Protective effect of phospholipid hydroperoxide glutathione peroxidase (PHGPx) against lipid peroxidation in mussels Perna perna exposed to different metals

Levels of antioxidant defenses and lipid peroxidation were evaluated in mussels exposed to lead (200 mg/l), iron (500 microg/l), cadmium (200 microg/l) and copper (40 microg/l), for 12, 24, 72 and 120 h. Glutathione S-transferase (GST) activity was unchanged with all treatments. Catalase (CAT) increased after 120 h of exposure to all metals. Mussels exposed to Cd for 12 h, and to Cu and Fe for 120 h had increased lipid peroxidation, which might be associated to decreased levels of reduced glutathione (GSH) and glutathione peroxidase (GPx) activity. Pb exposure caused GSH depletion after 12 h and increased GPx activity after 120 h. Negative correlations were observed between the enzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) and malonaldehyde (MDA) levels after Fe and Cu exposure, indicating a protective role of PHGPx against lipid peroxidation, and suggesting the use of this enzyme as a new potential biomarker of toxicity associated with contaminant exposure in mussels.     

Photosynthetic pigments and peroxidase activity as indicators of heavy metal stress in the Grey mangrove, Avicennia marina (Forsk.) Vierh

Mangroves have been observed to possess a tolerance to high levels of heavy metals, yet accumulated metals may induce subcellular biochemical changes, which can impact on processes at the organism level. Six month-old seedlings of the grey mangrove, Avicennia marina (Forsk.) Vierh, were exposed to a range of Cu (0-800 micrograms/g), Pb (0-800 micrograms/g) and Zn (0-1000 micrograms/g) concentrations in sediments under laboratory conditions, to determine leaf tissue metal accumulation patterns, effects on photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids), and the activity of the antioxidant enzyme peroxidase. Limited Cu uptake to leaves was observed at low sediment Cu levels, with saturation and visible toxicity to Cu at sediment levels greater than 400 micrograms/g. Leaf Pb concentrations remained low over a range of Pb sediment concentrations, up to 400 micrograms/g Pb, above which it appeared that unrestricted transport of Pb occurred, although no visible signs of Pb toxicity were observed. Zn was accumulated linearly with sediment zinc concentration, and visible toxicity occurring at the highest concentration, 1000 micrograms/g Zn. Significant increases in peroxidase activity and decreases in photopigments were found with Cu and Zn at concentrations lower than those inducing visible toxicity. Significant increases in peroxidase activity only, were found when plants were exposed to Pb. Positive linear relationships between peroxidase activity and leaf tissue metal concentrations were found for all metals. Significant linear decreases in photosynthetic pigments with increasing leaf tissue metal concentrations were observed with Cu and Zn only. Photosynthetic pigments and peroxidase activity may be applicable as sensitive biological indicators of Cu and Zn stress, and peroxidase activity for Pb stress in A. marina.     

Produced water extracts from North Sea oil production platforms result in cellular oxidative stress in a rainbow trout in vitro bioassay

Produced water (PW) discharged from offshore oil industry contains chemicals known to contribute to different mechanisms of toxicity. The present study aimed to investigate oxidative stress and cytotoxicity in rainbow trout primary hepatocytes exposed to the water soluble and particulate organic fraction of PW from 10 different North Sea oil production platforms. The PW fractions caused a concentration-dependent increase in reactive oxygen species (ROS) after 1 h exposure, as well as changes in levels of total glutathione (tGSH) and cytotoxicity after 96 h. Interestingly, the water soluble organic compounds of PW were major contributors to oxidative stress and cytotoxicity, and effects was not correlated to the content of total oil in PW. Bioassay effects were only observed at high PW concentrations (3-fold concentrated), indicating that bioaccumulation needs to occur to cause similar short term toxic effects in wild fish.     

Leaf biochemical parameters in Avicennia marina (Forsk.) Vierh as potential biomarkers of heavy metal stress in estuarine ecosystems

Sediment loadings and leaf accumulation of the heavy metals copper (Cu), lead (Pb) and zinc (Zn) with accompanying changes in leaf chlorophylls' (a + b), carotenoids and the antioxidant enzyme peroxidase were examined in the grey mangrove Avicennia marina (Forsk.) Vierh, in order to determine the applicability of these parameters as biomarkers of heavy metal stress under field conditions. Copper was found to show limited accumulation to leaf tissue, following a linear relationship at lower sediment concentrations, with saturation at higher sediment Cu concentrations. Copper accumulation relationships to leaf tissue were maintained temporally, and increases in sediment Cu, salinity, and decreases in sediment pH and Zn contributed to the accumulation of Cu to leaf tissue. Lead showed a significant relationship between sediment and leaf Pb levels, but accumulation was minimal. Accumulation relationships for Pb were not maintained temporally, and high sediment Pb, low pH and organic content increased bioavailability and accumulation of Pb. Zinc was the most mobile of all metals and was accumulated to the greatest quantities in leaf tissue in a dose-dependant relationship. Some temporal variation in Zn accumulation occurred, and higher sediment pH, organic content Zn and Pb promoted leaf Zn accumulation. Leaf Cu and Zn showed the strongest relationship with peroxidase activity and to a lesser degree Pb. Zinc was the only accumulated metal to show relationship maintenance with peroxidase activity over time. It was found that peroxidase activity best reflects the total phytotoxic effect from the combined metal stress of all three accumulated leaf metals. The only significant photopigment relationship evidenced was that of leaf Zn with the chlorophyll a/b ratio, but was not maintained temporally. Peroxidase activity may be an appropriate biomarker for Zn or total metal accumulation in leaf tissue, and the chlorophyll a/b ratio a suitable biomarker of Zn accumulation though requires temporal monitoring under field conditions.     

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.     

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.     

Application of oxidative stress indices in natural populations of Perna viridis as biomarker of environmental pollution

Oxidative stress indices were measured in gills and digestive glands of Perna viridis collected from three coastal locations in Goa i.e., Bambolim, Marmugao Harbour and Malim. In addition to lipid peroxidation, the activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase and two non-enzymatic antioxidants (ascorbic acid and reduced glutathione) were investigated in order to understand their variation with respect to pollution status of the sampling locations. We observed a significant increase in lipid peroxidation and antioxidant enzymes of both the tissues at Mormugao Harbour and Malim, suggesting that the animals at these two locations are at higher level of oxidative stress as compared to those at Bambolim. Conversely, low levels of non-enzymatic antioxidants such as ascorbic acid and reduced glutathione, observed at Mormugao Harbour and Malim indicate that the animals may use these compounds to counteract stress in the tissues. This study shows that changes in lipid peroxidation, superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase and reduced glutathione in tissues of P. viridis can be used as molecular biomarkers in environmental monitoring programs.     

Studies on the Acute Toxicity of Zinc to a Freshwater Teleost: Channa punctatus (Bloch)

Static bioassay tests were carried out on Channa punctatus Bloch in the laboratory after renewal of concentrations every 24 h. The LC₅₀ values and 95 per cent confidence limits were 45.21 (39.62 … 51.11) ppm at 12 h and 29.8 (27.49 … 31.95) ppm of Zn at 96 h of exposure. The actual dissolution values of zinc were also determined in test solutions using an atomic absorption spectrophotometer. Behavioural changes were the higher rate of opercular movements, surfacing, loss of equilibrium and haemorrhage near mouth and caudal fin. The LT₅₀ values indicate that survival was increased with decreasing Zn concentration. The histopathological changes observed due to Zn toxicity in the gills have been discussed in relation to fish mortality.     

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.     

Hg, Zn and Cu levels in the muscle and liver of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan: relationship between metal concentrations and body length

We analyzed Hg, Zn and Cu concentrations in the liver and muscle of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan. The Hg concentration in the muscle increased proportionally with body length in the tiger sharks, whereas that in the liver increased rapidly after maturity (defined by a length of over 2.7 m). Muscle Hg levels were higher than liver concentrations in immature sharks, with the inverse trend observed in mature sharks. Notably, the Zn and Cu concentrations in the liver tended to decrease with increasing body length. This rapid increase in hepatic Hg concentration concurrent with the onset of maturity in sharks may result from the continuous intake of Hg via food and the slower growth of mature sharks. The high concentrations of the essential metals Zn and Cu in immature sharks may be explained by the physiological demands related to rapid growth.     

Antioxidant Enzymes Response in Vetiver Grass: A Greenhouse Study for Chelant‐Assisted Phytoremediation of Lead‐Contaminated Residential Soils

In a previous study we have demonstrated the suitability of using vetiver grass (Vetiveria zizanioides L.) for the phytostabilization of lead‐based paint contaminated residential soils. Vetiver did not show any growth retardation or toxicity symptoms despite high soil Pb levels. Antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) reportedly allow plants to combat metal stress. Thus, we hypothesized that in vetiver, these antioxidant enzymes can play an important role in combating Pb induced stress, and that chelant‐bound Pb is less toxic to vetiver compared to free Pb in soil. The response of antioxidant enzymes was studied in vetiver grass grown in Pb paint‐contaminated residential soils collected from San Antonio, Texas and Baltimore, Maryland. Chelating agents such as ethylenediaminetetraacetic acid (EDTA) and ethylenediaminedisuccinic acid (EDDS) were used to mobilize Pb from bound fractions to the labile pool, facilitating Pb uptake by vetiver. Although the Pb concentration in vetiver from these treatments was significantly higher than those grown in the absence of a chelant, the antioxidant enzymes activities were lower compared to the latter. Antioxidant enzymes activity of vetiver plants grown in the presence of chelants is lower compared to those in without chelant treatment, while they tended to increase with dose in treatments with varying chelant concentrations. Data obtained support the proposed hypothesis.     

Aldrin Induced Changes in the Activities of Three Phosphatases of Macrobrachium lamarrei (H. Milne EDWARDS)

Macrobrachium lamarrei is exposed to 0.008, 0.017 and 0.021 mg/1 aldrin (0.4, 0.8 and 1.0 of the LC_50,96h) for 24… 96 h and after exposure the activities of the acid, alkaline and glucose-6-phosphatases are determined. With concentration and time of exposure the acid phosphatase shows an increasing activity up to 170 % compared with the control, which is interpreted as a part of prenecrotic changes in the cells. With concentration and time of exposure the alkaline phosphatase shows a decreasing activity down to 20 % of the control, which is indicative of considerable disturbances of the total metabolism. The glucose-6-phosphatase shows an increasing activity with the aldrin concentration up to 150 % during 72 h; then the activity drops to 60… 80 %. The initial rise in the glucose concentration of blood caused by that is the clear consequence of a stress reaction and the change of metabolism resulting from it.     

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.     

Effects of heavy metals on the survival and feeding behaviour of the sandy shore scavenging gastropod Nassarius festivus (Powys)

The effects of Cu, Cd, Zn and Cr on the survival and feeding behaviour of the sandy shore scavenging gastropod Nassarius festivus were compared. The 96-h LC₅₀ for Cu, Cd, Zn and Cr were 0.36, 1.52, 1.76 and 36.9 mg l⁻¹, respectively. Four sublethal concentrations of each metal plus a control were prepared and the snails were exposed to experimental solutions for 96 h. Feeding behaviour was studied after the snails were starved for five days. As compared with the control, the number of individuals feeding was significantly reduced by exposure to 0.05 mg l⁻¹ Cu, 0.2 mg l⁻¹ Zn, 0.5 mg l⁻¹ Cd and 5 mg l⁻¹ Cr. The time spent feeding was greater for individuals exposed to greater concentrations of Zn and Cd but no effect was found for Cu and Cr. Chemoreception of food was studied by placing the snails at a fixed distance of 15 cm from the bait. The success rate of reaching the bait was less for individuals exposed to Cr but no effect was found for Zn, Cu or Cd. The time required for an individual to reach the bait decreased as the concentration of Zn increased. In contrast, a longer time was required for individuals exposed to Cr whereas the effect of Cd and Cu was insignificant. The potential of using feeding behaviour and chemoreception in contaminant evaluations is discussed.     

Cyanobacterial blooms in estuarine ecosystems: characteristics and effects on Laeonereis acuta (Polychaeta, Nereididae)

In January of 2003, a cyanobacterial bloom in the Patos' Lagoon (Southern Brazil) (32 degrees 05'S-52 degrees 12'W) was observed. Water samples were taken to identify the composition and abundance of the bloom, as well as the occurrence of toxins. The effects of this occurrence on the estuarine worm Laeonereis acuta (Polychaeta, Nereididae) was also evaluated. Predominance of cyanobacteria, particularly Anabaena trichomes ( approximately 2.5.10(6) individuals per liter) was observed, and low concentrations of microcystins and anticholinesterasic toxins were detected. Augmented levels of lipid hydroperoxides (LPO) and glutathione-S-transferase activity, and lowering of total protein content were also observed in organisms collected during the bloom event. Although non-toxic, the cyanobacterial bloom could augment the cycle of hyper-oxygenation and hypoxia in the water. During hyperoxia, L. acuta, an oxyconformer, should consume more oxygen, thus augmenting the rate of reactive oxygen species generation. A repeated cycle of hyper-oxygenation and hypoxia would finally induce oxidative stress, as evidenced by the high levels of LPO and glutathione-S-transferase activity.     

Effects of heavy metals on the survival and feeding behaviour of the sandy shore scavenging gastropod Nassarius festivus (Powys)

The effects of Cu, Cd, Zn and Cr on the survival and feeding behaviour of the sandy shore scavenging gastropod Nassarius festivus were compared. The 96-h LC₅₀ for Cu, Cd, Zn and Cr were 0.36, 1.52, 1.76 and 36.9 mg l⁻¹, respectively. Four sublethal concentrations of each metal plus a control were prepared and the snails were exposed to experimental solutions for 96 h. Feeding behaviour was studied after the snails were starved for five days. As compared with the control, the number of individuals feeding was significantly reduced by exposure to 0.05 mg l⁻¹ Cu, 0.2 mg l⁻¹ Zn, 0.5 mg l⁻¹ Cd and 5 mg l⁻¹ Cr. The time spent feeding was greater for individuals exposed to greater concentrations of Zn and Cd but no effect was found for Cu and Cr. Chemoreception of food was studied by placing the snails at a fixed distance of 15 cm from the bait. The success rate of reaching the bait was less for individuals exposed to Cr but no effect was found for Zn, Cu or Cd. The time required for an individual to reach the bait decreased as the concentration of Zn increased. In contrast, a longer time was required for individuals exposed to Cr whereas the effect of Cd and Cu was insignificant. The potential of using feeding behaviour and chemoreception in contaminant evaluations is discussed.     

Spatial Variation in MTBE Biodegradation Activity of Aquifer Solids Samples Collected in the Vicinity of a Flow-Through Aerobic Biobarrier

The spatial variation in methyl tert-butyl ether (MTBE) biodegradation activity of aquifer solids samples collected in the vicinity of a flow-through aerobic biobarrier was assessed through use of standard laboratory microcosms. These were prepared by collecting soil cores at a range of locations and depths along different flow paths through the biobarrier. Sections of core samples were placed in sealed bottles with MTBE-free groundwater from the site. The groundwater was filtered to remove microbes, and sparged with O₂. The initial MTBE concentration in the microcosms was adjusted to about 1 mg/L. Biodegradation activity was characterized by the magnitude of MTBE concentration reductions occurred over 4 weeks relative to control microcosms. Sampling locations and depths were selected to allow investigation of relationships between MTBEdegrading activity and dissolved oxygen (DO) concentration, MTBE, soil type, and initial microbial conditions (biostimulated vs. bioaugmented). The results suggest a relatively wide-spread presence of MTBE-degrading microbial consortia, with varying levels of MTBE-degrading activity. Significant changes in activity were observed over 0.3-m vertical distances in the same location; for example, cores from the most upgradient sampling locations contained sections with no discernible MTBEbiodegradation over 4 weeks, as well as sections that achieved order-of-magnitude MTBE concentration reductions within 2 weeks. None of those cores, however, achieved MTBE biodegradation to nondetect levels (<0.005 mg/L), as was observed in some cores from downgradient locations. Cores from the bioaugmented regions had the highest frequency of MTBE biodegradation to nondetect levels among their sections suggesting a direct effect of the inoculum and its distribution when it was implanted. Most cores with no activity were associated with the upgradient, low-DO, and high-MTBE concentration field environments, but low-DO field environments also yielded MTBE-degrading samples. There were no other clear correlations between MTBE-degrading activity in the microcosms and the local field environment conditions at the time of sampling.     

The impact of the herbicide atrazine on growth and photosynthesis of seagrass, Zostera marina (L.), seedlings

The impact of the widely used herbicide atrazine on seedling growth and photosynthesis of eelgrass was determined. The long-term impact of the herbicide atrazine (1, 10 and 100 μg/L) on growth of eelgrass Zostera marina (L.) seedlings, maintained in outdoor aquaria, was monitored over 4 weeks. Exposure to 10 μg/L atrazine resulted in significantly lower plant fresh weight and total chlorophyll concentration and up to 86.67% mortality at the 100 μg/L concentration. Short-term photosynthetic stress on eelgrass seedlings was determined and compared with adult eelgrass using chlorophyll fluorescence. The effective quantum yield in eelgrass seedlings was significantly depressed at all atrazine concentrations (2, 4, 8, 16, 32 and 64 μg/L) even within 2 h and remained at a lower level than for adult plants for each concentration. These results indicate that atrazine presents a potential threat to seagrass seedling functioning and that the impact is much higher than for adult plants.