytilus galloprovincialis from the Spanish Mediterranean coast

Mussels (Mytilus galloprovincialis) were sampled in March 1996 from five stations along the Western Mediterranean coast (Barcelona, Ebro Delta, Alboraya, Cullera, Denia) corresponding to urban, industrial and agricultural areas. Different biochemical and cellular markers were determined in the mussels in order to assess the effects and/or exposure to pollutants. The cytochrome P450 system, acetylcholinesterase and metallothioneins were among the biochemical markers selected for the study. Histochemical analysis of ß-glucuronidase and catalase activity were performed as marker enzymes for lysosomes and peroxisomes. Chemical analyses indicated that mussels from Barcelona and Denia as highly exposed to polycyclic aromatic hydrocarbons (PAHs)(1.8-2.7 µg g(-1) w.w. against 0.02-0.10 µg g(-1) w.w.), and polychlorinated biphenyls (PCBs)(132-260 ng g(-1) w.w. against 8-24 ng g(-1) w.w.). This was in agreement with changes in lysosome structure and higher number of peroxisomes in those organisms. High levels of metals (particularly Cr and Cu) were recorded in the digestive gland of Alboraya mussels, which also had elevated metallothionein content (28 nmol g(-1) w.w.) in comparison with the other stations (15-20 nmol g(-1) w.w.). Benzo(a)pyrene hydroxylase (BPH) activity indicated Cullera and Barcelona as possibly polluted sites. The results support the usefulness of the biomarker approach to assess and diagnose environmental pollution. The use of a battery of biomarkers at different levels of biological organization coupled with chemical analysis is highlighted.Mussels (Mytilus galloprovincialis) were sampled in March 1996 from five stations along the Western Mediterranean coast (Barcelona, Ebro Delta, Alboraya, Cullera, Denia) corresponding to urban, industrial and agricultural areas. Different biochemical and cellular markers were determined in the mussels in order to assess the effects and/or exposure to pollutants. The cytochrome P450 system, acetylcholinesterase and metallothioneins were among the biochemical markers selected for the study. Histochemical analysis of ß-glucuronidase and catalase activity were performed as marker enzymes for lysosomes and peroxisomes. Chemical analyses indicated that mussels from Barcelona and Denia as highly exposed to polycyclic aromatic hydrocarbons (PAHs)(1.8-2.7 µg g-1 w.w. against 0.02-0.10 µg g-1 w.w.), and polychlorinated biphenyls (PCBs)(132-260 ng g-1 w.w. against 8-24 ng g-1 w.w.). This was in agreement with changes in lysosome structure and higher number of peroxisomes in those organisms. High levels of metals (particularly Cr and Cu) were recorded in the digestive gland of Alboraya mussels, which also had elevated metallothionein content (28 nmol g-1 w.w.) in comparison with the other stations (15-20 nmol g-1 w.w.). Benzo(a)pyrene hydroxylase (BPH) activity indicated Cullera and Barcelona as possibly polluted sites. The results support the usefulness of the biomarker approach to assess and diagnose environmental pollution. The use of a battery of biomarkers at different levels of biological organization coupled with chemical analysis is highlighted.Mussels (Mytilus galloprovincialis) were sampled in March 1996 from five stations along the Western Mediterranean coast (Barcelona, Ebro Delta, Alboraya, Cullera, Denia) corresponding to urban, industrial and agricultural areas. Different biochemical and cellular markers were determined in the mussels in order to assess the effects and/or exposure to pollutants. The cytochrome P450 system, acetylcholinesterase and metallothioneins were among the biochemical markers selected for the study. Histochemical analysis of s-glucuronidase and catalase activity were performed as marker enzymes for lysosomes and peroxisomes. Chemical analyses indicated that mussels from Barcelona and Denia as highly exposed to polycyclic aromatic hydrocarbons (PAHs)(1.8-2.7 µg g-1 w.w. against 0.02-0.10 µg g-1 w.w.), and polychlorinated biphenyls (PCBs)(132-260 ng g-1 w.w. against 8-24 ng g-1 w.w.). This was in agreement with changes in lysosome structure and higher number of peroxisomes in those organisms. High levels of metals (pa...

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

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

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.

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

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

Subcellular Distribution of Cadmium and its Cellular Ligands in Mussel Digestive Gland Cells as Revealed by Combined Autometallography and X-ray Microprobe Analysis

Autometallography (AMG) and electron probe X-ray microanalysis (EPXMA) were applied in combination to determine the subcellular distribution of Cd and its subcellular ligands in the digestive gland cells of Cd-exposed mussels Mytilus galloprovincialis. Black silver deposits (BSD), which reveal the presence of metals when AMG is applied, were only localized in digestive cell lysosomes. Digestive cell cytoplasm and basophilic cells were devoid of BSD. EPXMA (static probe and X-ray mapping) indicated that Cd, S (possibly associated with metallothioneins or metallothionein-like proteins) and autometallographical Ag ions are co-localized within digestive cell lysosomes. In addition, Cd and S co-occur in the absence of Ag in the cytosol of digestive cells. AMG does not reveal the presence of the Cd ‘pool’ strongly bound to cytosolic Cd-metallothionein complexes; only ‘free’ Cd or Cd supposedly loosely bound to (semi)digested metallothionein within lysosomes was revealed. The levels of lysosomal Cd were indirectly quantified by stereology as the volume density of BSD (VvBSD). Significantly higher values were recorded in Cd-exposed mussels compared with controls at all exposure times. However, VvBSD values were lower at days 7 and 21 than at day 1. This relative decrease in VvBSD reflected another (and confounding) response elicited by Cd-exposure in the digestive epithelium: the volume density of basophilic cells (VvBAS) increased significantly as exposure progressed. Due to this cell-type replacement, the net accumulative capacity of the digestive epithelium decreases at long exposure times.

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

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

Towards an integrative soil health assessment strategy: A three tier (integrative biomarker response) approach with Eisenia fetida applied to soils subjected to chronic metal pollution

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.

Autometallographic localization of protein-bound copper and zinc in the common winkle, Littorina littorea: a light microscopical study

SummaryCopper (Cu), zinc (Zn) and calcium (Ca) were demonstrated histochemically by means of conventional stains (rubeanic acid for copper, dithizone for zinc, and cobalt nitrare 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 Bouins 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 contribution of metal/shell-weight index in target-tissues to metal body burden in sentinel marine molluscs. 2. Mytilus galloprovincialis

Abstract The tissue distribution of Zn in mussels collected from a Zn-polluted site and the changes in the pattern of distribution for Zn and Cu in response to experimental exposures to Cu, Zn and Cd, as well as the tissue distribution of Cd have been studied. The organs of mussels were accurately separated avoiding mixtures except the kidney area that was included within the remainder tissues. Mussels exposed to intermediate dose (ID)-Cd showed the highest Cd burden first in gills and then more or less equally distributed between the various tissues examined with a dominance of the digestive gland. Mussels exposed to high dose (HD)-Cd, showed Cd equally distributed through all the organs at both exposure times. Below 0.8 μg Cd/l seawater, the gills of Zn-polluted mussels were able to either protect against Cd entry or mobilise uptaken Cd towards other tissues. On the other hand, Zn- and Cu-burdens did not rise in gill tissue, although in depurating mussels Zn-levels were higher than in any other exposure group. Similar conclusions were obtained for the digestive gland. However, the accumulative capacity of the digestive gland tissue for Cd was not exhausted and Cu burdens were depressed on exposure to Cd. Interactions between metals in the various organs studied could be explained by redistribution of haemocytes or brown cells and by enhanced release of metal-bearing digestive lysosomes in response to metal exposure. It is suggested that (a) the pattern of metal accumulation and tissue distribution as well as a part of the interactions between Cu, Zn and Cd cannot be explained solely by the availability of metallothioneins in specific organs; and (b) the specific cell type in which these ligands occur as well as the relative abundance and distribution of these cells are relevant factors controlling metal burdens in organs of mussels. Therefore, in addition to the chemical and the biochemical approaches, investigations on tissue sections are required to better understand these phenomena. Meanwhile, the digestive gland of mussels could be used as the target organ to monitor metal pollution, including essential and non-essential metals, on the basis of metal/shell-weight indices.

The contribution of metal/shell-weight index in target-tissues to metal body burden in sentinel marine molluscs. 1. Littorina littorea

Accumulation and tissue distribution of Cu and Zn in target organs of Littorina littorea have been investigated. Weight changes in particular organs were associated with either Cu or Zn exposures and therefore, metal/shell-weight indices were used instead of metal concentrations to investigate metal accumulation and mobilisation. A regulatory mechanism was observed on exposure to low levels of Cu, but regulation did not occur between 8 and 80 μg Cu/l seawater. Conversely, Zn was well-regulated up to a concentration of 80 μg Zn/l seawater. ZnCu index increased linearly at increasing Zn exposure levels and decreased linearly at increasing Cu exposure levels. The gills are not target tissues reflecting environmental levels of either Cu or Zn, however, copper levels were elevated in gills after exposure to 80 μg Cu/l seawater for 29 and 41 days. On the other hand, the highest concentration of Cu in the kidney was found after exposure to 8 μg Cu/l seawater for 41 days. Higher exposures resulted in lower Cu concentrations. According to metal/shell-weight indices, Cu and Zn were not significantly accumulated in the digestive gland/gonad complex (DGGC) until a certain threshold value was reached (exposure level > 400 μg·day/l). On the other hand, DGGC weight was significantly reduced and, concomitantly, Zn and Cu concentrations raised. However, the metal concentration values increased beyond those resulting simply from a weight reduction. In conclusion, dissimilar mechanisms performed by different cell types in the different organs, together with metal-induced weight changes, would account for the existence of different patterns of metal accumulation and tissue distribution. It is therefore suggested that the suitability of winkles as indicators of Cu and Zn pollution should be reconsidered on the basis of measurements of metal burdens at tissue and cell levels.