Biomarkers in an invasive fish species, Oreochromis niloticus, to assess the effects of pollution in a highly degraded Brazilian River

Paraiba do Sul watershed is one of the most important Brazilian water bodies (5.5 million people depend on the river). It is in a critical environmental situation, polluted by industrial discharges, non-treated urban wastes, and pesticides, which have had cumulatively negative effects. This study analyzes the effects of pollution, with a biomarker approach, by using the invasive fish species, Oreochromis niloticus, as a sentinel species. The approach comprehends a general biomarker of the health of individual fish, the condition factor, a biomarker of genotoxicity, the micronuclei test; and specific biomarkers of contaminant exposure such as metallothionein (MT) and acetylcholinesterase (AChE) activity. The results revealed different effects in fish from diverse locations with varying degrees of pollution. Low AChE activities were found in fish from the region with strong agriculture activity, showing the effects of pesticides. Fish from an industrialized and heavily environmentally degraded area presented high levels of MT and low AChE activities, indicating an intricate polluted condition. It is noteworthy that fish located just upstream of the main water-treatment plant of the metropolitan area Rio de Janeiro presented high levels of MT, showing to be affected by metals. This can be an alert to public health officials. O. niloticus has proven a suitable sentinel species to assess the effects of pollutions in an aquatic system with a complex and serious polluted situation. The present study also shows the usefulness of integrating a set of biomarkers to define the exposure and the effects of anthropogenic inputs among impacted and reference sites in this water body.     

Introduction of the land snail Eobania vermiculata as a bioindicator organism of terrestrial pollution using a battery of biomarkers

The present study aimed to enrich the group of sentinel organisms of terrestrial pollution biomonitoring, by investigating the efficacy of the land snail Eobania vermiculata. For this reason, a package of biomarkers was performed on land snails E. vermiculata collected from polluted areas in the field or treated with heavy metals in the laboratory. The biomarkers used were neutral red lysosomal retention assay of the haemocytes, acetylcholinesterase activity in the digestive gland and the haemolymph, and metallothionein content of the digestive gland. Moreover, the morphometric changes in the lysosomal system and the morphometric alterations of the neutral lipids were also investigated. In addition, the content of cadmium, lead and copper was evaluated in the digestive gland of the snails. The results revealed appreciable alterations in the biomarker values both in field- and laboratory-conditions, accompanied by significant correlations among the biomarkers. Therefore, this exploratory study suggests the utility of E. vermiculata as a sentinel organism for biomonitoring the biologic impact of terrestrial pollution, and supports the package's efficacy of the selected biomarkers.     

Imposex and butyltin body burden in Nassarius nitidus (Jeffreys, 1867), in coastal waters within the Basque Country (northern Spain)

Levels of imposex (superimposition of male characters, upon females) and the presence of sterile females are assessed in the gastropod Nassarius nitidus (Jeffreys, 1867), at 22 locations in the Basque Country (northern Spain). At 18 of these localities, butyltin bioaccumulation (tributyltin (TBT); dibutyltin; monobutyltin) was analysed using isotope dilution and GC-ICP-MS. Higher imposex levels and TBT body burden were found in confined harbours, with a large vessel traffic or the presence of a fishing fleet or a shipyard. For the first time, four apparently sequential types of aborted capsules are described in this species. Another novelty is the interspecific comparison between imposex intensities in sympatrically living populations of N. nitidus and N. reticulatus. This showed that sensitivity to TBT pollution of both species is relatively similar (in terms of Relative Penis Length Index). Since N. nitidus has a restricted habitat distribution due to its low presence in wave-exposed habitats, its complementary use with other species is recommended for its use in TBT monitoring programmes.     

Introduction of cAMP and establishment of neutral lipids alterations as pollution biomarkers using the mussel Mytilus galloprovincialis. Correlation with a battery of biomarkers

The results of the present study suggest that the signaling molecule cAMP could constitute a reliable biomarker of pollution monitoring. Increased levels of cAMP were observed in tissues of mussels Mytilus galloprovincialis exposed to PAHs, 2,4-dichlorophenoxyacetic acid and lindane for 15 days. cAMP levels were higher in mantle/gonad complex, in relation to other tissues studied (digestive gland and gills). In support of our suggestion, cAMP was significantly correlated with established biomarkers, such as lysosomal membrane stability, AChE activity and metallothionein content. In addition, our results support the establishment of morphometrical alterations of neutral lipids as biomarker of organic pollution, since high accumulation of neutral lipids in the digestive gland of mussels exposed to organic pollutants in relation to non-exposed mussels was noted. In addition, correlation analysis between cellular and biochemical biomarkers showed that the volume density of neutral lipids negatively correlated with both lysosomal membrane stability and AChE activity. Regarding micronucleus test, our results do not support its use as a biomarker of organic pollution, since no statistical differences were found between control and exposed mussels. On the other hand, significant correlations were observed among the battery of biomarkers, such as lysosomal membrane stability, AChE activity and metallothionein content after the mussels' exposure to the specific organic compounds, thus supporting the important role of these biomarkers as reliable indicators of organic pollution. In conclusion, our results support cAMP and morphometrical alterations of neutral lipids as biomarkers of environmental pollution.     

Seasonal responses to diesel oil and subsequent recovery of the cytocgrome P-450 monooxygenase system in the common mussel,Mytilus edulis L., and the periwinkle,Littorina littorea L

Mussels and periwinkles under natural environmental conditions were exposed long-term (4–8 and 16–24 months, respectively) to environmentally realistic levels of, on average, 30 and 129 ppb diesel oil and then allowed to recover for 2–9 months in the absence of oil. With oil exposure, increases were seen in digestive gland microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity, and the changes in the latter were particularly consistent in periwinkles, showing a stepwise elevation with oil dosage at all times of the year. The changes were less consistent in mussels, with a dependence on season or reproductive condition being indicated. Fewer changes were seen in cytochrome b₅ content and the associated NADH-dependent reductase activities of the digestive gland microsomes of both molluscs. The microsomal parameters tended to be higher in female than male mussels, but the responses of the sexes to oil-exposure were not obviously different. With recovery the elevated microsomal parameters declined and the changes paralleled the depuration of polynuclear aromatic hydrocarbons from the tissues. Few changes were seen in benzo[a]pyrene hydroxylase activity. It is concluded that some of the observed enzyme changes, particularly in periwinkles, could have a potential for application in pollution monitoring, but that the in vivo function of these enzymes must first be elucidated.     

Hepatic levels of metal and metallothioneins in two commercial fish species of the Northern Iberian shelf

Metal levels (Cd, Pb, Hg, Cr, Fe, Cu, Zn) together with metallothioneins (MTs) were determined in the liver of two commercial fish species collected along the Northern Iberian coast. The four-spotted megrim (Lepidorhumbos boscii) and the pouting (Trisopterus luscus) were selected as representative species of the middle/outer (200-500 m) and inner shelf (70-120 m), respectively. Metal pollution in the middle/outer shelf was mostly detected in the Asturias and Basque Country areas, whereas in the inner shelf, pollution was widely spread along the coast. Significantly high levels of Pb, Cd, Cr and Hg were detected in T. luscus from different sampling sites and associated to anthropogenic pressure. MTs were induced in fish that had higher amounts of Zn, Cd, Cr and Hg in their livers, supporting the use of MT as a biomarker of metal exposure. The study supports the usefulness of the selected fish species as sentinel organisms for future biomonitoring studies.     

Bioaccumulation and effects of PCBs and heavy metals in sea stars (Asterias rubens, L.) from the North Sea: a small scale perspective

Sea stars (Asterias rubens L.) were collected in different stations distributed in the Southern Bight of the North Sea. Concentrations of four heavy metals and six PCB congeners were measured in two body compartments (body wall and pyloric caeca). In order to assess the potential harm of these contaminants, two biochemical parameters were measured in sea stars, viz. reactive oxygen species (ROS) production by amoebocytes and cytochrome P450 immunopositive protein (CYP1A IPP) induction in pyloric caeca. Sea stars from stations located in the plume of the Scheldt river showed the highest contamination levels. Other stations, similarly located, displayed lower levels. No simple relationship could be established between ROS production by sea star amoebocytes and contaminant levels measured in sea star tissues. CYP1A IPP induction displayed more contrasted responses, and highly significant regressions were found between PCB concentrations measured in pyloric caeca and CYP1A IPP. Both biological parameters were found to vary significantly over the study area. On the whole, data indicated that contamination levels and subsequent effects in sea stars were comparable to those described in previous large-scale studies, but that working at a smaller scale highlighted the existence of patterns of contamination which can blur general trends due to major contamination sources like contaminated rivers.     

Effects of chemical pollution on the activities of hepatic xenobiotic metabolizing enzymes in fish from the River Rh?ne

Polychlorobiphenyl (PCBs) levels and hepatic xenobiotic metabolizing enzyme activities were measured in fish from three locations of the River Rh?ne to study the consequences of a constant loading of PCBs from a PCB incineration plant. Our results show that levels of PCBs and enzyme activities were higher in fish living downstream from the plant than in fish from two locations upstream, suggesting enzyme induction by PCBs (known to be potent inducers in laboratory conditions). Enzyme activities were studied in spring and autumn in three species: nase (Chondrostoma nasus), roach (Rutilus rutilus) and grayling (Thymallus thymallus). Induction was observed for three cytochrome P-450-dependent monooxygenase activities (MO), i.e. 7-ethoxyresorufin O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD). There was a close correlation between EROD and AHH activities (for all species). Glutathione S-transferase activities were also shown to be related to the PCB levels. Conversely, cytochrome P-450 content and benzphetamine N-demethylase activity were not "PCB level-dependent". This study clearly demonstrates a close relationship between PCB contamination and MO activities in fish from the field and thus clearly emphasizes the interest in MO as a monitoring tool for estimating water quality.     

Sources of variation in the assimilation of lead by a common gastropod sentinel Cantareus aspersus

The assimilation efficiency of Pb was measured in the candidate sentinel Cantareus aspersus, to assess its capacity to consistently represent the bioavailability of Pb. Three populations were compared from habitats with different levels of toxic and essential metals. For each, 36 snails were fed a laboratory diet with known concentrations of Pb, Ca and Mg over 64 days. Lead bioavailability was measured as the percentage assimilation by the soft tissues (mass in tissues as a fraction of that consumed), and its variation was assessed between populations, exposure periods, dietary Mg and replicates. The indicated bioavailability was consistent over time and Pb consumed, but differed between populations and diets. Population differences were due, in part, to their different growth rates: soft tissue growth was positively correlated with Pb mass and, in the most rapidly growing populations, this accounted for more than 40% of the variation in soft tissue Pb. Populations did not differ in their consumption of Pb, but the highest Mg diet induced higher food consumption and a reduced assimilation of Pb. Tissue growth determines retention and contributes to the variation in the bioavailability indicated by these populations, as do essential metals in the diet. Such variability has important implications for monitoring programmes using resident populations of sentinels to map the bioavailability of toxic metals or integrate a pollution signal over time.     

The use of imposex to assess tributyltin contamination in coastal waters and open seas

Imposex in muricid gastropods has been used to monitor tributyltin (TBT) contamination worldwide. Pollution was severe during the 1980s but regulations prohibiting the use of TBT-based antifoulants on vessels < 25 m in length have been highly effective in reducing TBT levels in coastal waters. Large vessels are still sources of TBT and major ports, especially those with dry docking and repair facilities, continue to be hot-spots of contamination. Measures of imposex suggest that severe pollution is normally localised to within a few kilometres of them. Mild imposex, possibly as a result of TBT pollution (although other causes have not been ruled out), has been described in whelks Buccinum undatum from offshore waters of the southern and central North Sea. However, the species is still abundant there and the occurrence of imposex does not seem to have affected its breeding performance. Imposex was mild or absent in populations of dogwhelks from open oceanic sites around the north Atlantic Ocean, suggesting that the oceans are free of contamination at biologically significant levels.     

Biomarker response in the earthworm Lumbricus terrestris exposed to chemical pollutants

Earthworms are important organisms for the soil ecosystem. They are sensitive to toxic chemicals and represent useful bioindicator organisms for soil biomonitoring. Recently the use of biomarkers in earthworms has been increasingly investigated for soil monitoring and assessment purpose. The aim of the preset paper was to analyze the pollutant-induced response of a suite of cellular and biochemical biomarkers in the earthworm Lumbricus terrestris exposed to copper sulphate or methiocarb in OECD soil at the maximal concentrations recommended in agriculture. These responses were compared to lifecycle parameters such as survival, growth and reproduction.Granulocyte morphometric alteration, lysosomal membrane stability, metallothionein concentration, and acetylcholinesterase activity were considered. In either copper sulphate or methiocarb exposure conditions the mean percentage variation of the pollutant-induced molecular and cellular biomarkers was consistent with the whole organism end-point responses. In particular pollutant-induced granulocyte enlargement, detected in either copper sulphate or methiocarb exposed organisms, showed to be a potential general biomarker that may be directly linked to organism health. Compared to the other biological responses to pollutants, it showed high sensitivity to pollutant exposure suggesting its possible applications as a sensitive, simple, and quick general biomarker for monitoring and assessment applications.     

Pesticides in the atmosphere of the Mississippi River Valley, part I--rain

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.     

Contaminant exposure and effects in pinnipeds: implications for Steller sea lion declines in Alaska

After nearly 3 decades of decline, the western stock of Steller sea lions (SSL; Eumetopias jubatus) was listed as an endangered species in 1997. While the cause of the decline in the 1970s and 1980s has been attributed to nutritional stress, recent declines are unexplained and may result from other factors including the presence of environmental contaminants. SSL tissues show accumulation of butyltins, mercury, PCBs, DDTs, chlordanes and hexachlorobenzene. SSL habitats and prey are contaminated with additional chemicals including mirex, endrin, dieldrin, hexachlorocyclohexanes, tetrachlorodibenzo-p-dioxin (TCDD) and related compounds, cadmium and lead. In addition, many SSL haulouts and rookeries are located near other hazards including radioactivity, solvents, ordnance and chemical weapon dumps. PCB and DDT concentrations measured in a few SSL during the 1980s were the highest recorded for any Alaskan pinniped. Some contaminant exposures in SSL appear to be elevated in the Gulf of Alaska and Bering Sea compared to southeast Alaska, but there are insufficient data to evaluate geospatial relationships with any certainty. Based on very limited blubber data, current levels of PCBs may not pose a risk to SSL based on comparison to immunotoxicity tissue benchmarks, but SSL may have been at risk from pre-1990 PCB exposures. While exposure to PCBs and DDTs may be declining, SSL are likely exposed to a multitude of other contaminants that have not been monitored. The impacts of these exposures on SSL remain unknown because causal effects have not been established. Field studies with SSL have been limited in scope and have not yet linked contaminant exposures to adverse animal health or population effects. Several biomarkers may prove useful for monitoring exposure and additional research is needed to evaluate their utility in SSL. We conclude that there are insufficient data to reject the hypothesis that contaminants play a role in the continued decline of SSL, and suggest that a coordinated monitoring program be developed which can be related to key biological, ecological and laboratory toxicity data.     

Cholinesterase activities in the scallop Pecten jacobaeus: characterization and effects of exposure to aquatic contaminants

Nearshore marine environments of industrialized countries are increasingly threatened by anthropogenic pollution. It is therefore a priority task to investigate the sensitivity of new ecotoxicological warning signals of the occurrence and effects of aquatic pollutants. The main aims of the present study were: 1) to characterize the biochemical properties of ChEs in tissues of the bivalve Pecten jacobaeus, using different specific substrates and selective inhibitors; 2) to measure sensitivity of ChE activities to in vitro exposure to the OPs azamethiphos and DFP and to the heavy metals cadmium and zinc. Our final aim was to carry out a preliminary evaluation of the suitability of ChEs measurement in tissues of the scallop for monitoring marine environmental quality and neurotoxic compounds contamination in the Mediterranean Sea. Responses to specific inhibitors have suggested that ChEs in adductor muscle share many characteristics with vertebrate acetylcholinesterase. Dose-dependent inhibition of ChE was observed in response to in vitro exposure to environmental contaminants such as cadmium and azamethiphos. Sensitivity to zinc and DFP was lower. ChEs in P. jacobaeus might therefore have potential as a sensitive biomarker for monitoring marine pollution. Results of the present study will be useful to focus further experiment of exposure to pollutants under in vivo conditions. CAPSULE: Cholinesterase activities in scallop Pecten jacobaeus were observed to be sensitive to contaminants in vitro and may therefore have potential as biomarkers for monitoring water pollution.     

Combined exposure to ambient UVB radiation and nitrite negatively affects survival of amphibian early life stages

Many aquatic species are sensitive to ambient levels of ultraviolet-B radiation (UVB) and chemical fertilizers. However, recent studies indicate that the interaction among multiple stressors acting simultaneously could be contributing to the population declines of some animal species. Therefore, we tested the potential synergistic effects between ambient levels of UVB and a contaminant, sodium nitrite in the larvae of two amphibian species, the common European toad Bufo bufo and the Iberian green frog Rana perezi. We studied R. perezi from both mountain and coastal populations to examine if populations of the same species varied in their response to stressors in different habitats. Both species were sensitive to the two stressors acting alone, but the interaction between the two stressors caused a multiplicative impact on tadpole survival. For B. bufo, the combination of UVB and nitrite was up to seven times more lethal than mortality for each stressor alone. In a coastal wetland, the combination of UVB and nitrite was four times more toxic for R. perezi than the sum of the effect on mortality for each stressor alone. One mg/L of nitrite killed half the population of R. perezi at Gredos Mountains at day 10 in the absence of UVB. In the presence of UVB, 50% of the tadpoles from the same experiment died at day 7. Similar toxic response were found for R. perezi in two highly contrasted environments suggesting this synergistic interaction can be a widespread phenomenon. The interaction of excess chemical fertilizers and manure with ambient UVB radiation could be contributing to the global decline of some amphibian species. We suggest that potential exposure to UVB radiation be accounted for when assessing water quality criteria regarding nitrite pollution.     

Evaluation of environmental levels and biological impact of TBT in Malta (central Mediterranean)

Chemical monitoring for butyltins in bulk seawater, surface microlayer and superficial sediments determined that the two main local sources of marine contamination by tributyltin (TBT) compounds in Malta are ship-repairing dockyards and marinas. In bulk seawater, TBT values ranged from below the detection limit of 5 ng Sn l(-1) to 300 ng Sn l(-1); in sediments derived from the most polluted areas, TBT concentrations as high as 1500 ng Sn g(-1) were measured. At TBT levels found in local harbours, several sublethal biological responses are possible and were observed, including a significant reduction in MFO enzyme system activities of fish; digestive cell atrophy in the oyster Ostrea edulis; and induction of imposex in the snail Hexaplex trunculus. The latter two responses are evident at TBT concentrations below the environmental quality standard (20 ng TBT l(-1)). The ecological implications of these results are discussed and it is concluded that exposure of marine organisms to TBT in local harbours may be expected to lead to a reduction in the long-term survival of a number of such organisms. For these reasons, both the use of TBT-based antifouling paints for pleasure boats as well as drydock practices need to be controlled by appropriate regulations and their enforcement.     

地下水污染监测井布设方案研究

为保护地下水资源,工业项目建设过程中需要制定出合理的地下水污染监控方案。特别是在地下水系统比较脆弱的低丘区,其松散层发育较薄,极易造成地下水污染。以某工场项目为例,利用水质模拟模型,模拟出该项目在非正常工况下可能污染物下渗到地下水系统中的运移扩散规律,并根据模拟结果制定出合理的观测井布设方案和监测频率。     

Survey of total and respirable suspended particulate matter in an iron foundry. Comparison of stationary sampling and personal monitoring

In the workshop of an iron foundry total and respirable suspended particulate matter was collected. The performances of (1) filtration systems with 47 mm membrane filters, (2) Andersen cascade impactors, and (3) personal total or respirable monitoring, were compared at a position away from intense sources of particulate debris.Using 15 stationary samplers a survey was made of the particulate levels in the workshop, over a period of 2 weeks. Very large concentration gradients and concentration variations as a function of time were measured for total suspended particulate matter.In the three major source areas, i.e. the pouring department, the core-making department and the shake-out department, special studies were performed to compare stationary and personal monitoring.In the immediate vicinity of intense point sources of coarse particles, such as core-making or shake-out, stationary sampling cannot be used to estimate the personal exposure to total suspended particulates. For respirable particles, however, one or two well-situated stationary size-selective samplers can provide a good estimate of the personal exposure as measured with a personal respirable monitor. The differences found are in the order of 10–20%.     

The use of Nucella lapillus (L.) transplanted in cages to monitor tributyltin (TBT) pollution

Specimens of the gastropod Nucella lapillus were transplanted from their natural habitat to sites affected by tributyltin (TBT) pollution to diverse degrees; individuals were held in cages that also included mussels as food. Subsamples were taken from the cages and the natural site at different time intervals (1, 2 and 5 months) to compare growth, imposex (a set of sexual anomalies caused by TBT) and butyltin concentrations accumulated in female tissues. Individuals in cages experienced negligible mortality and considerable growth. In addition, while the Relative Penis Size Index (RPSI, an index of imposex) remained consistent in the natural site through the experiment, it increased markedly at the implant sites; the same applies to the butyltin residues. These results are discussed to conclude that cage transplanting of N. lapillus is a suitable technique that can quickly (i.e. 1 month) detect differences in the level of TBT contamination among sites; however, extrapolation of experimental results to the field seems to require a longer exposure time (at least 5 months). Finally, the use of such an approach is deemed helpful to interpret the relationship between N. lapillus population dynamics and TBT pollution.