Preliminary results of a field study on some stress-related parameters in Tapes philippinarum naturally infected by the protozoan Perkinsus sp.

The aim of the present work was to investigate the possible use of the clam Tapes philippinarum—a filter-feeding bivalve living on soft bottoms—in the monitoring of sediment pollution in the Lagoon of Venice (North-East Italy). To this end, two clam populations, exposed to different environmental conditions (a relative clean site and an industrial polluted area), have been monitored for the prevalence of the protozoan parasite Perkinsus sp. as well as for several biological parameters commonly used as determinants of contaminants exposure. First results showed a low prevalence, higher in the clean site, but a very diffuse distribution of the parasite throughout the two populations. Nevertheless, greater digestive cells height and longer lysosomal latency time were observed in clams from the clean area. As for the physiological stress-related parameters, even if clearance rates were always higher in these clams, scope for growth values indicated a more complicated biological response.     

Implications of seasonal priming and reproductive activity on the interpretation of Comet assay data derived from the clam, Tapes semidecussatus Reeves 1864, exposed to contaminated sediments

We explore the use of the clam Tapes semidecussatus Reeves 1864 as an indicator for the presence of potentially genotoxic substances in estuarine sediments. The limitations associated with the interpretation of Comet assay data (expressed as % DNA in tail) in terms of clam reproductive state, size (age) and thermal exposure history following laboratory acclimation are discussed. Hatchery-reared clams, subjected to ambient temperature fluctuations during growth, were exposed in vivo under laboratory conditions for three weeks to sediment samples collected from a polluted site and a "clean" reference site. The DNA damage observed in haemocytes, gill and digestive gland cells was significantly higher in animals exposed to contaminated sediment compared to those exposed to sediment from the reference site. The extent of DNA damage recorded was not correlated with size (age). Spawning was not observed during the experiment. Nevertheless, clams with well-developed gonads showed a statistically higher degree of DNA damage in gill and digestive gland cells- but not haemocytes, demonstrating an increased sensitivity to potential genotoxic compounds, possibly caused by impaired DNA repair capacity due to reproductive activity. Furthermore, the degree of DNA damage in clams exposed to contaminated sediments was higher in autumn and winter compared to spring and summer, suggesting an effect of seasonal priming.     

Acetylthiocholine (ATC)--cleaving cholinesterase (ChE) activity as a potential biomarker of pesticide exposure in the Manila clam, Ruditapes philippinarum, of Korea

The acetylthiocholine (ATC) - cleaving cholinesterase (ChE) activity in Manila clam, which is widely distributed throughout the coastal environment of Korea, was assayed as a potentially useful biomarker of organophosphorous pesticides (OPs). A clear dose-response relationship was determined between inhibited ChE in adductor muscle of clams and four OPs (methidathion, chlorpyrifos, diazinon, IBP) which are heavily used OPs in Korea. The measured EC(50)-24?h values of methidathion, chlorpyrifos, diazinon, and IBP for Ruditapes philippinarum were 7.16?μg?l(-1), 0.34?mg?l(-1), 3.01?mg?l(-1), and 3.41?mg?l(-1), respectively. In field studies, ChE activity in Manila clams collected from 23 stations in the mid-western coastal region demonstrated spatial variation with statistical differences. These results suggest that ChE activity in R. philippinarum is a potential biomarker for assessing organophosphorous pesticide contamination in coastal environments.     

Tapes philippinarum seed exposure to metals in polluted areas of the Venice lagoon

The concentration changes of 12 metals (As, Hg, Cd, Cr, Co, Cu, Pb, Mn, Ni, V, Zn, Fe, and Hg) in the soft tissues of Tapes philippinarum during growth were investigated. Clams were seeded in two contaminated areas of the Venice lagoon (San Giuliano and Fusina) and in an area of the Marano lagoon (Lignano Sabbiadoro) close to the clam-farm where the seed was produced. Metal trends were very different according to the considered element, the study site and the growth period.Arsenic was always higher in clam tissues than in surface sediments and suspended particles in all the three stations. Mercury, Cd and Cu were higher in the clams from Marano and Fusina but not in those from San Giuliano. Zinc and Co in clams exceeded the concentrations in the sediment and suspended particles only at Marano. The other elements (Cr, Pb, Mn, Ni, and Fe) were always higher in SPM and sediments. In general metal concentrations in clams were more highly correlated to concentrations in the suspended particles rather than in the surface sediments and in suspended clams rather than in bottom clams, nevertheless significant differences between stations and contaminants were found. Metal concentrations in clams were always lower than the European regulatory limits.     

Field application of biochemical markers and a physiological index in the mussel,Mytilus galloprovincialis: transplantation and biomonitoring studies in the lagoon of Venice (NE Italy)

A number of biochemical markers and a physiological index were measured in mussels, Mytilus galloprovincialis, transplanted or native to five different contaminated sites in the lagoon of Venice. Mussels from Pellestrina, a reference site in the adjacent Adriatic Sea, were transplanted for 6 weeks to areas of the lagoon where indigenous mussels were also collected. As biochemical indices, superoxide dismutase (SOD), catalase, aldehyde dehydrogenase (ADH) and NADPH cytochrome c reductase (NADPHcred) were measured in mussel digestive gland; survival in air as a physiological index was also determined. Biomarker responses varied among sites and between indigenous and transplanted animals. Significant induction of catalase and SOD was shown in animals transplanted to the urban sites of Salute and Chioggia, respectively. In indigenous mussels, induction of SOD and NADPHcred was seen in animals from the polluted site of Treporti and the heavily contaminated industrial area of Marghera. The overall biochemical data indicate significantly higher activity for ADH in transplanted animals in comparison with indigenous ones which, in contrast, present an increase in SOD. As regard survival in air, control mussels did not seem to be healthier in comparison either with transplanted or indigenous ones, suggesting that pollution has no effect on this parameter.     

Biochemical responses in farmed mussel Perna perna transplanted to contaminated sites on Santa Catarina Island, SC, Brazil.

The effects of contaminants on the biochemical parameters of the intensively farmed mussel Perna perna, are unknown. The aim of this study was to compare biochemical responses in mussels held in clean and contaminated sites in Santa Catarina Island, Brazil. Mussels were transplanted from a farming area, Ratones Grande Island (RGI), to two contaminated sites, Itacorubi (ITAC) and Hercílio Luz Bridge (HLB). A reference group was kept at RGI. After 150 and 180 days of exposure, the digestive glands of the mussels were analyzed for catalase, glucose 6-phosphate dehydrogenase (G6PDH) and glutathione S-transferase (GST) activities. No changes were observed in the catalase activity, in both periods. Low G6PDH activity was observed in mussels transplanted for 150 days at the ITAC site. Increased GST activity was observed in mussels from ITAC and HLG sites after 180 days. These responses are probably related to the augmented discharges of domestic effluents associated with elevated rainfall index.     

胶州湾经济生物体内的重金属含量

于1987年和1988年春、秋两季分析测定了胶州湾24种经济生物体内的重金属铜、铅、镉、锌和铬含量。结果表明,受周围污染环境的影响,该湾经济生物体内的锌、铬、铅和铜含量明显高于受排污影响较小的黄海中部海域同种生物体内的含量;不同种类、体长和不同季节生物体内的重金属含量都有较显著差异。本调查还采用在湾内广泛分布且游动能力差的菲律宾蛤仔探讨了不同地点生物体内重金属含量,未发现潮下带蛤仔与距污染源远近有明显关系。鉴于该湾生物体内重金属含量均低于“海洋生物污染评价标准”和“人体消费标准”,可以认为,胶州湾经济生物体的可食部分对人类食用还是安全的。     

<Emphasis Type="Italic">Zostera marina</Emphasis> seed burial can be enhanced by Manila clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>: A microcosm study

Seagrass seed bank plays a key role in the regeneration of new vegetation when seagrasses are removed by the natural or man-made disaster. Various factors may affect the development of sediment seed bank. We conducted a microcosm experiment to test the effects of burrowing and feeding activities of Manila clam, Ruditapes philippinarum on the burial of Zostera marina seeds in sediments. The effects of lasting time (3-hour, 1-day, 3-day, 7-day, 14-day and 28-day), clam density (0, 2, 4 and 8 clams with shell length of 3 cm in each microcosm) and clam size (shell length of 2, 3 and 4 cm at 4-clam density) on seed burial were examined in plastic microcosm cores (30 cm high × 10 in inner diameter) in a 28-day period. Results showed that the seed burial depth significantly increased with time, the density and the size of clams. No seeds were buried in the sediment in the cores without clams during the whole experiment period. For the 3-cm clams, about 91.61% of the seeds were buried in the sediment at the end of the experiment in the high-density treatment (8 clams at each core); while in the medium and low-density treatments (4 and 2 clams in each core, respectively), about 76.93% and 60.61% of the seeds were buried in the sediment, respectively. For the size treatments, large (4 cm) clams buried 89.56% of the seeds at the end of the experiment, much more than those of medium (3 cm, 76.93%) and small (2 cm, 61.50%) size clams. During the whole experiment period, nearly all of the buried seeds were at a depth of from 0 cm to 5 cm. These results suggested that Manila clam Ruditapes philippinarum may play an important positive role in seagrass seed bank dynamics in the field.     

Effects of toxic dinoflagellate Alexandrium tamarense on the energy budgets and growth of two marine bivalves

Harmful algal blooms (HAB) may impose a serious threat to aquatic lives and human health. We determined the effects of a toxic dinoflagellate Alexandrium tamarense (clone ATCIO1, isolated from Hong Kong coastal waters) on the energy budget, quantified as scope for growth (SFG), and the growth rate of the manila clam Ruditapes philippinarum and the green-lipped mussel Perna viridis. To quantify the SFG, clams and mussels were dosed with different amounts of toxic A. tamarense for 6 days, resulting in different toxin burdens in the tissues. Clearance rate, absorption efficiency, and respiration rate were subsequently measured in these toxin containing bivalves. Clearance rate significantly declined at the highest toxin burden in the clams only, while there was no significant difference in the clearance rate among different treatments for the mussels. The respiration rate of either bivalve was not significantly affected by toxin accumulation in the tissues. In contrast, the absorption efficiency of both clams and mussels decreased, in a concentration-dependent manner for mussels but not for clams, when the tissue accumulated the toxins. With an increase in paralytic shellfish poisoning (PSP) toxin burden, SFG in both clams and mussels was significantly reduced, primarily because of the decrease of absorption efficiency. The growth rate of juvenile clam R. philippinarum, measured as an increment in tissue dry weight over a 15 d exposure period, was significantly lower during their feeding on toxic dinoflagellate than the growth rate of clams feeding on the diatom Thalassiosira pseudonana. The juvenile mussel P. viridis, however, exhibited similar growth rates after feeding on the toxic dinoflagellates and the nontoxic diatom. This study showed that SFG can provide a sensitive and integrated measure of the effect of HAB on the physiology of bivalves. Clam R. philippinarum may be more sensitive, in terms of their energy budget, to PSP toxin accumulation than the mussel P. viridis.     

Biomarkers and trace metals in the digestive gland of indigenous and transplanted mussels, Mytilus galloprovincialis, in Venice Lagoon, Italy.

The aim of this study was to investigate the cellular and biochemical response of mussels, Mytilus galloprovincialis, transplanted from a relatively pristine site to a polluted one and vice versa in the Lagoon of Venice (northeast of Italy) and to apply auto-metallography, a rapid and sensitive histochemical technique, to determine the bio-available fraction of heavy metals accumulated in the body tissues of organisms. Animal digestive glands have been used for morphological analyses (lysosomal volume, surface and numerical density, mean epithelial thickness, mean diverticular and luminar radius), autometallographical black silver deposits quantification and biochemical assays (superoxide dismutase and catalase activity). Furthermore, heavy metal content was determined by atomic absorption spectrophotometry (AAS) using standard procedures. The overall results indicate a direct influence of the environment in the thinning of the digestive cells and in the increasing number of lysosomes in mussels from the more polluted site. These data are in agreement with the metal content in digestive cell lysosomes as determined by autometallography, whereas AAS measurements show less significant differences.     

Comparison Between the Energy Flow Networks of Two Habitats in the Venice Lagoon

Abstract. The energy flows of two habitats are quantified and compared by using a trophic network model. The habitats are representative of two typical subsystems of the Venice Lagoon: the seagrass meadows, which represent a mature stage in the ecological succession of lagoon environments, and the Tapes philippinarum fishing grounds, which are subjected to mechanical clam harvesting.
The results obtained for the 'seagrass habitat'are consistent with other field studies, confirming the'key-stone'role played by phanerogams in a lagoon environment. The results for the 'Tapes habitat'show that the energy flow from the sediment to the water column is extremely important. Indeed, the high T. philippinarum biomass cannot be supported solely by the primary production of the local phytoplankton. Instead, this biomass could be supported by a supply provided by the sediment resuspension caused by intensive clam fishing. These findings suggest the presence of a positive feedback between fishing effort and T. philippinarum standing crop.     

江苏沿海贝类软体中麻痹性贝毒素种类及含量变化的多年观测研究

Paralytic shellfish toxins(PSTs) are notorious neurotoxins that threaten public health and food safety worldwide.Although PST monitoring programs have recently been established throughout China, the profiles and variation of PSTs in important commercial clams(e.g., Mactra veneriformis, Ruditapes philippinarum, and Meretrix meretrix) along the Jiangsu Province coastline remain largely unexplored. In this study, a validated hydrophilic interaction liquid chromatography–tandem mass spectrometry(HILIC-MS/MS) method was used to examine PST profiles and levels in 540 clam samples from natural production areas along Jiangsu Province coastline during2014–2016. Although the PST levels(≤6.38 μg saxitotoxin equivalents(eq)/kg) were consistently below European Union regulatory limits(≤800 μg saxitotoxin eq/kg) during this time period, saxitotoxin, decarbamoylsaxitotoxin,and gonyautoxins 1 and 4 were detected, and nearly 40% of the samples were saxitotoxin-positive. The PST levels also varied significantly by seasons, with peak values observed in May during 2014–2016. This is the first systematic report of PSTs in clams from Jiangsu Province, and additional research and protective measures are needed to ensure the safety of clams harvested in this area.     

Speciation, distribution, and potential ecological risk assessment of heavy metals in Xiamen Bay surface sediment

Based on the survey of surface sediment in Xiamen Bay in October 2011, the speciation, distribution, and potential ecological risk assessment of heavy metals （Cu, Pb, Zn, Cd, and Cr） in this area were studied us-ing the sequential extraction method and ecological risk assessment method. The results indicated：（1） the total concentrations of these heavy metals were influenced by runoff, industrial wastewater, and sewage, and were all higher in the coastal area than the offshore area while the highest area of Pb was a little far-ther away from the coastal water due to atmosphere deposition;（2） sequential extractions suggested that Cu was mainly composed with residual and Fe/Mn-oxide bound fractions, Pb was bound to Fe/Mn-oxide, Zn and Cr were dominated by residual, and Cd bound to exchangeable and carbonate fractions; （3） the order of migration and transformation sequence was Cd＆gt;Pb＆gt;Cu＆gt;Zn＆gt;Cr and the degree of pollution was Cd＆gt;Pb＆gt;Cu＆gt;Zn＆gt;Cr;and （4） the ratios of the secondary and primary phases showed that Zn and Cr were both clean, Cu may be polluted, Pb was moderately polluted, while Cd was heavily polluted.     

Neuroendocrine disruption in Mya arenaria clams during gametogenesis at sites under pollution stress

This study examined the neuroendocrine status of clams on intertidal mud flats in the St. Lawrence Estuary and Saguenay Fjord areas during late gametogenesis. The impact of pollution was determined by a test battery of early stress markers (metallothioneins, heme levels, glutathione S-transferase activity), tissue damage (lipid peroxidation and DNA damage) and morphologic characteristics (age, soft-tissue weight ratio and growth index). Neuroendocrinal status was examined by tracking serotonin and dopamine metabolism, monoamine adenylate cyclase activity in synaptosomes, monoamine oxidase and arachidonate cyclooxygenase activities in relation to gametogenetic activity: pyrimidine synthesis, (aspartate transcarbamoylase activity or ATC), vitellogenin-like proteins and gonado-somatic index. The results show that clam soft tissue weights were reduced at sites close to harbours and higher at sites near domestic wastewater outfalls. The age-to-length ratio of clams was generally higher at impacted sites, suggesting reduced growth. The biomarkers of stress or damage confirmed that oxidative stress, DNA damage, metallothioneins and glutathione S-transferase activity were significantly increased at varying degrees, at the polluted sites. Vitellogenin-like proteins and gametogenetic activity were significantly increased at sites influenced by domestic wastewaters. Furthermore, the clams were still in active gametogenesis and not ready for spawning, as indicated by the concordance of the serotonin/dopamine ratio with vitellogenin-like proteins and pyrimidine synthesis. However, gonadal cyclooxygenase activity was increased at polluted sites and significantly correlated with most of the stress biomarkers, suggesting that the clams were in a state of inflammation rather than at the spawning stage. Finally, a multivariate analysis revealed that the sites were readily discriminated with high efficiency (>71%) and that both neuroendocrine physiological markers and stress responses were identified as the major components, thus explaining the global physiological response of the clams. We conclude that the effects of pollution compromise the clams' health status and that the initiation of gametogenesis in environments contaminated by municipal wastewaters or harbours contributes to the toxic effects of pollution.     

胶州湾水生系统中Pb、Zn 的分布特征及其在生物体中的浓缩

利用在胶州湾采集的47组同站位多介质样品,进行了痕量金属Pb、Zn及其它环境因子的化学成分测试,讨论了胶州湾水-沉积物-生物系统中Pb、Zn的生物地球化学总体特征及其在各介质平面上的分布,揭示了胶州湾水-沉积物-生物系统对陆源物质输入的响应.垂向上表层沉积物是Pb、Zn组分的富集带,该系统中的生物相对于其所处水环境具有显著的富集作用,生物体中Pb、Zn生物浓缩系数分别为36、935;横向上,Pb、Zn在底层水和沉积物介质中的分布主要受控于河口,即高值区分布于胶州湾的各个主要河口区,特别是在沉积物中金属组分浓度的高值区主要集中分布于胶州湾的东部.而孔隙水中Pb、Zn的高值主要分布于水交替较弱的海域,如红岛前缘.但生物体中的痕量金属组分化学场空间分布规律与上述各介质的化学场均不吻合,亦即是生物体中痕量金属组分的浓度与其所处环境中的同名金属组分浓度无关.作者认为,生物对痕量金属组分的富集并不简单地取决于它所处环境介质中同名金属组分的总量,而存在形态上的选择性.通过回归分析揭示了孔隙水对生物体中Pb、Zn的富集贡献较大.     

A multibiomarker approach in the clam Ruditapes decussatus to assess the impact of pollution in the Ria Formosa lagoon,South Coast of Portugal

The Ria Formosa lagoon is an ecosystem whose water quality reflects the anthropogenic influence upon the surrounding areas. In this lagoon, the clam Ruditapes decussatus has a great economical importance and has been widely used as a biomonitor. A multibiomarker approach (δ-aminolevulinic acid dehydratase, metallothionein, lipid peroxidation, acetylcholinesterase, alkali-labile phosphates, DNA damage) was applied to assess the environmental quality of this ecosystem and the accumulation of contaminants and their potential adverse effects on clams. Clams were sampled in different shellfish beds in the period between July 2007 and December 2008 and abiotic parameters (temperature, salinity, pH and dissolved oxygen of seawater and organic matter in the sediment), condition index, metals (Cd, Cu, Zn, Ni, Pb), TBTs and PAHs concentrations were measured in clam tissues. Data was integrated using Principal Component Analyses and biomarker indices: IBR (Integrated Biomarker Response) and HSI (Health Status Index). This multibiomarker approach enabled discrimination of a time and space trend between sites with different degrees of anthropogenic contamination, identifying one of them (site 2) as the most stressful and summer months as the most critical period for clams due to an increase of environmental stress (anthropogenic pressure along with extreme environmental conditions, e.g. temperature, dissolved oxygen, organic matter in the sediments, etc). The selected biomarkers provided an integrated response to assess the environmental quality of the system, proving to be a useful approach when complex mixtures of contaminants occur.     

Effects of polynuclear aromatic hydrocarbons on fishes and shellfish: An overview of research in virginia

This paper discusses the following topics related to polynuclear aromatic hydrocarbon pollution in estuaries: (1) the use of oysters (Crassostrea virginica), hard clams (Mercenaria mercenaria) and brackish water clams (Rangia cuneata) in residue monitoring; (2) the effects of elevated PAH residues on oyster condition; (3) three years of field studies relating PAH sediment contamination to abnormalities in fishes; and (4) laboratory bioassays for effects and bioconcentration modeling. Oysters, hard clams and Rangia have been shown, from 3 years of field studies, to be good monitors of pollution inputs as one proceeds along salinity gradients from 25‰ to 0·5‰. Effects of increased body burdens of PAHs are shown by a lowering of the oysters' condition index, as measured by lipid levels. Fishes inhabiting the Elizabeth River, VA, which is highly contaminated with PAHs, have abnormalities (cataracts, skin lesions, abnormal fins, etc.). These abnormalities have higher incidence in regions of the river where the sediments are more heavily contaminated. Laboratory studies utilizing contaminated sediments have reproduced some of the abnormalities observed in the field. Bioconcentration of PAHs from sediments has been studied with oysters and hard clams. Oysters generally accumulate three times the body burdens of clams exposed to the same suspensions. A faster depuration rate (k₂) for clams appears to be responsible for the higher equilibrium body burden of oysters.     

The role of alkaline phosphatase in the sediments of Venice Lagoon on nutrient regeneration

Potential alkaline phosphatase activity and nutrient concentration have been determined in the seawater, interstitial water and sediments of the most polluted area in the Venice Lagoon, at 6 stations, during three cruises performed in the October/November 1982 period, when a minimum phytoplankton standing crop existed. The relationships between parameters were established by linear regression analysis.The results clearly indicate the important role of alkaline phosphatase (APA) in phosphorus regeneration. Indeed, APA appears to be a good indicator of the degree of nutrient regeneration occurring in surface sediments on a global basis.In the Venice Lagoon a significant nutrient release from the sediments to the overlying lagoon water takes place. This process appears to be at least as important as external contributions in both establishing and controlling the concentration of the nutrients in lagoon waters.     

Hepatic biomarkers of sediment-associated pollution in juvenile turbot, Scophthalmus maximus L

Hatchery-reared turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to sediment collected from polluted sites in Cork Harbour and a reference site at Ballymacoda, Co. Cork, Ireland. The potential of surficial sediment for inducing hepatic biomarkers was assessed at two levels of biological organisation: expression of cytochrome P450 [Western blotting analysis and 7-ethoxy-resorufin O-dealkylase (EROD), 7-benzoxy resorufin O-dealkylase (BROD), 7-methoxy resorufin O-dealkylase (MROD), 7-pentoxy-resorufin O-dealkylase (PROD) activities] and DNA integrity (Comet assay). Positive controls were generated, either by exposing turbot to cadmium chloride-spiked seawater (Comet assay) or to beta-naphthaflavone by intraperitoneal injection (cytochrome P450 induction). The induction of cytochrome P450 activity (EROD, MROD and PROD) in animals following a 7-day exposure to contaminated sediments was significantly higher than those exposed to reference site sediment and remained elevated thereafter; BROD was not induced. DNA single-strand breaks were also significantly higher following exposure to contaminated sediments throughout the experiment. Although no direct correlation between induction of alkoxyresorufin O-dealkylase activities and a particular chemical class was established, the induction of MROD and PROD activities in fish exposed to sediments containing complex contaminant mixtures, appeared to be more sensitive than conventional EROD activity assays. We conclude from the present laboratory study that S. maximus is a suitable sentinel species for the assessment of moderately contaminated sediments and therefore allows for the further development of this model for future, ecologically relevant, field studies.     

Seasonal variations of exposure biomarkers, oxidative stress responses and cell damage in the clams, Tapes philippinarum, and mussels, Mytilus galloprovincialis, from Adriatic sea

This work investigated the natural variability of several biomarkers in Tapes philippinarum and Mytilus galloprovincialis, sampled from Northern Adriatic where these organisms are important sentinel species for future environmental impact assessment. Levels of metallothioneins, peroxisomal enzymes and acetylcholinesterase, showed a significant seasonality and marked differences between clams and mussels. Among antioxidant enzymes, catalase and GST decreased during the warmer period, the latter enzyme activity resulting particularly high in clams. The total oxyradical scavenging capacity toward peroxyl radicals decreased in mussels from winter to summer, indicating a prooxidant challenge due to higher seawater temperature and intensity of light irradiance. Lysosomal membrane stability did not exhibit significant seasonal variations, while some variations were observed for DNA damages. Overall results indicated a significant influence of seasonal variability on several biomarkers and species-specific differences which should be considered to discriminate the appearance of anthropogenic disturbance.