Biological responses in edible crab, <Emphasis Type="Italic">Callinectes amnicola</Emphasis> that could serve as markers of heavy metals pollution

Responses of lagoon crab, Callinectes amnicola were explored as useful biological markers of heavy metal pollution. The toxicity level of the metals based on the 96-h LC50 values showed that copper with LC50 value of 0.018 mM was found to be two times more toxic than Lead (0.041 mM) against the lagoon crab, C. amnicola. The exposure of the lagoon crab to sublethal concentrations (1/100th and 1/10th of 96-h LC50 values) of Cu and Pb compound, respectively, resulted in the bioaccumulation of the test metals to varying degrees in the selected organs that were dependent on the type of metal and concentration of metal compound in the test media. The degree of metal (Cu and Pb) accumulation was generally in the following order: gills > muscle > heptopancrease. Exposure of the crabs to sublethal concentrations of the metals also caused pathological changes such as the disruption of the gill filaments and degeneration of glandular cells with multifocal areas of calcification in the hepatopancreas. A reduction in the weight of the exposed animals over a 14-day period of observation was also recorded. The significance of these results and the usefulness of the biological endpoints in monitoring programmes aimed at establishing the total environmental level of heavy metals in aquatic ecosystems were discussed.     

Urinary and serum metabolites of di-n-pentyl phthalate in rats

Di-n-pentyl phthalate (DPP) is used mainly as a plasticizer in nitrocellulose. At high doses, DPP acts as a potent testicular toxicant in rats. We administered a single oral dose of 500 mg kg⁻¹ bw of DPP to adult female Sprague-Dawley rats (N = 9) and collected 24-h urine samples 1 d before and 24- and 48-h after DPP was administered to tentatively identify DPP metabolites that could be used as exposure biomarkers. At necropsy, 48 h after dosing, we also collected serum. The metabolites were extracted from urine or serum, resolved with high performance liquid chromatography, and detected by mass spectrometry. Two DPP metabolites, phthalic acid (PA) and mono(3-carboxypropyl) phthalate (MCPP), were identified by using authentic standards, whereas mono-n-pentyl phthalate (MPP), mono(4-oxopentyl) phthalate (MOPP), mono(4-hydroxypentyl) phthalate (MHPP), mono(4-carboxybutyl) phthalate (MCBP), mono(2-carboxyethyl) phthalate (MCEP), and mono-n-pentenyl phthalate (MPeP) were identified based on their full scan mass spectrometric fragmentation pattern. The ω − 1 oxidation product, MHPP, was the predominant urinary metabolite of DPP. The median urinary concentrations (μg mL⁻¹) of the metabolites in the first 24 h urine collection after DPP administration were 993 (MHPP), 168 (MCBP), 0.2 (MCEP), 222 (MPP), 47 (MOPP), 26 (PA), 16 (MPeP), and 9 (MCPP); the concentrations of metabolites in the second 24 h urine collection after DPP administration were significantly lower than in the first collection. We identified some urinary metabolic products in the serum, but at much lower levels than in urine. Because of the similarities in metabolism of phthalates between rats and humans, based on our results and the fact that MHPP can only be formed from the metabolism of DPP, MHPP would be the most adequate DPP exposure biomarker for human exposure assessment. Nonetheless, based on the urinary levels of MHPP, our preliminary data suggest that human exposure to DPP in the United States is rather limited.     

Gonad differential proteins revealed with proteomics in oyster (Saccostrea cucullata) using alga as food contaminated with cadmium

As mercury and lead, cadmium (Cd) is one of the highly toxic metals in both the ocean and land environments, but its toxicological mechanism in organisms including human is still unclear because of the complex toxicological pathways in vivo. Here, the alga Chlorella vulgaris were cultivated at room temperature under the stress of cadmium (1 mg L⁻¹) to obtain a toxic food, and then the contaminated food were directly supplied to oyster (Saccostrea cucullata) in seawater. After feeding with C. vulgaris contaminated with Cd (C. vulgaris-Cd), the differential proteins in the oyster gonad (OG) were effectively separated and identified with proteomic approaches. Eleven protein spots were observed to be significantly changed in the OG feeding with C. vulgaris-Cd, which seven spots of these differential proteins were down-regulated while four spots were up-regulated. These altered spots were further excised in gels and identified by a combined technique of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and database searching. A portion of these differential proteins were further proofed by real-time PCR and Western blotting. The results indicate that the major functions of these differential proteins were described as follows: binding, protein translocation, catalysis, regulation of energy metabolism, reproductive function and skeleton structure. These differential proteins in part may effectively provide a few novel biomarkers for the evaluation of Cd pollution level via a food pathway for harming halobios, mammal and human health, and for understanding the complex mechanisms of Cd toxicity in vivo.     

A multidisciplinary weight of evidence approach for classifying polluted sediments: Integrating sediment chemistry, bioavailability, biomarkers responses and bioassays

Evaluation of chemical bioavailability and onset of biological alterations is fundamental to assess the hazard of environmental pollutants, particularly when associated to sediments which need to be removed. In the present work, five sediment samples were collected from the Venice Lagoon and data from sediment chemistry were integrated with those of bioaccumulation of chemicals in European eel (Anguilla anguilla) exposed under laboratory conditions, responses of a wide battery of biomarkers, and standardized ecotoxicological bioassays. The overall results were elaborated within a recently developed, software-assisted weight of evidence (WOE) model which provides synthetic indices for each of considered line of evidence (LOE), before a general evaluation of sediment hazard. Levels of chemicals in sediments were not particularly elevated when compared to sediment quality guidelines of Venice Protocol. On the other hand, bioavailability was evident in some samples for Cd, Cu, Zn and, especially, polycyclic aromatic hydrocarbons. The ecotoxicological approach provided further evidence on the biological and potentially harmful effects due to released contaminants, and oxidative-mediated responses appeared of primary importance in modulating sublethal responses and the onset of cellular alterations. Biomarkers variations were sensitive, and more evident variations included significant changes of cytochrome P450 biotransformation pathway, antioxidant responses, onset of oxidative damages, lysosomal membrane stability and genotoxic effects. The results obtained from the battery of bioassays indicated that responses measured at organism level were in general accordance but less marked compared to the onset of sublethal changes measured through biomarkers. Overall this study revealed differences when comparing evaluations obtained from different LOEs, confirming the importance of considering synergistic effects between chemicals in complex mixtures. Compared to a qualitative pass-fail approach toward normative values, the proposed WOE model allowed a quantitative characterization of sediment hazard and a better discrimination of on the basis of various types of chemical and biological data.     

Assessment of pollution along the Northern Iberian shelf by the combined use of chemical and biochemical markers in two representative fish species

Muscle concentrations of organochlorinated compounds as well as biliary levels of polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APEs) were determined in two different fish species, the four-spotted megrim (Lepidorhombus boscii) and the pouting (Trisopterus luscus) collected along the Northern Iberian coast. Additionally, a set of biochemical markers namely, 7-ethoxyresorufin O-deethylase (EROD), UDP-glucuronosyltransferase (UGT) and catalase (CAT) were measured in liver subcellular fractions. Chemical analysis indicated geographical differences in pollutant loads that were further reinforced by biomarker responses. Thus, EROD activity showed a good correlation with the amount of PCBs bioaccumulated in muscle tissue of both fish species. Elevated UGT activity was observed in those individuals highly exposed to APEs and 1-naphthol. The study reinforces the need to select representative sentinel species from different habitats for biomonitoring purposes and provides further support for the use of biomarkers in assessing the health of coastal areas.     

Changes in cell-type composition in digestive gland of slugs and its influence in biomarkers following transplantation between a relatively unpolluted and a chronically metal-polluted site

Changes in cell-type composition (CCTC) is a general phenomenon that takes place in the digestive gland epithelium of stressed molluscs. The aim of the present work was to determine whether CCTC is a reversible process in the digestive gland of sentinel slugs chronically exposed to metal pollution and how CCTC affects metal accumulation parameters and different cell and tissue biomarkers of exposure and effect. Slugs (Arion ater) from an abandoned zinc mine were transferred to a relatively unpolluted site and the other way around for 3, 10 and 28 d. The volume density of black silver deposits (Vv_BSD) after autometallography, and metallothionein (MT) levels were used as biomarkers of exposure to metals and CCTC and lysosomal responses were selected as effect biomarkers. Results indicated that slugs were sensitive to recent metal pollution; however, slugs chronically exposed to metals presented some characteristic features and were less responsive to pollution cessation without signs of CCTC reversal.     

Efficacy assessment of peracetic acid in the removal of synthetic 17α-ethinyl estradiol contraceptive hormone in wastewater

Increasing concerns have been raised on endocrine disrupting chemicals like the sex hormone 17α-ethinylestradiol (EE2), the more since traditional wastewater (WW) treatments appear to be ineffective for their removal. The efficacy of the relatively novel disinfectant peracetic acid (PAA) in EE2 removal was evaluated, as well as its potential effects on WW quality parameters. The treatments tested for EE2 removal were also evaluated in terms of toxicity, through the determination of biochemical responses (antioxidant enzymes, lipid peroxidation and vitellogenin induction) using zebrafish (Danio rerio) as a biological model. PAA contact times less than 20 min appeared insufficient regardless of the PAA dose tested, but a 100% EE2 removal was attained at a PAA concentration of 15 mg/L with a contact time of 20 min. Total suspended solids, chemical oxygen demand and pH in PAA treatments remained well within levels set in European legislation for WW discharge. EE2 induced significant increased vitellogenin (VTG) levels in both female and male fish, indicating increased estrogenic activity, especially in males suggesting an endocrine disruption effect. With the addition of PAA (15 mg/L), however, VTG levels in both sexes returned to control values. Although this PAA treatment showed increased levels of the antioxidant enzyme catalase, the lipid peroxidation levels were similar or even lower than in controls. Overall the results suggest that the use of PAA appears a promising way forward as a less toxic alternative to chlorine disinfection with high efficiency in the removal of EDC like EE2.     

Population level effects of reduced fecundity in the fish species perch (Perca fluviatilis) and the implications for environmental monitoring

A 40% reduction in relative gonad size in perch (Perca fluviatilis) has been observed over that past two decades at the Swedish national reference site Kvädöfjärden. This biomarker response could be interpreted as a reduction in fecundity and increased risk of local extinction. However, abundance estimates from the same area has not provided any evidence of a reduction in population size. In the present study, a matrix population model was developed to investigate if a reduction in fecundity can be expected to have long term effects on population viability for perch and to evaluate the probability to detect such effects through abundance estimates. The model was parameterized from 17 years of population data from Kvädöfjärden as well as from other studies on perch. The model included density dependence and environmental stochasticity. The results indicated that a reduction in fecundity that is in level with the observed reduction in relative gonad size in Kvädöfjärden will cause a substantial risk for local extinction. The risk that the population will fall below 20% of the carrying capacity within 50 years is 44% when the fecundity is reduced by 40%. However, due to variability in abundance measurements it will take some time before a reduction in gonad size leads to statistically significant effects on the population. If the fecundity is reduced by 40% successively over a 10-year period, the probability to detect this through abundance estimates within 10 years is less than 50%. The results of the present study clearly show that relevant biomarkers have an important role in environmental monitoring as early warning signals, preferably in combination with measurements at higher levels of biological organization.     

The changes of T-PAH levels and health status of mussels in Izmit bay (Turkey) after Marmara earthquake and subsequent refinery fire

As is well known, a powerful earthquake along the North Anatolian Fault struck the eastern part of the Marmara region on August 17, 1999. Izmit Bay, which is known as one of the most polluted sites of Turkey, was also affected by the quake and the subsequent refinery fire. The measurements performed just before and after the earthquake showed that T-PAH levels increased significantly after the event [Okay OS, Tolun L, Telli-Karako? F, Tüfek?i V, Tüfek?i H, Morko? E. Izmit Bay (Turkey) ecosystem after Marmara earthquake and subsequent refinery fire: the long-term data. Marine Pollution Bulletin 2001;42:361-9]. In the framework of ecotoxicological studies, the Bay ecosystem was continuously monitored for T-PAH levels in seawater, sediments and mussels (Mytilus galloprovincialis) to find out whether change occurred during the 2-year period following the earthquake. For that purpose, after the earthquake, the samples were collected six times between the period of September 1999 and March 2001 at coastal stations of the Bay situated away from the mouth of main discharges. The responses of the mussels were also measured by means of the lysosomal stability of the blood cells and feeding rate biomarker techniques at two different sites of the bay. Although the T-PAH levels in all matrices generally showed a decreasing trend, they were found to be still high especially at stations near the refinery. Both biomarker results showed that the health status of the mussels is very poor in the Bay ecosystem, based on the results obtained from the two sites monitored.     

Biomarkers and community indices as complementary tools for environmental safety

Research on biomarkers as early bioindicators of perturbation in populations and individuals has been gaining ground over the last decade. This ecotoxicological approach relies on the fact that changes occur at low levels of organization before the community is affected and thus they can be monitored to assess environmental safety. Changes may concern behavior, physiology, biochemistry, or genomic structure and functioning, and may impair population dynamics in the long-term.Ecotoxicity studies based on biomarkers allow us to measure the impact of environmental stressors and to easily follow the evolution of the systems towards degradation or restoration. Over and above their use as simple indices of exposure to specific pollutants, biomarkers can give an insight into ecosystem health.The results of our experience in field studies involving ecotoxicologists and ecologists will be presented in order to illustrate the relevance of such an integrating strategy for environmental quality assessment.