One-year variation in quantity and properties of microplastics in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from Aveiro lagoon.

As filter feeders, marine bivalves inhabiting estuarine and coastal areas are directly exposed to microplastics (MPs) in water. To assess whether MPs number, and their shape, size, colour, and polymer type present in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) varied over one year, bivalves were collected over the year of 2019 in the lower part of the coastal Aveiro lagoon, Portugal. After extraction from the bivalve's whole-body soft tissues, a subset of the visually inspected particles was randomly separated for identification using the Fourier-transform mid-infrared (FT-MIR) spectroscopy. A fraction of the inspected particles, 26-32% of particles >100 µm, and 59-100% of smaller ones were confirmed as MPs. Concentrations varied within the intervals of 0.77-4.3 items g-1 in mussels and 0.83-5.1 items g-1 in cockles, with the lowest values observed in January. In winter, the accumulation of large-sized fibers was composed of a mixture of plastic types, which contrasted against the most abundant MPs in summer consisting mainly of polyethylene of diverse size classes and shapes. Temperature decrease registered in winter might have triggered a lower filtration rate, resulting in lower MPs concentrations in the whole-soft body tissues of organisms. Different properties of MPs found in bivalves between January-February and August-September appear to reflect changes in the characteristics of MPs available in the Aveiro lagoon.

Two strategies to live in low chronic pollution estuaries: the potential role of lifestyle.

Although physiological and genetic adaptation to toxic stress has been the focus of recent research, the role of lifestyle in pollution tolerance has received less attention. In this study, copper tolerance of benthic and epi-benthic species of harpacticoid copepods was investigated. Concentration-response curves were generated for populations of both species but collected at contaminated and uncontaminated estuaries. The population of the benthic species from the contaminated site showed higher tolerance than its population from the uncontaminated site. The epi-benthic species showed no inter-specific differences in tolerance. The comparison of tolerance between field collected animals of the benthic species and animals from the same population but reared in the laboratory for two generations suggested the existence of a genetic inherited tolerance. Results revealed the importance that lifestyle may have on the generation of tolerance to toxic substances and highlight a potential unforeseen role of it in maintenance of biodiversity on contaminated habitats.

Biochemical characterization of cholinesterases in Enchytraeus albidus and assessment of in vivo and in vitro effects of different soil properties, copper and phenmedipham.

Enchytraeus albidus are important organisms of the soil biocenosis, used as standard test species in environmental risk assessment. The inhibition of cholinesterases (ChE) activity of several species has been widely used to assess the exposure and effects of anti-cholinesterase environmental contaminants. Several studies have shown the association between ChE activity inhibition and adverse effects on behaviour and survival. Extensive studies addressing survival and behavioural endpoints, as well as other biomarkers, have been done in E. albidus with different types of soil contaminants. The main objectives of this study were: (1) to characterize biochemically the ChE present in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, using different substrates and selective inhibitors; (2) to assess the in vivo effects of copper, phenmedipham and different soil properties (pH, organic matter, clay) on the ChE activity; (3) to assess the in vitro effects of copper and phenmedipham on the ChE activity. The results suggest the presence of one ChE in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, which displays properties of both acetylcholinesterase and pseudocholinesterase considering the typical mammalian enzymes. It is also shown that ChE activity is not inhibited by exposure to different soil properties and that copper and phenmedipham inhibited ChE activity both in in vivo and in in vitro conditions and therefore ChE inhibition seems to be a robust biomarker for this herbicide and this heavy metal. This study showed that ChE activity in E. albidus might be correlated to previously determined higher level effects like survival and reproduction, as well as avoidance behaviour.

Biochemical effects and polycyclic aromatic hydrocarbons (PAHs) in senegal sole (Solea senegalensis) from a Huelva estuary (SW Spain).

Relations between several stress oxidative biomarkers and aromatic polycyclic hydrocarbon (PAH) concentrations have been studied in wild sole, Solea senegalensis collected in the vicinity of a petrochemical industry. Antioxidant enzyme activities in eco-toxicological studies constitute excellent markers for exposure to a large variety of pollutants. The 16 PAHs in sediment as well as oxidative damage (LPO), activity of catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR) and PAHs type metabolites in sole liver were analysed. Significant correlations (p<0.05) were established between some biomarkers as GST, GPx and CAT and PAHs metabolites in liver (naphthalene, pyrene and phenanthrene) and PAHs concentrations in sediments (fluoranthene, acenaphthene, anthracene and chrysene). PAHs accumulated in the sediment and organisms are inducers of antioxidant defences. GST, GPx and CAT were robust biomarkers showing correlations with both PAHs in sediments and liver PAH metabolites showing different responses to low and high molecular weight PAHs.

Ecotoxicological tools for the tropics: Sublethal assays with fish to evaluate edge-of-field pesticide runoff toxicity.

This study proposes short-term sublethal assays for the tropics using the fish Poecilia reticulata. Assays were evaluated under realistic exposure scenarios by simulating a runoff over an agricultural area sprayed with deltamethrin (Decis). In situ assays were performed inside microcosms set up to simulate runoff water entrance in lentic systems. Laboratory assays were conducted with water samples from the microcosms. In both assays the biochemical parameters were similarly responsive to Decis, with a significant inhibition of acetylcholinesterase (30-46%) and induction of lactate dehydrogenase (33-67%) and glutathione S-transferases (48-176%) activities, observed as of the lowest Decis dose. Postexposure feeding was more sensitive in the laboratory assay than in situ. Among the additional potential stressors, only acetylcholinesterase was significantly influenced by the runoff per se. The proposed methodologies were well adapted for assays with P. reticulata, as organisms were easily deployed and retrieved and enzymatic activities and postexposure feeding were sensitive endpoints.

Blood delta-ALAD, lead and cadmium concentrations in spur-thighed tortoises (Testudo graeca) from Southeastern Spain and Northern Africa.

Mediterranean spur-thighed tortoise (Testudo graeca) is actually included in the IUCN as vulnerable species. Its main European population is located in southeastern Spain. Although a great deal of information has been acquired on the internal medicine and survey and even parasitological fauna on these animals, there are no references about contaminants levels in this species. The objectives of this study were to compare the levels of two metals (cadmium and lead) in the blood of spur-thighed tortoises from two different populations, one from Southeastern of Spain (n = 22) and the other from North of Africa (n = 39), kept in captivity at the Santa Faz Recuperation Centre (Alicante, Spain) and to investigate the relationship between their blood levels of lead and their blood delta-aminolevulinic acid dehydratase (delta-ALAD) activity. Blood lead and cadmium concentrations were higher in tortoises from African than in those from Spain. Moreover, a negative and significant correlation (P < 0.05) was found between delta-ALAD activity and blood lead levels, indicating the suitability of this enzyme as biomarker for lead in this species.

The influence of short-term experimental fasting on biomarker responsiveness in oil WAF exposed mussels.

Mussels are widely used in toxicological experimentation; however, experimental setups are not standardized yet. Although there is evidence of changes in biomarker values during food digestion and depending on the mussel nutritive status, the mode of feeding differs among toxicological experiments. Typically, mussels are fed with different diets in different long-term experiments, while fasting is the most common approach for short-term studies. Consequently, comparisons among experiments and reliable interpretations of biomarker results are often unfeasible. The present investigation aimed at determining the influence of fasting (against feeding with Isochrysis galbana) on biomarkers and their responsiveness in mussels exposed for 96 h to the water accommodated fraction (WAF) of a heavy fuel oil (0%, 6.25%, 12.5% and 25% WAF in sea water). PAH tissue levels in digestive gland and a battery of biomarkers were compared. WAF exposure led to decrease of cytochrome-C-oxidase activity, modulated glutathione-S-transferase activity, augmented lipid peroxidation, inhibited acetyl cholinesterase (AChE) activity, and led to lysosomal enlargement (VvLYS and S/VLYS) and membrane destabilisation, lipofuscin accumulation, and histopathological alterations (VvBAS, MLR/MET and CTD ratio) in the digestive gland epithelium; and were integrated as IBR/n (biological response index). Overall, no significant changes were recorded in AChE activity, S/VLYS and CTD ratio in any experimental treatment, while all the other biomarkers showed significant changes depending on the fasting/feeding condition, the exposure to WAF and/or their interaction. As a result, the integrated biomarker index IBR/n was higher at increasing WAF exposure levels both in fasted and fed mussels albeit the response was more marked in the latter. The response profiles were qualitatively similar between fasted and fed mussels but quantitatively more pronounced in fed mussels, especially upon exposure to the highest concentration (25% WAF). Therefore, it is highly recommended that mussels are also supplied with food during short-term, like during long-term toxicological experiments. This practice would avoid the interference of fasting with biological responses elicited by the tested chemicals and allow for reliable comparison with data obtained in long-term experiments and monitoring programmes.

Acute effects of Benzo[a]pyrene, anthracene and a fuel oil on biomarkers of the common goby Pomatoschistus microps (Teleostei, Gobiidae).

The objective of this study was to investigate the effects of two different PAHs and a complex petrochemical mixture on the common goby, Pomatoschistus microps, using selected biomarkers as effect criteria. Benzo[a]pyrene (BaP) and anthracene were used as reference substances, while the water accommodated fraction of #4 fuel-oil (#4 WAF) was used as an example of a petrochemical mixture. P. microps was used since it is both a suitable bioindicator and a good test organism. Groups of fish were exposed to different concentrations of each of the test substances for 96 h and the activities of several enzymes commonly used as biomarkers were determined at the end of the bioassays. All the substances inhibited P. microps acetylcholinesterase (AChE) indicating that they have at least one mechanism of neurotoxicity in common: the disruption of cholinergic transmission by inhibition of AChE. An induction of lactate dehydrogenase (LDH) activity was found in fish exposed to BaP or to anthracene, suggesting an increase of the anaerobic pathway of energy production. On the contrary, inhibition of LDH was found in fish exposed to #4 WAF, suggesting a distinct effect of the mixture. An induction of P. microps glutathione S-transferase (GST) activity was found in fish exposed to BaP or to #4 WAF, while an inhibition was observed after exposure to anthracene. These results suggest that GST is involved in the detoxification of BaP and #4 WAF, but not of anthracene. All the substances increased catalase activity and isolated PAHs also increased superoxide dismutase, glutathione reductase and glutathione peroxidase activities, while #4 WAF did not cause significant alterations on these enzymes. These results suggest that all the substances may induce oxidative stress on P. microps, with BaP and anthracene apparently having more oxidative stress potential than #4 WAF.

Transport and acclimation conditions for the use of an estuarine fish (Pomatoschistus microps) in ecotoxicity bioassays: effects on enzymatic biomarkers.

Acclimation of organisms for ecotoxicity testing is in general processed according to Organisation for Economic Co-operation and Development (OECD) and/or Environmental Protection Agency (EPA) guidelines, under controlled conditions. However, when organisms are collected in the field, their capture, transport and adaptation to laboratory conditions are factors of stress. In their natural habitat, estuarine fish are exposed to considerable fluctuations of environmental variables, while in laboratory they are acclimated to constant conditions and this can be per se a factor of stress that may influence biomarker responses. Therefore, it is important to investigate the effects of these procedures on estuarine fish performance before using them as test organisms in ecotoxicity bioassays. The goal of the present study was to investigate the effects of transporting the common goby, Pomatoschistus microps from the field (natural populations) to the laboratory and of its acclimation to laboratory conditions on the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and glutathione S-transferases (GSTs). Fish were collected in a reference site of the Minho River estuary (NW of Portugal) and the activities of the biomarkers were monitored before and after the transport of organisms to the laboratory and during the acclimation period (at 5, 10 and 15 days). The activities of all the enzymes indicated that capture and transport conditions had no effects on enzymatic activities. Furthermore, AChE, LDH and GST presented higher activities at the end of the acclimation period than at beginning, suggesting a physiological adaptation to laboratory conditions. This adaptation should be taken into consideration in the experimental design to avoid bias in the interpretation of effects of xenobiotics on biomarkers.

Behaviour and biomarkers of oxidative stress in Gambusia holbrooki after acute exposure to widely used pharmaceuticals and a detergent.

Pharmaceuticals are continuously dispersed into the environment, as a result of human and veterinary use, and have become a relevant environmental concern. In the present study, the acute toxicity of three therapeutic agents (diazepam, clofibrate, and clofibric acid) and a detergent, sodium dodecylsulphate (SDS), to the euryhaline fish Gambusia holbrooki was evaluated. Special attention was devoted to oxidative stress parameters. G. holbrooki males, captured in the estuary of the Minho River (NW Portugal), were exposed for 96 h to the selected compounds. The following oxidative stress biomarkers were evaluated in gills and liver tissues: reduced and oxidised glutathione, lipid peroxidation, and several antioxidant enzymes, namely (1) total and selenium-dependent glutathione peroxidase (GPx), (2) glutathione reductase (GRed), (3) copper-zinc superoxide dismutase (Cu-ZnSOD) and manganese superoxide dismutase (MnSOD), and (4) glutathione-S-transferases (GSTs). In the particular case of diazepam, swimming behaviour was also evaluated. The obtained results indicate an overall diminished oxidative stress response caused by SDS and diazepam. Oxidative-based alterations were observed after exposure to clofibrate and clofibric acid, with modifications of several enzymatic activities. Diazepam caused evident behavioural changes: animals showed dark pigmentation and also abnormal postures, namely lethargy and anomalous movement.

Ras gene in marine mussels: a molecular level response to petrochemical exposure.

Mussels are susceptible to numerous toxicants and are often employed as bioindicators. This study investigated the status of the ras proto-oncogene in Mytilus galloprovincialis following petrochemical exposure. A M. galloprovincialis homologue of the vertebrate ras gene was isolated, showing conserved sequence in regions of functional importance and a high incidence of polymorphic variation. Mutational damage was investigated in mussels chronically exposed to the water-accommodated fraction of #4 fuel-oil (WAF), and in mussels collected along the NW coast of Portugal in sites with different levels of petrochemical contamination. A ras gene point mutation was identified in the codon 35 of one individual exposed to 12.5% WAF. No mutations were detected in mussels from the WAF control or environmental samples. This represents the first report of a ras gene mutation, experimentally-induced by petrochemical exposure, in an invertebrate species.

Enzymatic biomarkers in the crab Carcinus maenas from the Minho River estuary (NW Portugal) exposed to zinc and mercury.

Zinc and mercury are common contaminants of estuaries and coastal ecosystems where they may induce adverse effects on the biota. Carcinus maenas is a key-species in several European estuaries, living in close association with the sediment where a considerable number of environmental contaminants, including zinc and mercury may accumulate. In the present study, the acute effects of zinc and mercury on C. maenas from the Minho River Estuary (NW Portugal) were investigated by using the activity of the enzymes cholinesterase (ChE), lactate dehydrogenase (LDH) and glutathione S-transferases (GST) as effects' criteria. Crabs were exposed for 96h to several concentrations of Zn(2+) (0, 1.84, 3.71, 7.39 and 14.79mg/l) or Hg(2+) (0, 0.09, 0.19, 0.37 and 0.74mg/l). The choice of Zn(2+) test concentrations was based on the LC50 value (14.86mg/l) determined in a first part of the study, while the choice of Hg(2+) concentrations was based on values from the literature. At the end of the bioassays, eye, muscle and hepatopancreas tissues were isolated and used for ChE, LDH and GST determinations, respectively. Zinc significantly inhibited ChE activity (p<0.05, EC50=14.68mg/l), caused significant alterations of GST activity (p<0.05) and induced LDH activity (p<0.05) at the exposure of 14.79mg/l. Mercury significantly inhibited ChE activity (p<0.001, LOEC=0.09mg/l, EC50=0.235mg/l) and increased both GST (p<0.05, LOEC=0.774mg/l) and LDH activities (p<0.05, LOEC=0.119mg/l). These results suggest that both metals interfere with cholinergic neurotransmission in C. maenas by inhibiting ChE activity. In addition, they also interfere with GST activity and this may reduce the capacity of detoxification of some chemicals and/or to increase the probability of oxidative stress to occur. Furthermore, both metals increase LDH activity, suggesting an interference with energy production pathways. Therefore, the presence of zinc and mercury in estuaries at concentrations in the mg/l or mug/l range, respectively, may have a negative impact on C. maenas.

Impact of chemical exposure on the fish Pomatoschistus microps Krøyer (1838) in estuaries of the Portuguese Northwest coast.

Juveniles of the estuarine fish Pomatoschistus microps were collected from autumn 2001 to summer 2002 in five stations along the Portuguese Northwest coast with different types and/or levels of environmental contamination: two reference sites with low levels of contamination (R1 and R2) and three differently impacted areas with higher levels of contamination. UI is located in an estuary under the influence of urban and industrial effluents, AA in a channel that receives intensive agriculture run-off and IE in a highly impacted industrial area. The activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferases (GST) were used as environmental biomarkers on P. microps. A significant seasonality effect on all the enzymatic activities was found, lower levels being registered in winter and spring on AChE, in autumn on LDH, and in winter on GST and EROD. The battery of biomarkers used was capable of discriminating sites with different types and/or levels of contamination, R1 and UI being the highest discriminated (91.7% and 66.7%, respectively). At R1 significantly lower levels of AChE and LDH were found, and EROD was significantly inducted at UI. Furthermore, IE presented higher levels of GST, and R2 and AA an inhibition of AChE in winter and spring. The results indicated that the battery of biomarkers used in this study seems to be a useful tool to distinguish between different types of environmental contamination in estuarine systems, and that P. microps is a suitable species to be used as bioindicator.

Transcriptional and biochemical analysis of antioxidant enzymes in the mussel Mytilus galloprovincialis during experimental exposures to the toxic dinoflagellate Prorocentrum lima.

The genotoxic and cytotoxic effects of Diarrhetic Shellfish Poisoning (DSP) toxins have been widely investigated in bivalve molluscs, representing the main vectors of these compounds in the Atlantic coast of Europe. DSP toxins are produced by Harmful Algal Blooms (HABs) of Dinophysis and Prorocentrum dinoflagellates, being subsequently accumulated by marine organisms and biomagnified throughout trophic webs. Yet, bivalves display increased resistance to the harmful effects of these toxins during HAB episodes. While previous reports have suggested that such resilience might be the result of an increased activity in the bivalve antioxidant system, very little is still known about the specific mechanism underlying the protective effect observed in these organisms. The present work aims to fill this gap by studying transcriptional expression levels and biochemical activities of antioxidant enzymes in different tissues the mussel Mytilus galloprovincialis during experimental exposures to DSP toxins produced by the dinoflagellate Prorocentrum lima. Results are consistent with the presence of a compensatory mechanism involving a down-regulation in the expression of specific genes encoding antioxidant enzymes [i.e., SuperOxide Dismutase (SOD) and CATalase (CAT)] which is counterbalanced by the up-regulation of other antioxidant genes such as Glutathione S-Transferase pi-1 (GST-pi) and Selenium-dependent Glutathione PeroXidase (Se-GPx), respectively. Enzymatic activity analyses mirror gene expression results, revealing high antioxidant activity levels (consistent with a protective role for the antioxidant system) along with reduced lipid peroxidation (increasing the defense against oxidative stress).

Effects of widely used pharmaceuticals and a detergent on oxidative stress biomarkers of the crustacean Artemia parthenogenetica.

Pharmaceuticals are continuously dispersed into the environment as a result of human and veterinary use, posing relevant environmental concerns. The present paper reports the acute toxic effects of three therapeutic agents (diazepam, clofibrate and clofibric acid) and a detergent, sodium dodecylsulphate (SDS), to the hypersaline crustacean Artemia parthenogenetica. This study specially focused on oxidative stress parameters, namely (1) total and selenium-dependent glutathione-peroxidase (GPx), (2) glutathione reductase (GRed), (3) total superoxide dismutase (SOD), and (4) glutathione-S-transferases (GSTs). The effects of tested substances on lipid peroxidation (thiobarbituric acid reactive substances, TBARS), and soluble cholinesterases (ChE) were also investigated. Diazepam caused a significant inhibition of ChE (LOEC = 7.04 mg/l) and total GPx activities. SDS was responsible for a decrease in the activity of both ChE (LOEC = 8.46 mg/l) and GRed (LOEC = 4.08 mg/l). Both fibrates (clofibrate and clofibric acid) were responsible for significant decreases in Se-dependent GPx, with LOEC values of 176.34 and 3.09 mg/l, respectively. Clofibrate also caused a slight increase of TBARS content of A. parthenogenetica homogenates. These results indicate that the exposure to all the tested compounds induced alterations on the cellular redox status in A. parthenogenetica. In addition, diazepam was shown to have the capability of interfering with A. parthenogenetica neurotransmission, through the inhibition of ChE.

Acute effects of 3,4-dichloroaniline on biomarkers and spleen histology of the common goby Pomatoschistus microps.

The aromatic amine 3,4-dichloroaniline (DCA) is a model environmental contaminant, precursor for synthesis and degradation product of several herbicides, which is commonly found in European estuarine ecosystems. In this work, the possibility of using biochemical and histological markers to assess sub-lethal effects of DCA in natural populations of Pomatoschistus microps juveniles was investigated. Alterations on the activities of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and glutathione S-transferase (GST) and histological alterations on spleen were investigated after 96 h of exposure to sublethal concentrations of DCA (0.50-1.49 mg/l). At the concentrations tested, DCA had no effect on AChE activity. LDH and GST activities were significant altered in treated animals when compared to control groups. As already described for mammals, DCA induced splenic histological alterations in P. microps, including expansion of red pulp and deposition of hemosiderin granules in a concentration-dependent manner. This suggests that DCA is a xenobiotic of concern in estuaries receiving agricultural effluents.

Environmental pollution and natural populations: a biomarkers case study from the Iberian Atlantic coast.

The degradation of estuaries is a result of human activities which overloads the environment with substances of both industrial and/or natural origins. Bioindicators have been consistently used to interpret effects of contaminants in the environment. In this study, the use of biomarkers (particular measurable characteristics of a bioindicator organism) was used to evaluate the contamination by xenobiotics of Crangon crangon natural populations. The central aim was to evaluate the capability of a battery of biomarkers to discriminate sites with different types of contamination. The activity of the enzymes cholinesterases (ChE), lactate dehydrogenase (LDH) and glutathione S-transferases (GST) were used as biomarkers. In addition, the ChE form(s) present in the cephalotorax of C. crangon were characterised. Organisms were seasonally sampled from winter 2001/2002 to autumn of 2002, at "reference" sites and at sites that receive agricultural, industrial and/or urban effluents. Results obtained in the characterisation of ChE with different substrates and selective inhibitors demonstrate that the form of ChE present in the cephalotorax of C. crangon shows proprieties of vertebrates' AChE and therefore it may be classified as true AChE-like ChE. The battery of biomarkers exhibited seasonal and local variations, apparently related to agricultural, industrial or urban effluent contamination. The tested biomarkers proved to be able to discriminate sources of environmental contamination, and confirms C. crangon as a sensitive species suitable to be used as a bioindicator.

In vivo evaluation of three biomarkers in the mosquitofish (Gambusia yucatana) exposed to pesticides.

In this study, the acute toxicity and the in vivo effects of commercial chlorpyrifos, carbofuran and glyphosate formulations on cholinesterase (ChE), glutathione S-transferase (GST) and lactate dehydrogenase (LDH) activities of the mosquitofish (Gambusia yucatana) were investigated. In a first phase of the study, head and muscle ChE were characterized with different substrates (acetylthiocholine iodide, s-butyrylthiocholine iodide and propionylthiocholine iodide) and the selective inhibitors eserine hemisulfate, 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284C51), and N,N'-diisopropylphosphorodiamic acid (iso-OMPA). The results obtained suggest that the enzyme present in both head and muscle of G. yucatana is mainly acetylcholinesterase (AChE). Acute toxicity was evaluated by exposing fish to several concentrations of single pesticides and of a mixture of chlorpyrifos/glyphosate. LC50 values were determined after 96 h of exposure, except in the case of carbofuran for which LC50 was calculated after 24 h since almost all the fish died within this period. LC50 values were 0.085 mg/l for chlorpyrifos, 17.79 mg/l for glyphosate, 0.636 mg/l for carbofuran and 0.011 mg/l for the chlorpyrifos/glyphosate mixture. A Toxic Unit approach was used to compare the toxicity of chlorpyrifos and glyphosate when occurring in a mixture with their toxicities as single compounds. Synergistic effects of chlorpyrifos and glyphosate when present in a mixture were found. At the end of each bioassay (24 h for carbofuran, 96 for the other substances/mixture), effects on biomarkers were analyzed. Muscle LDH activity was not altered by any of the three pesticides tested. Gill GST activity was significantly inhibited (40%) by carbofuran after 24 h of exposure to concentrations equal or higher than 0.06 mg/l. ChE muscle and head activity were significantly inhibited (50% and 30%, respectively) by carbofuran at concentrations equal or higher than 0.25 mg/l. Chlorpyrifos induced a significant inhibition of both muscle and head ChE (80% and 50%, respectively) after 96 h of exposure to concentrations equal or higher than 0.05 mg/l. Carbofuran did not induce significant alterations of fish ChE. The ChE EC50 determined for chlorpyrifos/glyphosate mixture (0.070 mg/l) was higher than the correspondent value calculated for chlorpyrifos alone (0.011 mg/l) suggesting an antagonistic effect of glyphosate on ChE inhibition by chlorpyrifos. ChE activity of G. yucatana seems to be a good biomarker to diagnose the exposure of wild populations of this species exposed to anticholinesterase pesticides.

Quantitative concentration-toxicity relationship for the injury of rat thymocytes by chemical compounds used in inter-laboratory toxicity ring-tests.

Rat thymic lymphocytes were used to determine the cytotoxic effect of the two toxicants, 3,4-dichloroaniline (DCA) and sodium bromide (NaBr), at concentrations that have effects in the 21-day reproduction test with Daphnia magna. To evaluate the effect of the chemical compounds on cell membrane integrity, we measured the permeability of the thymocytes to trypan blue and the leakage of lactate dehydrogenase (LDH) from the cells. The viability and metabolic activity of the thymocytes were quantified by the MTT assay. The DCA concentrations tested (from 31 to 310 nm) did not affect significantly the viability of thymocytes, as determined by trypan blue exclusion or by LDH leakage; however, with the MTT assay we could detect cytotoxicity at 62 nm. Both the trypan blue assay and the LDH assay indicated that concentrations of NaBr above 50 mm significantly affected the viability of the thymocytes. However, the MTT assay revealed a more pronounced toxic effect of NaBr, with significant cell damage being detected at 1 mm.

Glutathione-S-transferase activity of Fucus spp. as a biomarker of environmental contamination.

Coastal zones are important areas from both ecological and economical points of view. However, in the last decades, in several regions of the globe, they have been increasingly impacted by complex discharges of contaminants and by marine traffic accidents. The Portuguese Atlantic coast is particularly exposed to these contaminants due to the proximity of important navigation routes. Several rocky shore organisms have been tested and used as bioindicators of environmental contamination. However, to the best of our knowledge Fucus spp., which are key species in rocky shore communities, have not been used as bioindicators in monitoring studies based on biomarkers. The objective of this study was to investigate the potential of glutathione-S-transferase (GST) activity of several Fucus species (Fucus ceranoides, Fucus spiralis var. platycarpus, Fucus spiralis var. spiralis and Fucus vesiculosus var. vesiculosus) to discriminate sites with different contamination levels along the Portuguese Northwestern coast, between the Minho river estuary and the Aveiro's Lagoon, as an environmental biomarker. With the exception of F. spiralis var. spiralis, for which a confusing pattern of activity was found requiring further analysis, all the other species and varieties showed higher GST levels in more contaminated sites than in less contaminated ones, indicating that Fucus spp. are suitable for use as bioindicators and their GSTs as biomarkers of environmental contamination in coastal zones and estuaries.