The glutathione antioxidant system as a biomarker suite for the assessment of heavy metal exposure and effect in the grey mangrove, Avicennia marina (Forsk.) Vierh

Alterations in the glutathione antioxidant system and lipid peroxidation in Avicennia marina were studied under laboratory and field conditions. The activity of glutathione peroxidase (GPx) was found to respond to Zn exposure, and a significant positive relationship between leaf Zn concentration and GPx activity was observed after 96 h and 8 weeks. Lipid hydroperoxides increased proportionally with increasing leaf Zn concentration after 2 and 8 weeks, while no changes in total glutathione were observed. Induction of GPx at 96 h predicted effects at the individual level at a later time interval (reduced biomass at 8 weeks). Results from the field revealed that increasing leaf metal concentration (Zn, Cu or Pb) produced a proportional increase in GPx activity whereas lipid hydroperoxides and total glutathione were not affected. The utility of GPx as an early warning biomarker is suggested, since GPx activity increases in a dose-dependant fashion in response to accumulated leaf metals, and is predictive of later effects on growth.     

Comparative effects of the blue green algae Nodularia spumigena and a lysed extract on detoxification and antioxidant enzymes in the green lipped mussel (Perna viridis)

Nodularia spumigena periodically proliferates to cause toxic algal blooms with some aquatic animals enduring and consuming high densities of the blue green algae or toxic lysis. N. spumigena contains toxic compounds such as nodularin and lipopolysaccharides. This current work investigates physiological effects of exposure from bloom conditions of N. spumigena cells and a post-bloom lysis. Biochemical and antioxidative biomarkers were comparatively studied over an acute 3-day exposure. In general, a post-bloom N. spumigena lysis caused opposite physiological responses to bloom densities of N. spumigena. Specifically, increases in glutathione (GSH) and glutathione peroxidase (GPx) and decreases in glutathione S-transferase (GST) were observed from the N. spumigena lysis. In contrast, N. spumigena cell densities decreased GSH and increased GST and lipid peroxidation (LPO) in mussels. Findings also suggest that at different stages of a toxic bloom, exposure may result in toxic stress to specific organs in the mussel.     

Antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (Perna viridis): do mussels "integrate" biomarker responses?

Polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCs) are generally present in the marine environment in complex mixtures. The ecotoxicological nature of contaminant interactions, however, is poorly understood, with most scientific observations derived from single contaminant exposure experiments. The objective of this experiment was to examine dose-response relationships between antioxidant parameters and body contaminant levels in mussels exposed to different exposure regimes under laboratory conditions. Accordingly, the green-lipped mussel, Perna viridis, was challenged with a mixture of PAHs (anthracene, fluoranthene, pyrene, benzo[a]pyrene) and OC pesticides (alpha-HCH, aldrin, dieldrin, p,p'-DDT) over a 4 week period. Contaminants were delivered under four different dosing regimes, with all treatments receiving the same total contaminant load by the end of the exposure period. Antioxidant biomarkers were measured after 1, 2, 3 and 4 weeks, including glutathione (GSH), gluathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and lipid peroxidase (LPO). GST and CAT were induced in hepatic tissues in most of the exposure regimes, with the majority of significant induction occurring in a constant exposure regime and a two-step alternate exposure regime. Significant differences among exposure regimes were detected in the body burden of contaminants after 28 days. Hepatic CAT and GSH are proposed as potentially useful biomarkers as they showed good correlation with target contaminants and were not readily affected by different dosing patterns.     

Antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (Perna viridis): Do mussels “integrate” biomarker responses?

Polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCs) are generally present in the marine environment in complex mixtures. The ecotoxicological nature of contaminant interactions, however, is poorly understood, with most scientific observations derived from single contaminant exposure experiments. The objective of this experiment was to examine dose-response relationships between antioxidant parameters and body contaminant levels in mussels exposed to different exposure regimes under laboratory conditions. Accordingly, the green-lipped mussel, Perna viridis, was challenged with a mixture of PAHs (anthracene, fluoranthene, pyrene, benzo[a]pyrene) and OC pesticides (α-HCH, aldrin, dieldrin, p,p′-DDT) over a 4 week period. Contaminants were delivered under four different dosing regimes, with all treatments receiving the same total contaminant load by the end of the exposure period. Antioxidant biomarkers were measured after 1, 2, 3 and 4 weeks, including glutathione (GSH), gluathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and lipid peroxidase (LPO). GST and CAT were induced in hepatic tissues in most of the exposure regimes, with the majority of significant induction occurring in a constant exposure regime and a two-step alternate exposure regime. Significant differences among exposure regimes were detected in the body burden of contaminants after 28 days. Hepatic CAT and GSH are proposed as potentially useful biomarkers as they showed good correlation with target contaminants and were not readily affected by different dosing patterns.     

Antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (Perna viridis): Do mussels “integrate” biomarker responses?

Polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCs) are generally present in the marine environment in complex mixtures. The ecotoxicological nature of contaminant interactions, however, is poorly understood, with most scientific observations derived from single contaminant exposure experiments. The objective of this experiment was to examine dose-response relationships between antioxidant parameters and body contaminant levels in mussels exposed to different exposure regimes under laboratory conditions. Accordingly, the green-lipped mussel, Perna viridis, was challenged with a mixture of PAHs (anthracene, fluoranthene, pyrene, benzo[a]pyrene) and OC pesticides (α-HCH, aldrin, dieldrin, p,p′-DDT) over a 4 week period. Contaminants were delivered under four different dosing regimes, with all treatments receiving the same total contaminant load by the end of the exposure period. Antioxidant biomarkers were measured after 1, 2, 3 and 4 weeks, including glutathione (GSH), gluathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and lipid peroxidase (LPO). GST and CAT were induced in hepatic tissues in most of the exposure regimes, with the majority of significant induction occurring in a constant exposure regime and a two-step alternate exposure regime. Significant differences among exposure regimes were detected in the body burden of contaminants after 28 days. Hepatic CAT and GSH are proposed as potentially useful biomarkers as they showed good correlation with target contaminants and were not readily affected by different dosing patterns.     

Relationships between tissue concentrations of chlorinated hydrocarbons (polychlorinated biphenyls and chlorinated pesticides) and antioxidative responses of marine mussels, Perna viridis

Marine mussels, Perna viridis, were transplanted from a reference site to various polluted sites around Hong Kong. After 30 d of exposure, antioxidative responses in the gills and hepatopancreas and tissue concentrations of chlorinated hydrocarbons [polychlorinated biphenyls (PCBs) and chlorinated pesticides (CPs)] were determined for individual mussels. Glutathione S transferase (GST) and glutathione (GSH) were positively correlated with tissue PCB concentrations. Only one of the enzymatic antioxidants, glutathione peroxidase (GPx), showed significant response to tissue PCB. No significant correlation was found between tissue concentrations of chlorinated hydrocarbons and other enzymatic antioxidants (superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and NADPH DT-diaphorase (DT-d). Oxidative stress, measured as thiobarbituric acid reactive substances, was correlated with chlorinated pesticide concentrations in tissues. This study demonstrated a correlation between GST/GSH and chlorinated hydrocarbons. The apparent lack of correlation between trace organic pollutants and some of the enzymatic antioxidants may be due to the inhibitory effects caused by these chemicals. The above results suggest that more investigations are needed before these enzymes can be used as biomarkers.     

Relationships between tissue concentrations of chlorinated hydrocarbons (polychlorinated biphenyls and chlorinated pesticides) and antioxidative responses of marine mussels,Perna viridis

Marine mussels, Perna viridis, were transplanted from a reference site to various polluted sites around Hong Kong. After 30 d of exposure, antioxidative responses in the gills and hepatopancreas and tissue concentrations of chlorinated hydrocarbons [polychlorinated biphenyls (PCBs) and chlorinated pesticides (CPs)] were determined for individual mussels. Glutathione S transferase (GST) and glutathione (GSH) were positively correlated with tissue PCB concentrations. Only one of the enzymatic antioxidants, glutathione peroxidase (GPx), showed significant response to tissue PCB. No significant correlation was found between tissue concentrations of chlorinated hydrocarbons and other enzymatic antioxidants (superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and NADPH DT-diaphorase (DT-d). Oxidative stress, measured as thiobarbituric acid reactive substances, was correlated with chlorinated pesticide concentrations in tissues. This study demonstrated a correlation between GST/GSH and chlorinated hydrocarbons. The apparent lack of correlation between trace organic pollutants and some of the enzymatic antioxidants may be due to the inhibitory effects caused by these chemicals. The above results suggest that more investigations are needed before these enzymes can be used as biomarkers.     

Oxidative stress in the mussel Mytella guyanensis from polluted mangroves on Santa Catarina Island,Brazil

Digestive glands of the mangrove mussel Mytella guyanensis, collected at one non-polluted site (site 1) and two polluted sites (sites 2 and 3), were analysed for different antioxidant defenses, lipid peroxidation and DNA damage. Thiobarbituric acid-reactive substance (TBARS) and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels were enhanced at the polluted sites. With the exception of superoxide dismutase, the activities of catalase and glutathione peroxidase were also higher at the polluted sites. Greater increases were observed in glutathione reductase, glutathione S-transferase and etoxyresorufine-O-deethylase activities at the polluted sites. Conversely, reduced glutathione content was higher at the control site. Trace metal contents in mussels collected at polluted sites were increased compared to the control site, and there were strong positive correlations between TBARS and Cu and Pb contents. M. guyanensis is routinely exposed to an oxidative stress condition at both polluted sites, and considering xenobiotic bioaccumulation in bivalve molluscs, the mangrove mussel represents an excellent bioindicator for environmental monitoring studies.     

Effects of sewage pollution and bacterial load on growth and oxidative balance in the freshwater mussel Diplodon chilensis

The effect of chronic exposure to Escherichia coli on morphometrical parameters, different antioxidant defenses, lipid peroxidation and protein oxidation has been studied in digestive gland of the freshwater mussel Diplodon chilensis in the laboratory during 6 weeks. In, a parallel field study, we evaluated morphometrical and oxidative stress parameters in digestive glands of mussels collected from a sewage polluted area (SMA) and from a pristine area (control). Both sites were characterized by analyzing bacteriological and physic-chemical parameters. In the laboratory D. chilensis was able to filter bacteria at high concentrations with a clearing rate of 0.510 ± 0.036 L/h per gram of dry soft tissue mass. The digestive gland mass to shell length ratio (DGM/SL), reduced glutathione (GSH), lipid peroxidation, as concentration of thiobartituric acid reactive substances (TBARS), and glutathione-S-transferase (GST) activity of mussels fed with bacteria were significantly higher than those of control mussels after the fourth week. Fecal bacteria in lake water samples were undetectable in the control, and higher than 24,000 MPN (most probable number)/100 mL in SMA. DGM/SL was higher in SMA. No differences between sites were observed in total lipid and protein content, neither in superoxide dismutase activity. GSH content was higher in SMA, with no difference in the oxidized form. GST activity and MDA were significantly higher in SMA but protein oxidation was not affected.     

Field validation of antioxidant enzyme biomarkers in mussels (Perna viridis) and clams (Ruditapes philippinarum) transplanted in Hong Kong coastal waters

Green-lipped mussels, Perna viridis, and Manila clams, Ruditapes philippinarum were sourced from “clean” sites in the Hong Kong region, depurated in a laboratory using uncontaminated filtered seawater for 8 days, and transplanted to a suspected gradient of chemically polluted sites in Hong Kong. After 14- and 28-days of field exposure, several antioxidant parameters including glutathione S transferase (GST), catalase (CAT), glutathione peroxidase (GPx), and glutathione (GSH) were quantified in gill and hepatopancreas tissues. Whole body tissue concentrations of polycyclic aromatic hydrocarbons (PAHs), petroleum hydrocarbons (PHCs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs) were determined in pooled site samples. Chemical analyses indicated that: (a) clams had higher levels of PAHs, PHCs, DDTs and PCBs, whereas mussels had higher hexachlorocyclohexane (HCHs) and there was no difference between species for dieldrin and remaining OCs; (b) Kat O should not be continued as a “clean” reference site for Hong Kong, because of the levels of contaminants measured and (c) PAH concentrations in the current survey were similar to those previously measured. Toxicological conclusions were: (a) antioxidant responses were different between species; (b) CAT and GST have highest utility in clams for field use in Hong Kong, whereas CAT in both gill and hepatopancreas tissue showed most potential in mussels; (c) significant induction of antioxidant responses over day 0 (excluding GPx in both tissues, and GST in mussel hepatic tissue); (d) groups of contaminants do not consistently induce antioxidant responses and (e) organochlorines and PCBs correlated significantly with CAT and GST in clam hepatopancreas and with CAT in mussel gill and hepatic tissue. Multivariate statistical techniques indicated little relationship between the site patterns for antioxidant responses and the contaminant gradients identified in body burden analysis.     

Biomarkers of exposure and effect in flounder (Platichthys flesus) exposed to sediments of the Adriatic sea.

The modifications of several biomarkers were investigated in flounder (Platichthys flesus) when exposed in the laboratory to sediment samples collected from the Northern Adriatic Sea. Besides clean sand used as a control substrate, fish were exposed to sediments sampled offshore the delta of the Po River, the harbour of Trieste, and from the industrial harbour of Venice (Porto Marghera). After six days of exposure, the enzyme activities ethoxyresorufin-O-deethylase (EROD), UDP glucuronyltransferase (UDPGT), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx), were assayed in fish liver. In addition, the contents of reduced glutathione (GSH), nonprotein thiols (SH), total sugars and extractable lipids were also determined in hepatic tissue, as well as the number of micronucleated hepatocytes and biliary concentrations of fluorescent aromatic compounds (FAC). Despite some variability within treatment groups, differences among exposed organisms were evident and consistent with known contaminant levels of sampled areas. Microsomal activities (EROD, UD-PGT) and FAC levels were the most sensitive exposure indicators. Variations in the other biomarkers showed only tendencies which although not statistically significant were generally consistent with the contamination pattern.     

Field validation of antioxidant enzyme biomarkers in mussels (Perna viridis) and clams (Ruditapes philippinarum) transplanted in Hong Kong coastal waters

Green-lipped mussels, Perna viridis, and Manila clams, Ruditapes philippinarum were sourced from “clean” sites in the Hong Kong region, depurated in a laboratory using uncontaminated filtered seawater for 8 days, and transplanted to a suspected gradient of chemically polluted sites in Hong Kong. After 14- and 28-days of field exposure, several antioxidant parameters including glutathione S transferase (GST), catalase (CAT), glutathione peroxidase (GPx), and glutathione (GSH) were quantified in gill and hepatopancreas tissues. Whole body tissue concentrations of polycyclic aromatic hydrocarbons (PAHs), petroleum hydrocarbons (PHCs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs) were determined in pooled site samples. Chemical analyses indicated that: (a) clams had higher levels of PAHs, PHCs, DDTs and PCBs, whereas mussels had higher hexachlorocyclohexane (HCHs) and there was no difference between species for dieldrin and remaining OCs; (b) Kat O should not be continued as a “clean” reference site for Hong Kong, because of the levels of contaminants measured and (c) PAH concentrations in the current survey were similar to those previously measured. Toxicological conclusions were: (a) antioxidant responses were different between species; (b) CAT and GST have highest utility in clams for field use in Hong Kong, whereas CAT in both gill and hepatopancreas tissue showed most potential in mussels; (c) significant induction of antioxidant responses over day 0 (excluding GPx in both tissues, and GST in mussel hepatic tissue); (d) groups of contaminants do not consistently induce antioxidant responses and (e) organochlorines and PCBs correlated significantly with CAT and GST in clam hepatopancreas and with CAT in mussel gill and hepatic tissue. Multivariate statistical techniques indicated little relationship between the site patterns for antioxidant responses and the contaminant gradients identified in body burden analysis.     

Validation of Arenicola marina in field toxicity bioassays using benthic cages: biomarkers as tools for assessing sediment quality

Sediment toxicity assessments using caged organisms present advantages over using laboratory and native community studies. The use of caged Arenicola marina in sediment toxicity assessments was evaluated. Lugworms were exposed in situ to sediments from coastal and port areas in Spain for seven days, and the activities of the biotransformation enzymes ethoxyresorufin O-deethylase, dibenzylfluorescein dealkylase and glutathione S-transferase, the activities of the antioxidant enzymes glutathione reductase and glutathione peroxidase and lipid peroxidation were then analyzed as biomarkers. Biomarker results and sediment physicochemical data were integrated. Cádiz Bay (SW Spain) sediments presented metal contamination that was not linked to a biochemical response. In LPGC Port (SW Spain), Pb contamination exhibited a moderate toxic potential, while PAHs, and presumably pharmaceuticals, provoked biochemical responses that efficiently prevented lipid peroxidation. In Santander Bay (N Spain), exposure to PAHs and, presumably, pharmaceuticals induced biomarker responses, but lipid peroxidation occurred nevertheless. These results indicated that caged A. marina were effective for the assessment of sediment quality and that the selected biomarkers were sufficiently sensitive to identify chemical exposure and toxicity.     

Sublethal effect of naphthalene on lipid peroxidation and antioxidant status in the edible marine crab Scylla serrata

The effect of naphthalene on lipid peroxidation and antioxidants status was studied in hepatopancreas, haemolymph and ovary of the Scylla serrata with reference to the active vitellogenic stage. There was an overall increase in lipid peroxidation activity in the tested samples. In contrast, the enzymatic (catalase, glutathione peroxidase, superoxide dismutase) and non-enzymatic antioxidants (vitamins C, E and glutathione) showed decreased activities for hepatopancreas, haemolymph and ovary.     

Copper accumulation by bacteria and transfer to scallop larvae

A bacterial copper-resistant strain was isolated from a hatchery-conditioned adult of the scallop Argopecten purpuratus and was identified as Vibrio sp. according to its physiological characteristics. The lowest concentration of Cu2+ required for its complete inhibition in VNSS medium was 50 microg ml(-1). The Vibrio strain was found to accumulate copper, exhibiting cellular and loosely bound copper levels of 201.14 and 493.21 microg g(-1) dry weight, respectively, after 24 h of incubation in VNSS medium supplemented with 15 microg ml(-1) of Cu2+, with cellular concentration factors of 10.17 and 14.35 after 12 and 24 h of exposure. When a scallop larvae culture was exposed to a concentration of 6.46 x 10(2) CFU ml(-1) of Cu-enriched Vibrio, they accumulated 20.42 +/- 1.12 and 30.96 +/- 1.85 microg Cu2+ g(-1) dry weight after 12 and 24 h, respectively. This study suggests that bacterial copper accumulation could be very active in marine environments increasing the occurrence of copper transfer to marine food chains.     

The toxicity and oxidative stress of TiO2 nanoparticles in marine abalone (Haliotis diversicolor supertexta)

The increasing use of nanotechnology highlights the need to understand and clarify the environmental impacts of nanomaterials. In this study, the acute toxicity and oxidative stress of titanium dioxide nanoparticles (nTiO2) in mature marine abalone (Haliotis diversicolor supertexta) were assessed. No acute effect was found in any of the treatment groups with nTiO2 concentration gradients ranging from 0.1 to 10 mg/L. However, the activity of an antioxidant enzyme superoxide dismutase (SOD) significantly increased in the group that was exposed to 1.0 mg/L nTiO2. The content of a non-enzymatic antioxidant, glutathione (GSH), significantly decreased in the groups with an nTiO2 concentration≥1.0 mg/L. The level of lipid peroxidation (LPO) was found to increase as the nTiO2 dose increased. Furthermore, NO was produced in excess in abalone. These results demonstrated that, although nTiO2 is not acutely toxic to abalone, it does exert oxidative stress on abalone.     

Effects of 4-nonylphenol exposure in mussels (Mytilus galloprovincialis) and crabs (Carcinus aestuarii) with particular emphasis on vitellogenin induction

Since it is often difficult to estimate possible adverse effects due to contamination in selected ecosystems, multi-species biomonitoring may provide more information, taking into account different routes of exposure, ecological roles and metabolic capabilities of animals. In this context, we exposed for 7 days the mussel Mytilus galloprovincialis and the crab Carcinus aestuarii to 4-nonylphenol (NP), a well-known xenoestrogen. In mussels (0-0.2 mg NP l(-1)), we measured NP bioaccumulation in soft tissues and vitellogenin (Vg)-like protein levels in digestive glands from both males and females by the alkali-labile phosphate assay (ALP). As no reference data were available for crab exposure, the NP 96-h LC(50) value was previously determined. Then, in sublethally exposed (0-1.0 mg NP l(-1)) male crabs, NP bioaccumulation and Vg levels were measured in hemolymph, gonads and digestive gland. Bioaccumulation of NP increased from 43 to 371 microg g(-1) d.w. in mussels, and from 3.6 to 37 microg g(-1) d.w. in crabs, depending on the NP concentration in water. Dose-dependent Vg-like protein induction was observed in both species, appearing to be related to NP bioaccumulation, although a partial decrease was recorded at the highest concentration tested. A similar trend was observed in both digestive gland and gonad of exposed crabs; Vg increased to a lesser extent, although significantly, in hemolymph. Results demonstrated that NP induces Vg synthesis both in male and female mussels, as well as in male crabs. On the basis of the responsiveness of both species investigated, a multi-species approach is indicated in biomonitoring programmes.     

Effects of the pharmaceuticals gemfibrozil and diclofenac on the marine mussel (Mytilus spp.) and their comparison with standardized toxicity tests

Human pharmaceuticals, like the lipid lowering agent gemfibrozil and the non-steroidal anti-inflammatory drug diclofenac are causing environmental concern. In this study, the marine mussel (Mytilus spp.) was exposed by injection to environmentally relevant and elevated (1 μg/L and 1000 μg/L) concentrations of both compounds and biomarker expression was observed. Gemfibrozil exposure induced biomarkers of stress (glutathione S-transferase and metallothionein) at both concentrations 24 h and 96 h after exposure, respectively. Biomarkers of damage (lipid peroxidation (LPO) and DNA damage) were significantly affected, as well as the biomarker for reproduction, alkali-labile phosphate assay, indicating the potential oxidative stress and endocrine disrupting effect of gemfibrozil. Diclofenac significantly induced LPO after 96 h indicating tissue damage. Additionally standard toxicity tests using the marine species Vibrio fischeri, Skeletonema costatum and Tisbe battagliai showed differences in sensitivity to both drugs in the mg/L range. Results indicate a suite of tests should be used to give accurate information for regulation.