Abstract: Enchytraeids are important organisms of the soil biocenosis. They improve the soil pore structure and the degradation of organic matter. These organisms are used in standardized testing, using survival and reproduction (6 weeks) as endpoints. The use of biomarkers, linked to ecologically relevant alterations at higher levels of biological organization, is a promising tool for Environmental Risk Assessment. Here, enchytraeids were exposed for different time periods (two days and three weeks) to different soils (OECD artificial soil, different compositions in its organic matter, clay or pH value, and LUFA 2.2 natural soil) and different chemicals (Phenmedipham and copper). The main question addressed in the present study was if the effects of chemicals and different soil properties are preceded by alterations at the sub-cellular level, and if these endpoints may be used reliantly as faster screening tools for the assessment of different stress conditions in soils. The parameters measured in E. albidus whole body were: lipid peroxidation (LPO), total glutathione (TG), as well as the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST). The results showed that biomarker responses in E. albidus were significantly affected by the soil type (GST, CAT, GPx, GR and LPO) and the duration of exposure in OECD artificial soil (GST, GPx, GR, CAT and LPO) but not in LUFA 2.2 natural soil. For the abiotic factors studied, after 2 days, low pH decreased significantly the TG levels and the activities of CAT and GR,and low OM also significantly decreased CAT and GR activities. After 3 weeks, differences in soil properties caused a decrease in GR and GPx activities, whereas increased GST activity was observed due to low organic matter and pH. Copper significantly increased the activities of CAT, GPx and GR, and decreased the activity of GST after 2 days as well as inscreasing LPO levels after 3 weeks. Phenmedipham increased LPO levels, associated with increased levels of TG as well as increased activities of CAT and GPx and decreased GST activity after 3 weeks exposure. This study shows that both abiotic and chemical stresses could be followed through biomarker analysis and that some of these determinations are potential endpoints in a quick soil contamination assessment procedure.

Abstract: Environmental factors affecting aquatic invertebrate communities were assessed using Daphnia magna in situ bioassays and biological indices based on community assemblages of benthic macroinvertebrates. Investigations were carried out in two heavily industrialized and urbanized river basins from the NE of Spain (Llobregat and Besós). Measures of energy consumption (i.e. algal grazing), and of specific biochemical responses (biomarkers) were conducted on individuals transplanted upstream and downstream from effluent discharges of sewage treatment plants. In both rivers there was a clear deterioration of the ecological water quality parameters and benthic communities towards downstream reaches. In all but one of the 19 locations studied, transplanted organisms were affected in at least one of the five measured responses. In three of them, significant effects were detected in most of the traits considered. Principal Component and Partial Least Square Projections to Latent Structures regression analyses indicated that the measured responses in D. magna in situ bioassays and those of macroinvertebrate assemblages were affected by distinct environmental factors. From up to 20 environmental variables considered, seven of them including habitat degradation, suspended solids, nitrogenous and conductivity related parameters affected macroinvertebrate assemblages. On the other hand, levels of organophosphorus compounds and polycyclic aromatic hydrocarbons were high enough to trigger the responses of D. magna in situ bioassays. These results emphasize the importance of combining biological indices with biomarkers and more generalized and ecologically relevant (grazing) in situ responses to identify ecological effects of effluent discharges from sewage treatment plants in surface waters.

Abstract: Environmental contamination is often characterised by a combination of stress factors of various sources (biological, physical and chemical). The predictability of their joint effects is an important stage in environmental risk assessment procedures. In this study, the two main conceptual models for mixture evaluation based on the effect of individual compounds, concentration addition (CA) and independent action (IA) and deviations to synergism/antagonism, "dose ratio" and "dose level" dependency were used. The single and combined effects of cadmium, carbendazim and low dissolved oxygen levels were assayed for life-cycle parameters (survival and feeding) of the water flea Daphnia magna Straus. The results of single exposures revealed an increase of acute and chronic toxicity as concentrations of cadmium and carbendazim increases. At low dissolved oxygen levels both survival and feeding parameters were significantly affected (P< or =0.05). In the acute mixture exposure of cadmium and carbendazim a "dose ratio" dependency was observed with a higher toxicity when cadmium was dominant whereas at high concentrations of carbendazim a lower effect on survival was observed. At chronic exposures an antagonistic deviation from IA model was observed for this mixture. The IA model showed to be adequate for toxicity prediction on acute exposure combinations with low DO levels where a synergistic behaviour was observed. However, at sublethal exposures IA and CA models failed by underestimation. Validation from toxicokinetic and toxicodynamic modelling studies should be made in the future as a way to understand toxicological pathways involved in complex mixture/combination exposures.

Abstract: In this study we evaluate the ability of an in situ bioassay with Chironomus riparius larvae, using larval development and growth as endpoints, to biomonitor water quality and to assess the biological recovery of metal contaminated freshwater ecosystems of mine areas that are subject of restoration measures. The bioassay was carried out in streams located near an abandoned goldmine in North Portugal, throughout an environmental rehabilitation of the mine (2002-2004). During this period, a decrease in the inhibition of larval growth in the metal contaminated stream was observed. The bioassay was also performed in streams located near an active tungsten mine in Central Portugal. Larval growth and development were highly inhibited in the stream that receives acid drainage from the tungsten mine and treated water from the AMD treatment station. The results indicate that the bioassay can be used to evaluate the efficiency of environmental restoration measures in mining areas.

Abstract: The present study examined how subcellular partitioning of Cd in plants with different strategies to store and detoxify Cd may affect trophic transfer of Cd to the isopod, Porcellio dilatatus. The plant species used were Lactuca sativa, a horticultural metal accumulator species; Thlaspi caerulescens, a herbaceous hyperaccumulator species; and the non-accumulator, Thlaspi arvense. Taking into account that differences in subcellular distribution of Cd in plants might have an important role in the bioavailability of Cd to a consumer, a differential centrifugation technique was adopted to separate plant leaf tissues into four different fractions: cell debris, organelles, heat-denatured proteins (HDP) and heat-stable proteins ([HSP], metallothionein-like proteins). Plants were grown in replicate hydroponic systems and were exposed for 14 d to 100 muM Cd spiked with (109)Cd. After a 14-d feeding trial, net assimilation of Cd in isopods following consumption of T. caerulescens and T. arvense leaves reached 16.0+/-2.33 and 21.9+/-1.94 mug/g animal, respectively. Cadmium assimilation efficiencies (AE) were significantly lower in isopods fed T. caerulescens (10.0+/-0.92%) than in those fed T. arvense (15.0+/-1.03%). In further experiments, Cd AEs were determined among isopods provided with purified subcellular fractions of the three plants. On the basis of our results, Cd bound to HSP was the least bioavailable to isopods (14.4-19.6%), while Cd bound to HDP was the most trophically available to isopods (34.4-52.8%). Assimilation efficiencies were comparable in isopods fed purified subcellular fractions from different plants, further indicating the importance of subcellular Cd distribution in the assimilation. These results point to the ecological relevance of the subcellular Cd distribution in plants, which directly influence the trophic transfer of Cd to the animal consumer.

Abstract: The drainage of abandoned mines leads to several ecological problems, particularly the acidification of surface freshwater systems and heavy metal contamination. In order to study the possibility of using the behavioural early warning responses of Portuguese indigenous benthic invertebrates to detect an acute short-term pulse of Acid Mine Drainage (AMD), experiments with the Multispecies Freshwater Biomonitor MFBtrade mark were performed and locomotion and ventilation were measured as endpoints. AMD was collected from the "São Domingos" mine (Southeast Portugal) and the following species were selected: Echinogammarus meridionalis (Pinkster, 1973), Hydropsyche pellucidula (Curtis, 1834) and Choroterpes picteti (Eaton, 1870). For simulating the pulsed exposure, AMD was added to river water where invertebrates were collected and pH was lowered until reaching 3.5. The effects of H(+) and heavy metals were discriminated using HCl positive controls. In addition to behaviour, mortality was registered. E. meridionalis was the most sensitive species in terms of mortality and behavioural endpoints, followed by C. picteti and H. pellucidula. E. meridionalis early warning responses consisted of increased locomotion with subsequent increase in ventilation, whereas for C. picteti only an increase in locomotion was observed. H. pellucidula showed no early warning responses. This work demonstrates the suitableness of using benthic invertebrates' behavioural early warning responses for detecting spikes of pollutants like AMD.

Abstract: Endosulfan sulphate is the transformation product of endosulfan and it is the most frequent form of surface water contamination with endosulfan. The aim of this study was to evaluate the possible effects promoted by endosulfan sulphate in changes on the life cycle, embryo development and sex differentiation of Daphnia magna. The endpoints used were moulting frequency, fecundity, growth, developmentally abnormalities and sexual differentiation. The nominal concentrations of endosulfan sulphate tested ranged from 9.2 to 458.7mugL(-1). Endosulfan sulphate promoted a significant decrease of the offspring number in all concentrations. Results showed a reduction of the size of females, together with a decrease in moulting frequency. Furthermore, an increase in embryo deformities was observed at all concentrations tested. Above a concentration of 91.7mugL(-1) there was an increased production of males. The results suggest that endosulfan sulphate interferes with the life cycle and sex determination of the crustacean D. magna.

Abstract: The present study provides the first data on inorganic element levels (As, Cd, Co, Cr, Cu, Hg, Mn, Pb, Se, and Zn) in juvenile, immature, and adult razorbills (Alca torda) collected along the central coast of Portugal. Element concentrations were assessed by ICP-MS in kidney, liver, muscle, and feathers of 28 razorbills, including 4 juveniles, 17 subadults, and 7 adults. The effect of age and tissue on element accumulation was also assessed. The detected levels in razorbills may indicate a possible contamination risk by Hg and Cr. With respect to bird tissues, higher accumulation of Se and Cd was detected in kidney, Zn and Pb in feathers, and As and Mn in liver. Age was found to affect the accumulation of Cd, Cr, Cu, Hg, and Mn, juveniles presenting higher levels of Cu and Mn than older individuals. In razorbill kidney, Zn-Hg constituted the most significant relationship among metal concentrations. Liver presented the highest number of significant relationships (mostly involving Zn and Co). With regard to feathers, the most significant relationships involved Se, Zn, Cr and Cu concentrations. Positive linear relationships were detected among kidney, liver, and muscle, with emphasis on relationships involving Se and Hg, which may be indicative of similar accumulation/regulation mechanisms in those organs. Element concentrations are discussed in view of possible detoxification mechanisms in seabirds.

Abstract: An integrated multilevel phytoassessment of an acid mine drainage (AMD, pH range 3.3-6.8) in southern Portugal was performed. A 7-day phytotoxicity bioassay with the duckweed Lemna gibba (chlorosis, necrosis, growth) was carried out, both in the laboratory and in situ, combined with an analysis of the resident epilithic diatom community. The toxicity test was performed with water from the AMD gradient, an unpolluted river control and acidified control water, in order to discriminate potential pH-effects from combined pH- and metal-effects. Diatom communities discriminated well among the sites (alkalophilic species versus halobiontic, acidobiontic and acidophilic species), showing inter-site differences to be larger than intra-site seasonal variations. In L. gibba exposed to AMD, necrosis and growth inhibition were higher in situ compared to the laboratory experiments. L. gibba was more sensitive to AMD than to acidified water. Already after 4 days, growth rate inhibition in L. gibba proved to be a reliable indicator of AMD-stress. Ecotoxicological thresholds obtained with L. gibba corresponded with those obtained previously with animals of intermediate tolerance to AMD. The results were summarised in a multimetric index.

Abstract: Enchytraeids (Enchytraeus albidus) directly improve the pore structure of the soil and are indirectly involved in regulating the degradation of organic matter. Due to their behavior they are able to avoid unfavorable environmental conditions. Avoidance tests allow a first assessment of toxicity of a contaminated or spiked soil within 48 h, by using the reaction of the enchytraeids as measurement endpoint. In this period, the organisms can choose between the control soil and the test soil. In the tests reported here, enchytraeids were exposed to LUFA 2.2 soil spiked with the following set of toxic substances: copper chloride, zinc chloride, cadmium chloride, phenmedipham, benomyl, carbendazim, dimethoate, atrazine, pentachlorophenol, chlorpyriphos, lindane, TBTO, Linear Alkylbenzene Sulfonates (LAS) and boric acid. Different chemical concentrations were tested. EC50s ranged from 8 mg/kg (Carbendazim) to >1000 mg/kg (e.g. LAS). While the tested heavy metals showed clear dose-response relationships, the effect pattern differed considerably in the tests with organic chemicals, e.g. no avoidance behaviour was observed in LAS, even at very high doses. Here we proposed to standardize the Enchytraeid avoidance test in a way similar to what is currently done for the earthworm and collembolan avoidance tests by the International Standardisation Organisation (ISO).

Abstract: 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.

Abstract: The aims of this study were to verify the suitability of in situ tests using the tropical midge Kiefferulus calligaster and to evaluate the most sensitive endpoint for the assessment of aquatic pesticide contamination. In situ tests were carried out in freshwater drainage channels (farm channels) that supply vegetable crops and receive considerable pesticide spray drift, and at channels outside farms (main channels). Moreover a pesticide-free farm was used as reference site. The endpoints analysed were: survival of the larvae, body length increment, capsule width increment, cholinesterase activity and glutathione S-transferase activity. Seasonal change was investigated as rainy season and dry season. Deleterious effects were observed at some farms especially during the rainy season when farmers apply heavier doses of pesticides. However, high mortality rates observed in main channels suggest that these water bodies are also affected by other impacts besides pesticide use. This work shows the potential of the in situ assay with K. calligaster as a tool for the environmental quality assessment of tropical aquatic ecosystems.

Abstract: 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.

Abstract: Steroid compounds have been globally detected in surface waters. The ecological impacts of these biologically active chemicals are largely unknown. Toxicity of testosterone and 4-hydroxyandrostenedione was assessed for the freshwater cladoceran Daphnia magna. Acute toxicity tests showed that 6.20mgL(-1) of testosterone, the highest concentration tested, did not have effect on the daphnids, whereas 4-hydroxyandrostenedione had an EC(50) of 4.26mgL(-1). Chronic toxicity tests were carried out using survival, body length, fecundity, and fertility as endpoints. Long-term testosterone exposure reduced D. magna fecundity and fertility at concentrations ranging from 0.31 to 2.48mgL(-1). The significant decrease in fecundity was associated with an increase in aborted eggs. Long-term 4-hydroxyandrostenedione exposure at 0.84mgL(-1) increased the mortality of the neonates. The chronic toxicity effects were observed at concentrations higher than the measured environmental concentrations of these compounds. Nevertheless, the reproductive impairment of the daphnids is likely to occur at environmental levels as an ultimate response to long-term exposure.

Abstract: In this study, a tree (Populus nigra L.) has been presented as data logger of mercury release in aquatic environments using tree rings chemistry to provide chronological historical monitoring of mercury discharge from a chlor-alkali industrial effluent to a coastal lagoon. Tree rings (Populus nigra L.) as mercury data logger is suggested by mercury accumulation trends in the tree rings reflecting the industrial plant capacity increments in the early stages of mercury discharges and enhancing industrial plant controls to minimize mercury discharges in the last two decades after imposed global regulations on mercury emissions.

Abstract: The present study provides the first data on inorganic element levels in juvenile, subadult, and adult Atlantic gannets (Morus bassanus). Physiological and potentially toxic elements (As, Cd, Co, Cr, Cu, Hg, Mn, Pb, Se, and Zn) were assessed by ICP-MS in kidney, liver, muscle, and feathers of 31 gannets, including 18 juveniles, 7 subadults, and 6 adults. The effect of age and tissue on element accumulation was also assessed. Mercury was roughly above the minimum level for adverse effects in birds. A higher accumulation of Se and Cd was detected in kidney, Pb in feathers, and Mn in liver. Age was found to affect the accumulation of Cd, Co, Hg, Mn, Se, and Zn. Adults presented significantly lower levels of Mn, Se, and Zn than subadults. Linear positive relationships within tissues were detected involving Se-Cd and Se-Hg. Also, positive linear relationships were detected among kidney, liver, and muscle, with emphasis on relationships involving Cd, Hg, Se, and Zn, which may be indicative of analogous regulation mechanisms in those organs. Atlantic gannets occurring in the study area leave their reproduction sites as juveniles. During their development process, several molting cycles occur and thus the possible contamination risk by Hg should reflect levels in the development areas rather than contamination levels in reproduction areas. The present study provides basic information on multielement accumulation in Morus bassanus, which may help us to understand the behavior and toxicity of various elements in marine birds.

Abstract: Ecotoxicological effects of the herbicide atrazine and the insecticides endosulfan sulphate and chlorpyrifos were evaluated using a test battery comprising aquatic organisms from different trophic levels. According to the categories established in the EU legislation, atrazine can be considered non-harmful for the species tested, while the insecticides can be considered very toxic for the crustaceans. The results of acute toxicity tests showed that the sensitivity of organisms were as follows: Thamnocephalus platyurus > Daphnia magna > Vibrio fischeri. Chlorpyrifos may act as a toxic compound in the aquatic environment of Guadiana River, as it may be detected in water at levels that promote toxic effects.

Abstract: Enchytraeids are ecologically relevant soil species and are commonly used in standardized toxicity tests. Their rapid reaction to a chemical exposure can be used as a toxicological measurement endpoint that assesses the avoidance behavior. The objectives of this investigation were to determine the effects of soil properties on the avoidance behavior of Enchytraeus albidus and to optimize the duration of avoidance test. The avoidance tests included (1) exposures in OECD artificial soil with standard or modified properties (pH, clay or peat content), and (2) exposures to copper chloride, cadmium chloride, and to the organic pesticides dimethoate and phenmedipham for different time periods. Results showed that alteration of OECD soil constituents significantly affected the avoidance behavior of enchytraeids, and that the 48-h exposure was the optimal duration of the test. Consideration of soil properties is important for selecting appropriate experimental design and interpreting the results of the enchytraeid avoidance test.

Abstract: 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.

Abstract: Following the development of urban and industrial centres petrochemical products have become a widespread class of contaminants. The aim of this study was to investigate the effects of petrochemical contamination in wild populations of mussels (Mytilus galloprovincialis) along the NW Atlantic coast of Portugal by applying antioxidant and energetic metabolism parameters as biomarkers. For that, mussels were collected at five sampling sites presenting different petrochemical contamination levels. To evaluate the mussels' antioxidant status, enzymatic activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione S-transferases, as well as glutathione redox status were evaluated in gills and digestive glands of mussels collected from the selected sites. Lipid peroxidation was determined in the same tissues to quantify cellular oxidative damage. Furthermore, to investigate how energetic processes may respond to these contaminants, the activity of NADP(+)-dependent isocitrate dehydrogenase was determined in mussels' digestive glands, and octopine dehydrogenase was determined in mussels' posterior adductor muscle. Furthermore, the concentrations of aliphatic hydrocarbons, unresolved complex mixture and polycyclic aromatic hydrocarbons (PAHs) were quantified in mussels' tissue, and abiotic parameters were quantified in water samples collected at each site. Several biomarkers showed statistically significant differences among sampling sites. The redundancy analysis (RDA) used to perform the integrated analysis of the data showed a clear separation of the sampling sites in three different assemblages, which are in agreement with the PAHs levels found in mussels tissues. In addition, the RDA indicated that some of the selected biomarkers may be influenced by abiotic parameters (e.g. salinity, pH, nitrates and ammonia). The approach selected for this study seems to be suitable for monitoring petrochemical contamination.

Abstract: A bioassay with Chironomus riparius larvae, using larval development and growth as endpoints, was carried out inside a rice field and in the adjacent wetland channel in Portugal, during pesticide treatments (molinate, endosulfan and propanil) to determine impact caused by pesticide contamination in freshwater ecosystems. The bioassay was also performed under laboratory conditions, to assess whether in situ and laboratory bioassays demonstrated comparable results. Growth was inhibited by concentrations of endosulfan (2.3 and 1.9 microgL(-1) averages) in water from rice field in both the field and laboratory, and by concentrations of endosulfan (0.55 and 0.76 microgL(-1) averages) in water from the wetland channel in the laboratory bioassay, while development was not affected. C. riparius larvae were not affected by molinate and propanil concentrations. The results indicate that endosulfan treatments in rice fields may cause an ecological impairment in adjacent freshwater ecosystems. The results also indicate that laboratory testing can be used to assess in situ toxicity caused by pesticide contamination.

Abstract: This study was conducted to investigate the potential use of biomarkers in bioassays with chironomids to assess contamination by pesticides in temperate and tropical climates. Two species of midge were studied, the widespread Chironomus riparius and the tropical Kiefferulus calligaster (Kieffer, 1911). Preliminary studies included investigations of the effects of temperature on larval development and the influence of larval age on normal variability of cholinesterase (ChE) and glutathione S-transferase (GST) activities and protein content. In the second phase, the influence of two abiotic factors particularly important in tropical conditions (temperature and oxygen concentration) and of the organophosphorous (OP) insecticide dimethoate on biochemical and conventional endpoints was investigated. Results showed that K. calligaster is morphologically and physiologically similar to C. riparius and for both, the time of larval development decreases with the increase of temperature. Moreover, 3rd and 4th instars appeared to be the most suitable for biomarkers determinations. ChE activity seems to be valuable biomarker regarding temperature and dissolved oxygen (DO) variations, while some caution should be taken when using GST as an environmental biomarker, since it shows some dependence of these parameters. C. riparius was more sensitive to dimethoate than K. calligaster suggesting that the use of bioassays with the former species in tropical conditions may overestimate the toxicity of OP pesticides to autochthonous species. When testing sub-lethal effects of dimethoate to C. riparius, ChE activity showed to be a very sensitive parameter detecting significant effects at the lowest concentration that caused emergence delay of larvae, suggesting that it is an ecologically relevant parameter.

Abstract: Resistance to fenitrothion and enzyme activities associated with the toxicity and metabolism of organophosphorus insecticides were measured in three genetically unique Daphnia magna clones collected from rice fields of Delta del Ebro (NE Spain) during the growing season and a lab sensitive clone. The studied clones showed up to sixfold differences in resistance to fenitrothion. The lack of correlation between in vitro sensitivity of acetylcholinesterase (AChE) to fenitrooxon and resistance to fenitrothion indicated that insensitivity of AChE to the most active oxon metabolite was not involved in the observed differences in resistance. Inhibition of mixed- function oxidases (MFOs) by piperonyl butoxide (PBO) increased the tolerance to fenitrothion by almost 20-fold in all clones without altering their relative ranking of resistance. Conversely, when exposed to fenitrooxon, the studied clones showed similar levels of tolerance, thus indicating that clonal differences in the conversion of fenitrothion to fenitrooxon by MFOs were involved in the observed resistance patterns. Despite that resistant clones showed over 1.5 higher activities of carboxilesterase (CbE) than sensitive ones, toxicity tests with 2-(O-cresyl)-4H-1,3,2-benzodioxaphosphorin-2 oxide, which is a specific inhibitor of these enzymes, evidenced that this system only contributed marginally to the observed clonal differences in tolerance. Glutathione-S-transferases activity (GST) varied across clones but not under exposure to fenitrothion, and was only related with tolerance levels in the field clones. In summary, our results indicate that MFO mediated differences on the bio-activation of the phosphorotionate OP pesticide to its active oxon metabolite contributed mostly in explaining the observed moderate levels of resistance, whereas the activities of CbE and GST had only a marginal role.

Abstract: Mercury has been considered as one of the most important pollutants in coastal and estuarine areas. Efforts have been made to detect, as early as possible, the effects of this and other metals in several species. Macroalgae, particularly Fucus spp., have been widely used as biomonitors of metal pollution. In this study, three Fucus species (F. spiralis, F. vesiculosus and F. ceranoides) were collected from several sampling sites in Portugal. The concentrations of mercury were determined in three structural parts (holdfast, stipe and receptacles). Two different techniques were used to determine mercury concentrations. Almost all mercury concentrations (in sediments and in water) were below national and international standards. Mercury concentration in the specimens (0.012-0.061 microg g(-1) for receptacles, 0.028-0.221 microg g(-1) for stipe and 0.029-0.287 microg g(-1) for holdfast) was always higher that those obtained for the sediment (0.001-0.112 microg g(-1)). With few exceptions the contrary was found for receptacles. In general, a good agreement between concentrations of mercury in sediment and Fucus was found. The results indicate that Fucus accumulate mercury and may be a suitable species for use in risk assessment for coast and estuarine areas, by providing valuable information regarding the levels of mercury that will be available for the consumers of Fucus spp.

Abstract: In aquatic environments, many factors determine the overall impact of a toxic event, constituting "exposure scenarios" that must be understood so that risk assessment strategies can be successfully applied. In this work, an experimental design was planned to investigate the influence of the organism age, duration of exposure and subsequent recovery period (after cessation of toxic exposure) on chlorpyrifos or carbendazim toxicity to Kiefferulus calligaster. The endpoints studied were cholinesterase (ChE) activity and glutathione S-transferase (GST) activity. The influence of age and exposure time and the existence of recovery depend on the chemical and the biomarkers tested, suggesting that no general rule can be applied when trying to assess overall toxicity of pesticides and that detailed knowledge has to be acquired on a case-to-case basis.

Abstract: Two prevailing theoretical models: concentration addition (CA) and independent action (IA), predict mixture toxicity on the basis of known toxicities of the mixture components. To date, both models have been in most occasions evaluated using unicellular in vivo responses or biochemical in vitro responses. However, when considering more complex models such as the whole organism physiology or life-history traits, the dominant ecotoxicological mode of action, based on the exposure concentrations at which various toxicological effects become operative at the level of whole organism, should be considered. Offspring production in Daphnia magna is driven by the resources acquired from food, and the number of live offspring produced by an organism is the result of two independent factors: the number of eggs produced and the percentage of eggs that survive egg development. In this study joint toxicity effects on offspring production in D. magna were tested using binary mixtures of toxic contaminants known to specifically impair food acquisition (lambda-cyhalothrin and cadmium) or to cause egg mortality during development (3,4 dichloroaniline). Tests were performed using a simplified 10-day reproduction assay initiated with gravid females. The results obtained indicate that irrespective of their primary pharmacological mode of action, the joint toxicity of cadmium and lambda-cyhalothrin was predicted by the CA model, whereas the joint toxicity of cadmium and 3,4 dichloroaniline was predicted by the IA model. Thus, the results obtained indicate that for life-history traits such as reproduction responses, the dominant ecotoxicological rather than the pharmacological mode of action should be used for predicting joint mixture effects.

Abstract: We used bioassays employing head capsule width and body length increase of Chironomus riparius larvae as end points to evaluate metal contamination in streams. Bioassays were performed in situ near an abandoned Portuguese goldmine in the spring of 2003 and 2004. Bioassays also were performed under laboratory conditions with water and sediment collected from each stream to verify if laboratory bioassays could detect in situ toxicity and to evaluate the relative contribution of sediment and water to overall toxicity. We used field sediments with control water and control sediments with field water to discriminate between metal contamination in water and sediment. Field water with dry and sieved, organic matter-free, and nontreated sediments was used to determine the toxicity of heavy metals that enter the organism through ingested material. In both in situ and laboratory bioassays, body length increase was significantly inhibited by metal contamination, whereas head capsule width was not affected. Body length increase was more affected by contaminated sediment compared to contaminated water. The lowest-effect level of heavy metals was observed in the dry and sieved sediment that prevented ingestion of sediment particles by larvae. These results suggest that body length increase of C. riparius larvae can be used to indicate the impact of metal contamination in rivers. Chironomus riparius larvae are more affected by heavy metals that enter the organism through ingested sediment than by heavy metals dissolved in the water column. Nevertheless, several factors, such as the particle size and organic matter of sediment, must be taken into account.

Abstract: Tropical regions' economy is usually based on agriculture, which involves an intensive use, and even frequent overuse, of pesticides. Nevertheless, not much research has been done on the impact of pesticides on tropical aquatic ecosystems, which are often contaminated by runoff-related pesticide inputs due to unpredictable and torrential rainfalls. This study aimed to: (i) adapt and evaluate a short-term sublethal in situ assay using post-exposure feeding as an endpoint, to the tropical cladoceran species Diaphanosoma brachyurum (collected at the Pedra do Cavalo dam in the Paraguaçu River basin, Bahia, Brazil), and, (ii) assess the role of the standard species Daphnia magna as an adequate laboratory surrogate. Lethal and sublethal (post-exposure feeding) responses were assessed for the two species. To evaluate these responses under environmentally realistic exposure conditions, a runoff event was simulated in an agricultural area previously contaminated with different deltamethrin concentrations. The resultant runoff water was used to set up microcosms with different dilutions, simulating the entrance of runoff water in an adjacent lentic system. An in situ assay with D. brachyurum was performed inside the microcosms, allowing to discriminate the effects due to deltamethrin from those due to other potential stressors associated with the experimental design (e.g. organism handling, load of suspended particles, microcosm design). Water samples were collected from microcosms to conduct a laboratory assay with D. magna. The in situ methodologies revealed to be suitable to conduct assays with D. brachyurum under tropical conditions, since all exposed organisms were successfully retrieved from the chambers. Furthermore, none of the potential stressors associated with the experimental design influenced the daphnids' performance. The tropical cladoceran species, exposed under more realistic conditions, revealed to be more sensitive than the laboratory standard species: lethal effects were only observed for D. brachyurum and sublethal effects were noticed at a lower deltamethrin concentration for this species than for D. magna.

Abstract: As a result of mining activities the exposure of metal sulphides to oxidation takes place with consequent release of acid mine drainage (AMD). Biomonitoring instruments have been proven to have the best deterrent effect upon polluters. A new approach in online biomonitoring, with the Multispecies Freshwater Biomonitortrade mark (MFB), was developed combining behavioural and feeding responses of the Portuguese indigenous benthic shredder, Echinogammarus meridionalis (Pinkster, 1973) (Crustacea, Amphipoda). These endpoints, along with mortality, were measured and analyzed for a gradient of sublethal doses of AMD. Original river water was used as the control and three doses of treatments were attained by adding increasing volumes of AMD to the control. The increase in AMD concentration and concomitant decrease in pH and increase in the concentration of most metals was followed by an overall increase of the mortality, decrease of locomotion/feeding activity and inhibition of the feeding rate. Mortality was observed in the two highest concentrations of AMD. Significant decrease in average locomotion/feeding activity took place in the second treatment. Although an inhibition of feeding was observed along the gradient of AMD concentration only in the highest concentration the feeding rate was significantly reduced.

Abstract: Lethal and sublethal effects of Cadmium and Zinc on two freshwater crustaceans were investigated. The 96-h LC50 for Cadmium and Zinc were 42.20 microg l(-1) and 5.43 mg l(-1) for Atyaephyra desmarestii, and 36.17 microg l(-1) and 4.61 mg l(-1) for Echinogammarus meridionalis, respectively. An increase in metal concentrations at sublethal levels resulted in significant reductions of the feeding rate of both species. The LOECs in the chronic feeding assays for Zinc were 1.29 mg l(-1) for A. desmarestii and 0.4 mg l(-1) for E. meridionalis. For Cadmium the LOEC was 6.53 microgl(-1) for both species. The results suggest the potential use of these two crustacean species as test organisms in ecotoxicological tests. Feeding assays appear to be a rapid, cheap and effective tool to be used in biomonitoring studies of Portuguese freshwaters.

Abstract: Apart from analyses for elemental contaminants in field collected specimens, very little is known about the assimilation, accumulation and toxic effects of inorganic contaminants in reptiles. This study examined the chronic accumulation of Cd in a European lacertid lizard (Podarcis carbonelli) following dietary provision of an environmentally realistic concentration of Cd for 21 weeks. Lizards were provided with Cd that had either been biologically incorporated into crickets, or as Cd(NO3)2 added superficially to crickets just prior to feeding. Among both treatment groups Cd accumulated in tissues in the following order of concentration: gut>liver>kidney>carcass. The majority of the Cd was retained within the gut, and transfer to internal organs was low. Morphological indices, brain and plasma cholinesterase activities, gut and liver metallothionein content, and standard metabolic rate were measured as biomarkers of exposure and effect; however, no differences between control lizards and Cd-treated lizards were observed.

Abstract: 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.

Abstract: 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.

Abstract: Single species responses have the potential to measure impacts at earlier stages than more traditional methods based in community structure. This study evaluates a bioassay with biological (survival, development, growth) and functional (post-exposure feeding rate) responses of Chironomus riparius larvae to assess water quality and contamination in rivers. The bioassay with C. riparius third instar larvae was performed, in autumn and spring, in reference sites and in organic and metal contaminated sites in Portuguese rivers. Biotic, physical and chemical parameters were determined for each site. The relationship between both bioassays responses and biotic indices (IBMWP and IASPT) and the physical and chemical parameters of respective sites were determined. In general biotic indices were able to discriminate between contaminated and not contaminated sites although they demonstrated a poor ability to detect low level of metal contamination during autumn. IASPT was negatively related to ammonia concentrations in both seasons. No significant differences in survival and post-exposure feeding rate were found between sites. Development was inhibited in the most metal contaminated site during autumn, but pH and ammonia concentrations in water accounted for 82% of developmental variation during this season. Growth was highly inhibited in the most metal contaminated site during both seasons. In autumn, growth was also inhibited in the low metal contaminated site and, during this season, pH and Mn and Fe concentrations in water samples accounted for 97% of growth variation between sites. The results suggest that in situ bioassay with C. riparius larvae using growth as the endpoint is a responsive and suitable tool that can be used as bioindicator of metal pollution and to biomonitor water quality in metal contaminated rivers.

Abstract: Soil decomposition is mainly dependent on the nature and characteristics of organic matter within the soil, the environmental conditions and the activity of microorganisms and soil fauna. Isopods play an important role in decomposition through litter fragmentation and stimulating and/or ingesting fungi and bacteria. The aim of this study was to jointly evaluate the effect of different food types and the effect of heavy metal contamination of those foods through isopod feeding performance assays. These studies used the terrestrial isopod Porcellionides pruinosus. After feeding with different leaf types for the study on feeding performance, alder leaves were chosen for the contamination experiments. Feeding parameters like consumption, assimilation, egestion and growth ratios were calculated and compared among treatments and food type. Lower quality food decreased isopods performance. Exotic food types were shown to be less preferred than alder or oak leaves. Contaminated food also resulted in a decrease in performance among the feeding parameters studies, although isopods can tolerate in certain cases high amounts of heavy metals. For this reason it is possible that in future this crustacean can be used as bioindicators of soil contamination or in the evaluation of contaminated sites or remediation processes.

Abstract: Two different concepts, termed concentration addition (CA) and independent action (IA), describe general relationships between the effects of single substances and their corresponding mixtures allowing calculation of an expected mixture toxicity on the basis of known toxicities of the mixture components. Both concepts are limited to cases in which all substances in a mixture influence the same experimental endpoint, and are usually tested against a "fixed ratio design" where the mixture ratio is kept constant throughout the studies and the overall concentration of the mixture is systematically varied. With this design, interaction among toxic components across different mixture ratios and endpoints (i.e. lethal versus sublethal) is not assessed. In this study lethal and sublethal (feeding) responses of Daphnia magna individuals to single and binary combinations of similarly and dissimilarly acting chemicals including the metals (cadmium, copper) and the pyrethroid insecticides (lambda-cyhalothrin and deltamethrin) were assayed using a composite experimental design to test for interactions among toxic components across mixture effect levels, mixture ratios, lethal and sublethal toxic effects. To account for inter-experiment response variability, in each binary mixture toxicity assay the toxicity of the individual mixture constituents was also assessed. Model adequacy was then evaluated comparing the slopes and elevations of predicted versus observed mixture toxicity curves with those estimated for the individual components. Model predictive abilities changed across endpoints. The IA concept was able to predict accurately mixture toxicities of dissimilarly acting chemicals for lethal responses, whereas the CA concept did so in three out of four pairings for feeding response, irrespective of the chemical mode of action. Interaction effects across mixture effect levels, evidenced by crossing slopes, were only observed for the binary mixture Cd and Cu for lethal effects. The analysis of regression residuals showed that interaction effects across mixture ratios were restricted to feeding responses in binary mixtures that included Cu. These results indicate that the ability of the CA and IA concept to predict mixture toxicity effects varies from lethal to sublethal endpoints irrespective of their primary mode of action. This suggests that when considering complex responses, the pharmacological notion of mode of action should be extended to encompass an ecotoxicological mode of action, based on the concentration at which various toxicological effects become operative in the biological system under consideration.

Abstract: Terrestrial isopods have become important tools for the ecotoxicological assessment of metal-contaminated soils. Their value as an invertebrate model is partly because of their extraordinary capacity to bioaccumulate toxic metals from the environment. Replication of this accumulation process in the laboratory has in the past relied on the amendment of organic food substrates through the addition of inorganic metal salts. However, the bioavailability of the metals when presented through doping regimes may differ from the bioavailability of metals in nature, because over time metals become biologically compartmentalised and form complexes with organic molecules. This study examines the differential bioavailability of Cd to the terrestrial isopod, Porcellio dilatatus, when presented as either a Cd-amended diet or pre-incorporated biologically into lettuce (Lactuca sativa). Isopods were either provided with lettuce contaminated superficially with Cd(NO(3))(2) or lettuce grown hydroponically in growth media containing 100 microM Cd(NO(3))(2). Assimilation efficiency of Cd was greater among isopods that were fed the amended diet (71%, S.E.=7%), than among isopods feeding on biologically contaminated lettuce (52%, S.E.=5%) and demonstrates that speciation of Cd is likely to influence the rate of Cd assimilation and accumulation in a laboratory test.

Abstract: Short-term 48 h laboratory bioassays with water from an acid mine drainage (AMD: pH 3.3, 4.4, 5.0, 5.5, 6.4, control) and water from an arsenic containing reservoir were performed with the freshwater shrimp Atyaephyra desmaresti Millet, validated in situ and compared to acidified control water (ACID). Behaviour, mortality and time to death were monitored with the Multispecies Freshwater Biomonitor (MFB). The shrimps had equal 24 h-LC50s at pH 4 in AMD and ACID. However, after 48 h AMD proved more toxic (48 h-LC50 at pH 5.2) than ACID (48 h-LC50 at pH 4.5). Stress behaviour in AMD consisted at pH < or = 6.4 of a pH-dependent decrease in activity, with disappearance of circadian rhythmicity, and at pH 4.4 a clear increase of ventilation. At pH 5.5 bioaccumulation of metals was higher and locomotion lower than at pH 5.0. In ACID, only at pH < or = 4.4 locomotion became abated and arythmic. Locomotion in the field was equal or higher compared to the laboratory, whereas the ventilation was higher in the laboratory. A. desmaresti is a valuable species to be used in short term behavioural bioassays of AMD in Europe.

Abstract: Contaminants in soils can enter food chains through primary producers. Bioavailable contaminants can induce growth, and reproductive or biochemical changes in plants. To evaluate the bioavailability of heavy metals in two soils from Jales mine surroundings, bioassays with the plants Brassica rapa (RCBr) and Avena sativa were performed. Biochemical parameters (protein and malondialdehyde [MDA] content, and catalase and peroxidase activities) were also measured. The soils had different heavy metal contents: JNC soil contained low heavy metal concentrations, whereas JC soil had high heavy metal contents. Results stressed the difference between species sensitivity, with A. sativa showing no toxicity effects when exposed to both soils. On the other hand, B. rapa presented a decrease in growth parameters when exposed to JNC soil and no changes when exposed to JC soil. A Life Cycle Bioassay confirmed this trend for B. rapa exposed to JNC soil, but also evidenced that JC soil was affecting B. rapa in terms of flower and seed pod production. Biochemical assays showed that plants affected by heavy metals also displayed oxidative stress, with an increase in MDA production, reduction of protein content, and reduction of catalase and peroxidase activities. All bioassays revealed that JNC soil, although with a lower heavy metal content, had a higher bioavailable fraction when compared to JC soil, which consequently increased its toxicity to plants.

Abstract: Water quality and the contamination in relation to land use in the fluvial-lagoon system of the Palizada River (FLSPR; State of Campeche, Mexico) was investigated using an integrated approach including 21-d in situ bioassays with the native mosquito fish Gambusia yucatana, determination of cholinesterase (ChE) activity in wild populations of this species, and water-quality variables. The present study was performed over 19 months at three sites with different types of anthropogenic impact. Significant differences in a water-quality index for aquatic life (WQI) were found among sites. Fish mortality was significantly and negatively correlated with WQI, dissolved oxygen, and sulfates. High survival rates (> or =80%) were found in preliminary exposures and in some of the bioassays performed at all sites. Therefore, test chambers and the cabinet seemed to be suitable for use in toxicity bioassays with G. yucatana. The in situ bioassay was able to discriminate levels of water contamination in both time and space, indicating that it is suitable for use in conditions similar to those found in the FLSPR. In the biomonitoring study, a ChE inhibition of greater than 20% in wild fish was found in some periods of the year at all sampling sites. This indicates the presence of anticholinesterase agents in the water. Fish from two of the sites had a ChE inhibition of greater than 40% in some sampling months, suggesting that deleterious effects already may have been induced in fish. Furthermore, at these sites, the pattern of ChE inhibition was in good agreement with the probable runoff of pesticides from agricultural fields.

Abstract: Apart from analyses for elemental contaminants in field-collected specimens, very little is known about the assimilation and accumulation of inorganic contaminants in reptiles. Recent dietary studies with reptiles (and some other vertebrates and invertebrates) have taken care to incorporate the principles of trophic transfer by pre-exposing prey items to the elemental contaminant of interest. However, there are conflicting data in the literature as to whether biologically incorporated metals are more bioavailable to consumers in a food chain than simple salts added to the diet, and this study examines this issue in a lacertid lizard. Adult individuals of Podarcis carbonelli were exposed to cadmium (Cd) in a 21-week dietary study to determine whether the form in which the Cd is provided influences assimilation efficiency for this metal. Lizards were provided with Cd that had either been biologically incorporated into crickets or as Cd(NO3)2 added superficially to crickets just prior to feeding. Radiospectrometric analysis for 109Cd in animal tissues and fecal material was used to follow Cd accumulation over the duration of exposure. The highest levels of accumulation were found within the gut. This, combined with the observation of higher rates of assimilation over the first five weeks, strongly suggests a rapid accumulation of Cd within the gut tissue followed by a slower redistribution to other tissues. No statistically different levels of Cd assimilation efficiency were detected between the two treatment groups, nor were there any statistically significant differences between the two treatment groups in relation to the proportional distribution to the gut, liver, or kidneys.

Abstract:

Abstract: Rice is the main crop in the subbasin of the fluvial lagoon system of Palizada River (FLSPR) in the state of Campeche, Mexico. The pesticides used to control pests of this crop mainly are carbofuran, chlorpyrifos, and glyphosate. Black-bellied whistling duck (Dendrocygna autumnalis) is an ecologically and economically important species in the area. This duck is consumed by local inhabitants throughout the year, despite its potential exposure to pesticides. Due to its feeding habits, abundance, and nutritional value, D. autumnalis is a good indicator of environmental contamination and a potential route of human exposure to organophosphate and carbamate pesticides. In this study, the brain cholinesterase (ChE) in the frontal cerebral cortex of autochthonous ducks was characterized. In addition, the potential of the three locally used pesticides and mixtures to inhibit ChE activity was investigated and the exposure of the wild duck population during intensive pesticide applications in rice fields was evaluated. We found that acetylcholinesterase (AChE) seems to be the predominant ChE form in the biological fraction analyzed. Carbofuran was the most potent ChE inhibitor of D. autumnalis brain ChE activity from the three pesticides analyzed. Cholinesterase inhibition after exposure to pesticide mixtures predominantly was due to carbofuran. A decrease (p < 0.05) in AChE activity (>30%) was apparent in wild ducks compared to reference ducks, with recovery of ChE inhibition in wild ducks occurring months later when no pesticides were applied in the field. Dendrocygna autumnalis brain ChE is a suitable parameter for inclusion in biomonitoring programs for both environmental protection and human safety.

Abstract: Mesopodopsis slabberi is a euryhaline and suprabenthic mysid with a wide geographic distribution and one of the most important mysid species in coastal shallow waters. Nevertheless, no references were found to its uses in ecotoxicological experiments (TOXNET, AQUIRE, ISI Web of Knowledge). This work is part of an ecotoxicological study, representing the first step in establishing a description of this species' tolerance to chemical pollutants typical of estuarine environments. It is possible to assert that acclimation was achieved, for it occurred during a period of time, with a low mortality. Due to the short life cycle of M. slabberi it is strongly believed that 12 days of acclimation is a far too long time, when experiments are to be made after this period. This species is suitable for acclimation in the laboratory, tolerant, and easy of handling. Logistics and materials used to maintain the acclimation system as described are simple and not costly and could easily be used in other laboratories.

Abstract: Locomotor behavior in terrestrial organisms is crucial for burrowing, avoiding predators, food seeking, migration, and reproduction; therefore, it is a parameter with ecological relevance. Acetylcholinesterase (AChE) is a nervous system enzyme inhibited by several compounds and widely used as an exposure biomarker in several organisms. Moreover, changes in energy reserves also may indicate an exposure to a stress situation. The aim of this study is to link biomarkers of different levels of biological organization in isopods exposed to increasing doses of dimethoate in semifield conditions. Locomotor parameters, AChE activity, and energy reserves (lipid, glycogen, and protein contents) were evaluated in the isopod Porcellio dilatatus after 48-h and 10-d exposure to dimethoate-contaminated soil. Results showed a clear impairment of both locomotor and AChE activity during the entire study, although effects were more pronounced after 48 h. Most locomotor parameters and AChE activity showed a clear dose-response relationship. By contrast, no clear trend was observed on energetic components. A positive and significant relationship was found between AChE activity and those locomotor parameters indicating activity, and the opposite was observed with those locomotor parameters indicating confusion and disorientation. The results obtained in this study enhance the importance of linking biochemical responses to parameters with ecological relevance at individual level, the value of locomotor behavior as an important marker to assess effects of toxicants, and also the usefulness and the acquisition of ecological relevance by AChE as a biomarker, by linking it with ecologically relevant behavioral parameters.

Abstract: 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.

Abstract: Survival and behavior of the mayfly Choroterpes picteti (Leptophlebiidae) exposed to acid mine drainage (AMD: pH 3.3-6.4) and a reservoir polluted with arsenic (pH 6.8) from Sao Domingos mine (Portugal) were studied in laboratory and in situ bioassays (48 h) with the Multispecies Freshwater Biomonitor, and compared with water from a reference river and acidified reference water (acid only). Metal body-burdens showed a negative pH dependency for Mn and As, a positive one for Pb, and for Zn, Cu, Co, and Cd a decrease at pH < 4.4. Generally, survival decreased with decreasing pH. The 48-h LC(50) (pH) for AMD and for acid only were similar (pH 4.8-4.9); however, the LT(20) (h) at pH 3.3 revealed AMD to be less toxic than acid only. C. picteti show diurnal rhythm with increased locomotor activity in the night. The circadian rhythm was weakened by acid exposure, but less so by AMD exposure. Compared to reference river water, ventilation was stimulated at pH < 6.0 in acid only and in reservoir water. Locomotion was stimulated at pH 5 in acid only and reservoir; however, it was reduced in all other treatments, when compared to reference river water. Under acid-only exposure, both locomotion and ventilation were significantly higher compared to AMD exposure at the corresponding pH values. The laboratory results were field validated.

Abstract: This study proposes assays with a freshwater chironomid, Chironomus xanthus, distributed over South America, based on subindividual (acetylcholinesterase activity) and individual (survival and postexposure feeding) level endpoints. Sediment and aquatic-rooted floating macrophyte assays were developed, due to the importance of both substrates for toxicant exposure in subtropical/tropical environments. Assays were evaluated under realistic exposure scenarios by simulating a runoff over an agricultural field dosed with deltamethrin. In situ assays were performed within microcosms to discriminate the effects of deltamethrin from those of additional potential stress factors (organism handling and caging, microcosms, use of aquatic roots, and runoff per se). A laboratory sediment assay based on feeding was conducted with samples from the microcosms. In all assays, both sublethal endpoints were responsive to deltamethrin and more sensitive than survival. Survival and feeding were more sensitive in situ than in the laboratory. In the in situ sediment assays, both sublethal endpoints were within a similar range of sensitivity; they were significantly inhibited as of the lowest Decis dose, from approximately 20 to 70%. In situ feeding was more sensitive in the sediment than in the macrophyte assay, where it was inhibited significantly only at the two highest Decis doses (up to approximately 60%). Larval performance was not influenced significantly by any of the other potential stress factors.

Abstract: In recent years biomarkers have been widely used for the assessment of effects and/or exposure to environmental contaminants. One of the most frequently used biomarkers is the inhibition of cholinesterases (ChE), which is a useful indicator of organophosphate and carbamate exposure and/or effects. Recent studies indicated that more than one ChE may be present in tissues of fish and that different forms may vary in their sensitivity to anticholinesterase agents. Cholinesterase activity of the juvenile of the common goby (Pomatoschistus microps), a widespread fish in estuaries of the Atlantic coast of northwestern Europe, was characterized using four substrates (acetylthiocholine iodide, acetyl-beta-metylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulfate, BW284C51, and iso-OMPA) in different tissues of the fish head. In addition, the range of ChE activity that may be considered as "normal" for non-exposed P. microps was determined. The results suggest the presence of two types of ChE in the whole-head homogenate. The present study underscores the relevance of ChE characterization before its use as a biomarker in biomonitoring studies.

Abstract: Soil ecotoxicology studies are usually performed in standard soils such as Organization for Economic Cooperation and Development artificial soil or LUFA ST. 2.2, a natural soil. When assessing the toxic effects in the environment, soil properties are often different from those in standard soils, which might lead to different exposure situation for the test species and therefore to misleading conclusions. Selected to cover a broad range of properties and based on the Euro-Soils concept, 17 different soils were studied regarding their suitability to two tes species: Folsomia candida and Hypogastrura assimilis (Collembola). In reproduction tests, the test species reacted differently to the soils. F. candida was less affected by soil properties: 500 to 1200 juveniles/vessel were found in untreated soils (i.e. controls). These differences can be attributed to normal interindividual variability. H. assimilis showed a significant correlation with maximum water-holding capacity and also a tendency to lower the reproductive output in soils with a low pH (<4). Therefore, some soils were revealed to be inappropriate for tests with H. assimilis. In the main tests, the effect of the reference test substance Phenmedipham (formulation Betosyp) was studied in those soils where sufficient reproduction was determined beforehand. Clearly, the chronic end point was more sensitive than survival when testing Phenmedipham. In H. assimilis, because of high variability and low effects of tested dosages, no conclusions could be drawn. In F. candida, different soils caused different toxic effects: Juveniles preferred soils with high C-to-N ratios. Higher microbial activity might support a quicker metabolization of the test substance. In general, the toxic response is caused by a synergistic action of several soil properties with each of them exerting an effect too small to be clarified with the available set of data.

Abstract: Soil ecotoxicology studies are usually performed in standard soils, such as the OECD (Organization for Economic Cooperation and Development) artificial soil or the LUFA St. 2.2, a natural soil. When assessing the toxic effects in the environment, soil properties are often different from those in standard soils, which might lead to a different exposure situation for the test species and, therefore, to a different evaluation of the risk of the test substance. Selected to cover a broad range of properties and based on the Euro-Soils concept, 18 different soils were studied regarding their suitability to two test species: Enchytraeus albidus and Enchytraeus luxuriosus (Enchytraeidae). In reproduction tests, the test species reacted differently to the tested soils, but both enchytraeids did not survive in acid soils (i.e. pH<or=5). Results indicate that although a combined effect of soil properties seems to occur, certain soil properties are causing specific toxic effects. In the present study, EC50s in enchytraeids changed by a factor of 9 for juveniles and nearly 30 for the adults of E. luxuriosus (maximum values; slightly lower values were found for E. albidus), which shows how important the test soil can become for the environmental risk assessment of chemicals. More data using more soils and species are required to understand the effect of soil properties in soil toxicology. Nevertheless, it was clear that certain soil properties such as OM (Organic Matter) and WHC (Water Holding Capacity) or pH, CEC (Cation Exchange Capacity), C/N (Carbon/Nitrogen ratio) and clay content did interact with the chemical and the organisms.

Abstract: In the present study, the effect of the heavy-metal salt copper chloride (CuCl2.2H2O) in soils freshly spiked (3 d) and aged (70 +/- 10 d; mean +/- SD) was studied in the test species Enchytraeus albidus, E. luxuriosus, and Folsomia candida. Up to nine soils were used: Besides the Organisation for Economic Co-operation and Development (OECD) artificial soil and the Agricultural Testing and Research Agency (Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Speyer, Germany) 2.2 natural standard soils, the others were selected based on the EURO Soil approach, taking into account the effect of different soil parameters (pH, organic matter, grain size distribution, and carbon to nitrogen ratio). Additionally, the effect of the chloride ions was studied separately. The results revealed the following: First, a soil effect was observed; for example, in F. candida, median effective concentrations (EC50s) varied between 262 mg/kg in a sample from the same site as the original EURO Soil 5 soil and greater than 1,000 mg/kg in OECD soil. Second, an aging effect was observed, mainly in F. candida. For example, toxicity of offspring survival was increased twofold in the OECD soil and approximately eightfold with aging in the EURO Soil 7 soil, whereas the enchytraeid species did not react differently after aging. Third, an effect of chloride ions on reproduction of the animals was found; however, this effect was independent of the aging period. Fourth, species variation was seen in terms of sensitivity (EC50), decreasing in the following order: E. luxuriosus > E. albidus >> F. candida. Differences in toxicity of offspring survival between enchytraeids and F. candida might be explained by the different routes of uptake.

Abstract: Soil contamination can be one path for streams and groundwater contamination. As a complement of chemical analysis and total contaminants determination, bioassays can provide information on the bioavailable fraction of chemical compounds, focusing on the retention and habitat function of soils. In this study the evaluation of the toxicity of two soils from the abandoned Jales Mine (Portugal) regarded both functions. The buffer capacity of soils was tested with bioassays carried out using the cladoceran Daphnia magna and the marine bacteria Vibrio fischeri. The habitat function of soils was evaluated with the reproduction bioassay with the collembolan Folsomia candida. The Microtox solid-phase test was performed with V. fischeri using soil as test medium, and soil elutriates were extracted to perform the Microtox basic test, and an immobilization and reproduction bioassay with D. magna. The marine bacteria showed high sensitivity to the soil with low heavy metal content (JNC soil) and to JNC soil elutriates, while the soil with highest heavy metal content (JC soil) or soil elutriates exposure did not cause any toxic effect. In the bioassays with D. magna, organisms showed sensitivity to JNC and also to JC soil elutriates. Both mobilization and reproduction features were inhibited. The bioassay with F. candida did not reflect any influence of the contaminants on their reproduction. Although JNC soil presented lower heavy metal contents, elutriates showed different patterns of contamination when compared to JC soil and elutriates, which indicates different retention and buffer capacities between soils. Results obtained in this study underlined the sensitivity and importance of soil elutriate bioassays with aquatic organisms in the evaluation strategy in soil ERA processes.

Abstract: To assess soil quality and risk assessment, bioassays can be useful tools to gauge the potential toxicity of contaminants focusing on their bioavailable fraction. A rapid and sublethal avoidance behaviour test was used as a screening tool with the earthworm Eisenia andrei and the isopod Porcellionides pruinosus, where organisms were exposed during 48 h to several chemicals (lindane, dimethoate and copper sulphate, for isopods and carbendazim, benomyl, dimethoate and copper sulphate for earthworms). Both species were also exposed to soils from an abandoned mine. For all bioassays a statistical approach was used to derive EC50 values. Isopods and earthworms were able to perceive the presence of toxic compounds and escaping from contaminated to clean soil. Furthermore the behaviour parameter was equally or more sensitive then other sublethal parameters (e.g. reproduction or growth), expressing the advantages of Avoidance Behaviour Tests as screening tools in ERA.

Abstract: An approach in determining ecosystem integrity and stress on ecosystem level is to assess processes within ecosystems. The aim of the present study was to evaluate the potential use of an in situ assay with immobilized Chlorella vulgaris as an indicator of effects on ecosystem functioning with regard to primary production. The herbicide linuron, applied in concentrations of 20, 60, and 180 microg linuron/L, was used to induce direct effects on primary producers. Direct and indirect changes in structure and function within outdoor model ecosystems of 3 m3 were monitored. The intermediate and highest concentration of linuron caused a decline in growth of the macrophyte Elodea sp., resulting in a significant increase of concentrations of nutrients. The increase in concentrations of nutrients caused an indirectly stimulated growth of immobilized C. vulgaris at the intermediate concentration, whereas similar concentrations of nutrients, at the highest treatment, did not stimulate Chlorella growth. It appeared that the direct effect of linuron on C. vulgaris growth was masked by nutrient availability at the intermediate but not at the highest linuron concentration. The observed immobilized algal growth was an accumulated effect of toxic and trophic pressures within the ecosystem, resulting in an integrative endpoint to detect actual impairment of ecosystem function.

Abstract: The Stepwise Stress Model (SSM) states that a cascade of regulative behavioral responses with different intrinsic sensitivities and threshold values offers increased behavioral plasticity and thus a wider range of tolerance for environmental changes or pollutant exposures. We tested the SSM with a widely introduced fish Gambusia holbrooki (Girard) (Pisces, Poeciliidae) and the standard laboratory test species Daphnia magna Straus (Crustacea, Daphniidae). The stress was simulated by short-term exposure to acid mine drainage (AMD) and to acidified reference water (ACID). Recording of behavioral responses with the multispecies freshwater biomonitor (MFB) generated continuous time-dependent dose-response data that were modeled in three-dimensional (3D) surface plots. Both the pH-dependent mortalities and the strong linear correlations between pH and aqueous metals confirmed the toxicity of the AMD and ACID gradients, respectively, for fish and Daphnia, the latter being more sensitive. AMD stress at pH < or = 5.5 amplified circadian rhythmicity in both species, while ACID stress did so only in G. holbrooki. A behavioral stepwise stress response was found in both species: D. magna decreased locomotion and ventilation (first step) (AMD, ACID), followed by increased ventilation (second step) (AMD). G. holbrooki decreased locomotion (first step) (AMD, ACID) and increased ventilation at intermediate pH levels (second step) (AMD). Both species, although from different taxonomic groups and feeding habits, followed the SSM, which might be expanded to a general concept for describing the behavioral responses of aquatic organims to pollution. Stepwise stress responses might be applied in online biomonitors to provide more sensitive and graduated alarm settings, hence optimizing the "early warning" detection of pollution waves.

Abstract: A short-term, sublethal, and cost-effective in situ sediment toxicity assay for routine assessments with the midge Chironomus riparius Meigen, based on postexposure feeding, was developed and evaluated. An inexpensive and easy-to-use assay chamber was designed. A sediment toxicity assay was successfully performed at a lentic system impacted by acid mine drainage, at sites with different types of sediment. It consisted of a 48-h exposure period followed by a 1-h postexposure feeding during which the larvae were fed on Artemia franciscana nauplii. Methodologies for feeding quantification of fourth-instar larvae (10-d old) were first developed and optimized under laboratory conditions. A. franciscana nauplii were shown to be more suited than fish flake food for postexposure feeding quantification, allowing higher precision and cost-effectiveness. It also required a shorter postexposure feeding period, thus minimizing the chances for an eventual organism physiological recovery from toxicant exposure. The influence of several environmental conditions during exposure on postexposure feeding was also evaluated: temperature, dissolved oxygen, pH, salinity, hardness, substrate, humic acids, light, and food availability. Only temperature was found to significantly influence postexposure feeding rates; exposure at 5 degrees C led to reduced feeding activity compared to 30 degrees C. Recovery rates of 87% were obtained after the 48-h field exposure at all sites (except site R2). A statistically significant postexposure feeding depression was observed at the three sites impacted by acid mine drainage. Therefore, the proposed short-term in situ assay is a potentially useful tool to assess sediment sublethal toxicity on a routine basis.

Abstract: Enchytraeids are typical inhabitants of many soils, contributing to vital processes of this environmental compartment. Indirectly they are involved in regulating the degradation of organic matter, as well as improving the pore structure of the soil. Due to their behaviour, they are able to avoid unfavourable environmental conditions. Avoidance tests with enchytraeids, initially developed with earthworms by several authors, are quick and easy to perform. With these tests a first assessment of the toxicity of a (contaminated or spiked) soil is possible in just 48 h by using the reaction of the enchytraeids as measurement endpoint. In this period of time the organisms can choose between the control soil and the other soil (a contaminated or spiked or another soil with different physico-chemical properties). In the tests reported here, the enchytraeids were exposed to control soils spiked with the fungicides Benomyl and Carbendazim and the herbicide Phenmedipham. Several chemical concentrations were tested in order to evaluate the avoidance behaviour to toxic substances. In fact, often these short-term screening tests gave results showing avoidance at concentrations in a range similar to the acute test results but, higher than in chronic tests. Further tests are needed to decide whether the results gained in this study can be extrapolated to other chemicals. It is proposed to standardize the Enchytraeid Avoidance Test as it is currently done for the Earthworm Avoidance Test by the International Standard Organization (ISO).

Abstract: During spring and summer 1999 a ring-test and field-validation study with an open, intact Terrestrial Model Ecosystem (TME) was conducted at four different European sites (Amsterdam, The Netherlands; Bangor, U.K.; Coimbra, Portugal; Flörsheim, Germany). The objective of the study was to establish a standardised method which allows the impact of chemical stressors on terrestrial compartments at ecosystem level to be investigated and possible uses of such data in existing Environmental Risk Assessments (ERAs) for chemicals to be evaluated. This issue of Ecotoxicology presents in a series of papers the results of the TME ring-test and field-validation study. Additionally, results derived from an open-homogeneous terrestrial microcosm (Integrated Soil Microcosm, ISM) are included in this series as a separate paper. In this first paper of the series background information on the planning and organisation of the study are given. The conceptual approach and the design of the study with TMEs are briefly outlined, based on the scientific discussion on the use of terrestrial microcosms in ecology and applied environmental sciences during the last 25 years. Further, some suggestions are presented on the selection of measurement endpoints to quantify structural and functional aspects of terrestrial ecosystems. Finally, the main results of the TME-study are summarised and conclusions are drawn on the technical feasibility of TMEs, their comparability with field studies and the potential use of TMEs in ERA.

Abstract: This study was designed to evaluate the suitability of an in situ microalgal bioassay with Pseudokirchneriella subcapitata to be used in freshwater environments. The assay potential was investigated by deploying it in a system impacted by acid mine drainage. Water samples were collected to perform a laboratory assay also. P. subcapitata was viewed to be a good option for the in situ assay because it grew well and according to control acceptability criteria when immobilized in calcium alginate beads. A reduction in algal growth was apparent at both impacted sites demonstrating assay sensitivity: the site closest to the effluent discharge was clearly impacted and the one further downstream appeared to be moderately impacted. Results from the laboratory assay, designed to distinguish effects of nutrient differences across sites from those due to the effluent, confirmed the in situ responses. Results are discussed in light of the significance of combining information from different assessment tools, namely in situ and laboratory assays, as well as water-quality parameters, particularly at sites that are moderately impacted.

Abstract: The effects of carbendazim on substrate induced respiration (SIR), dehydrogenase activity (DHA), phosphatase activity and thymidine incorporation by bacteria were evaluated in an experiment with an open intact Terrestrial Model Ecosystem (TME) and in a simultaneous field-validation study. Experiments were performed on four different European soils in Germany, The Netherlands, United Kingdom and Portugal. Data analysis focused on (i) detecting differences between experiments, especially in control values, (ii) checking similarity in data variability at each treatment level between experiments and (iii) analysing the resemblance of response to the model chemical in both experiments. Results obtained showed that control values from TME experiments were similar to those obtained on the respective field site, in most of the comparisons made for SIR, DHA and thymidine incorporation. Phosphatase activity revealed more differences, but values of both experiments had the same order of magnitude. At least part of the variation could be explained from the correlation of the microbial parameters with soil moisture content. Comparisons on data variability also revealed the absence of significant differences between experiments in all parameters in most cases, indicating that TMEs were able to represent the spatial variability found in the field. Effects of carbendazim, when occurring, were observed at treatment levels exceeding the highest recommended application rate of 0.36 kg a.i./ha. Effects on SIR and DHA were observed early in time, but effects on phosphatase activity and thymidine incorporation rate were found 8 or 16 weeks after chemical application. These effects were mild, and rarely a 50% inhibition on any of these parameters was seen at carbendazim dosages up to 87.5 kg a.i./ha. The response to the model chemical in TMEs and field plots was similar in most cases. These results give promising prospects for the use of TMEs as an integrative tool in higher tier levels of different assessment schemes.

Abstract: The abandoned Säo Domingos mine (Portugal) offers a pH and metal gradient of acid mine drainage (AMD), and is an ideal model for ecotoxicological studies. Short-term 24-h bioassays with water from the AMD (pH 3.3, 4.4, and 5.5, and control) were performed with fourth instars of Chironomus in the laboratory and in situ (AMD at pH 5.5) in artificial flow-through channels. This was compared to reference water acidified to the respective pH values (acid only). Behavioral stress responses were monitored online with the Multispecies Freshwater Biomonitor (MFB). The exposure to AMD and acid only was in the sublethal range (mortality 0-20%). The use of MFB chambers did not affect survival. Stress behavior of Chironomus consisted mainly of decreased locomotory activity in AMD and increased activity in acid-only tests, indicating that the metals in the AMD played a role as stress factor. Field exposure in the AMD mixing zone (pH 5.5) generated similar activity as in the corresponding laboratory exposure.

Abstract: The hypothesis is tested that toxicity of acid mine drainage can be detected by a selection of existing macroinvertebrate community and bioindicator metrices supplemented by toxicity tests with the local mosquitofish Gambusia holbrooki Girard and the shrimp Atyaephyra desmaresti Millet. The behavioural responses of A. desmaresti to acid mine drainage were recorded in the Multispecies Freshwater Biomonitor, based on behaviour and survival as parameters. Bioassessment methods were based on community diversity, structure, function, and bioindicators and supplemented by chemical analysis (temperature, pH, metals). The Biological Monitoring Working Party adapted for the Iberian Peninsula, the number of predators (Coleoptera, Hemiptera) and the number of Ephemeroptera and Trichoptera taxa differentiated the sites well. The on-line toxicity test revealed pH-dependent acute toxicity of the acid mine drainage for the shrimp (LC(50)-48 h: pH-AMD=5.8) and a pH- dependent decrease in locomotory activity with the lowest-observed-response-times (LORTs) within 5 h of exposure. Shrimp were more sensitive to acid mine drainage than fish (LC(50)-48 h: pH-AMD=4.9). A new multimetric index combining toxicity testing and bioassessment methods is proposed.

Abstract: The effects of the fungicide carbendazim (applied in the formulation Derosal) on soil microarthropod communities was determined in three Terrestrial Model Ecosystem (TME) tests and a field-validation study for a period of 16 weeks after application. TMEs consisted of intact soil columns (diameter 17.5 cm: length 40 cm) taken from a grassland field. The TMEs for the two tests (pre-test and ring-test) in Amsterdam, The Netherlands, were taken from the same site where the field-validation study was performed, the third TME test (pre-test) was performed in Bangor, Wales. Collembola communities showed large variations in numbers and no effects of carbendazim on species diversity were seen. Mites were not determined to species but only to four main taxonomic groups (Astigmata, Cryptostigmata, Mesostigmata, Prostigmata). Mite numbers in both TME and field soils also showed rather large variations and it was difficult to find consistent effects of carbendazim treatment. Principal response curve (PRC) analysis was performed to further evaluate effects of carbendazim on Collembola and mites in the TME and field tests. This multivariate technique demonstrated significant effects of carbendazim on Collembola communities in one TME test and the field-validation study but not in the other two TME tests, while mite communities showed significant effects in two TME tests but not in the third TME test and the field-validation study. NOECs for the effect of carbendazim on Collembola and mite communities derived from these PRC analyses ranged from 0.36 to 87.5 kg a.i./ha.

Abstract: This study proposes an ecologically relevant and cost-effective phytoplankton growth assay for routine in situ toxicity assessments. Assay procedures were developed applying, to the extent possible, the rationale behind the design of standard algal assays. Chlorella vulgaris was selected as test species because it grows well immobilized in alginate beads and has a wide geographic distribution. The performance of the assay in a freshwater system impacted by acid mine drainage demonstrated the suitability of assay chambers and procedures. The test system, made of inexpensive materials, allowed the rapid and easy deployment of the assay. The deployment of extra chambers at reference sites provided the ability to periodically check whether algal growth had already reached recommended growth criteria (time at which the assay should end). By deploying chambers filled with control medium at all sites, temperature was identified to explain 95% of the variation in growth. By using an artificial nutrient source shown capable of promoting algal growth according to recommended standards, toxicity from the mine effluent was distinguish from in situ nutrient limitation effects. The very good agreement (r2 = 90%) between mean in situ growth rates estimated by microscopy and by spectrophotometry and their similar coefficient of variation showed the latter to be a suitable straightforward methodology for assay endpoint estimation.

Abstract: 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.

Abstract: The authors surveyed agricultural production methods and pesticide use among subsistence farmers (campesinos) in 4 rural communities of Campeche, Mexico. Self-reports of symptoms of poisoning resulting from occupational pesticide exposure were elicited by questionnaire (N = 121), and acetylcholinesterase (AChE) activity during insecticide use was evaluated from blood samples (N = 127). In individuals from 2 of the 4 communities, AChE activity was significantly lower (p < 0.05) than the mean of activity determined for individuals in a reference group. Results of this study show that erythrocyte AChE inhibition provides a good biomarker of exposure to organophosphate pesticides in field studies with human populations. Carbamates, particularly carbofuran, seem to be more associated with exuberant and diversified symptomatology of pesticide exposure than organophosphates. Studies in field communities where both carbamates and organophosphates are suspected to exist should include blood AChE determinations, symptomatology surveys, and socioeconomic questionnaires. The authors recommend that the Mexican National Health Ministry authorities specify additional provisions regarding the use of protective equipment and the adoption of other safety practices during field work, increase information campaigns about the risks of pesticide use and the value of safety practices, and increase programs of medical monitoring and assistance for rural communities dealing with pesticides.

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Abstract: Preliminary acute soil toxicity tests are a starting point for risk assessment. These tests are performed at one exposure time and are used to estimate the lethal concentration for 50% of the population (LC50). However, it is known that LC50 varies over time, following an exponential decay model. Here, we present the variation of LC50 over time in the isopod Porcellionides pruinosus exposed to [14C]lindane when considering the concentrations measured in bulk soil and in extracted water. Using a wide range of concentrations, the percent mortality was recorded over various time intervals. Higher concentrations strongly influenced isopod survival. The LC50 infinity value for P. pruinosus was 3.57 micrograms/g for bulk soil concentration, suggesting a great sensitivity of this species to lindane. Simultaneously, the values estimated for the lethal body concentration (LBC) were 2.36 micrograms/g animal for bulk soil concentrations and 2.79 micrograms/g animal when extracted water concentrations are considered. An alternative to the LC50 determination is the estimation of LBC, which is proposed as a better way to describe the acute toxicity of chemicals. Kinetic-based toxicity models were fitted to the data and revealed uptake rate constants of 1.1 g soil/g animal/week and 84.3 ml extracted water/g animal/week for bulk soil and extracted water concentrations, respectively. Elimination rate constants of 1.7 per week were found for both pathways of exposure.

Abstract: This study aimed at evaluating the potential of an in situ algal bioassay for routine toxicity estimates of potentially contaminated estuarine environments using the microalga Phaeodactylum tricornutum immobilized in alginate beads. The influence of the initial cell density in the beads and of salinity on algal growth was first investigated. The potential of the proposed bioassay was evaluated by comparing laboratory with in situ results. A good growth performance of P. tricornutum was observed at all starting densities of beads. Although the growth rate of P. tricornutum was significantly affected by salinity, acceptability criteria currently adopted in algal assays were met, indicating the suitability of P. tricornutum as a test species for bioassays in estuaries. The large differences observed between the laboratory and in situ responses of P. tricornutum were most probably due to the temperature and light conditions less favorable for algal growth in the field and to the lack of representativeness of water samples compared to the field fluctuating conditions. These results showed the need for in situ assessments, especially in estuarine environments influenced by tides. To a lesser extent, the bioassay itself may also have been responsible for the laboratory and field differences. Further improvements in the bioassay chambers and procedures were also discussed.

Abstract: Lack of recognition of the value of wetlands has led to the loss of considerable areas of these ecosystems in the past. Linhos lake (Figueira da Foz, Portugal) is a good example of one of these ecosystems, in which human intervention was responsible for its environmental degradation and led to its precocious terrestrialization. Physico-chemical conditions and zooplankton community structure were studied in Linhos lake, in order to evaluate ecosystem functioning and to acquire baseline information. The system is characterised by high oxygen depletion. Spatial heterogeneity was confirmed by the existence of significant differences in total densities for the three zooplanktonic groups. Rotifers were the most abundant group attaining their maximum density in April (2251 x 1 ind/l). Keratella quadrata, K. cochlearis, Polyarthra vulgaris, Filinia terminalis and Hexarthra mira were the main abundant species. Correspondence analysis suggested temperature as the main controlling factor in species seasonality. In order to prevent the precocious disappearance of the lake some restoration measures were proposed based on zooplankton community structure.

Abstract: Bioaccumulation and elimination of 14C-lindane in Enchytraeus albidus was studied in artificial OECD soil and a silty loam from an agricultural field in Central West Portugal. Results showed that enchytraeids were able to bioaccumulate the chemical with a kinetic pattern similar to that of earthworms: fast uptake within a few days and a biphasic elimination pattern. A 10 day period to study uptake was sufficient, but a few more days were probably necessary for elimination. Bioaccumulation was influenced by soil type. The authors suggest that higher organic matter (OM) content and also the higher content on sand particles in the OECD soil may have led to a faster elimination: hydrophobic chemicals tend to adsorb to OM being in this way less bioavailable and therefore less bioaccumulated having bioaccumulation factor value around 6 while in natural soil is 10; the sand could act as abrasive particles (helpers) in the elimination process leading to an elimination of 90% of the chemical in two days while in natural soil 67% was eliminated in the same period of time.

Abstract: An achievable way to evaluate the bioavailability of a certain toxic in the environment is to measure the concentration inside soil organisms. Non-target saprotrophic organisms like isopods are often exposed to agrochemicals or other kind of persistent chemicals. In this study the isopod Porcellionides pruinosus was exposed to a constant concentration of Lindane (gamma-HCH) via food. Using toxicokinetic models the bioaccumulation and fate of the pesticide by isopods was assessed and compared with previous studies, where an unexpected decrease in gamma-HCH concentration was observed. Animal body burdens showed higher values, and a lower assimilation rate constant, although the elimination rate constant was twice the value previously observed. It was also observed that a significant amount of gamma-HCH had an unknown fate. To discover its possible destiny, a factorial experiment was carried out using two types of CO2 traps and contaminated leaves in the presence and absence of isopods. It was concluded that isopod activity might have been responsible for a more rapid biotransformation of gamma-HCH in leaves, since the amount of the pesticide is reduced in their presence.

Abstract: Knowledge of the transport pathways of metals into aquatic organisms is paramount in determining the metal's potential mechanism of toxicity. To determine the relative importance of water and food as cadmium (Cd) sources for the cladoceran Daphnia magna grazing on the algae Chlorella vulgaris, we measured cadmium accumulation and toxicity (feeding inhibition and survival) in three genetically different clones of D. magna subsequent to water, food, and water and food exposures. We found that Cd uptake from water and food was independent of source and additive in effect, with D. magna juveniles accumulating twice as much Cd from water than from food (algae). However, the efficiency with which Cd was assimilated by D. magna from its algal diet was much higher (10%) than from water (0.3%). Uptake and toxic responses were inversely related: tolerant clones accumulated more Cd. As a consequence, models based on uptake of metals from the combined routes of water and food may be reliable to predict metal dynamics in the field, but may fail to predict toxic effects since tolerance to metals is not necessarily linked to reduced total uptake of metals.

Abstract: To date, studies on genetic variability in the tolerance of aquatic biota to chemicals have focused on exposure to single chemicals. In the field, metals occur as elemental mixtures, and thus it is essential to study whether the genetic consequences of exposure to such mixtures differs from response to single chemicals. This study determined the feeding responses of three Daphnia magna Straus clones exposed to Cd and Zn, both individually and as mixtures. Tolerance to mixtures of Cd and Zn was expressed as the proportional feeding depression of D. magna to Cd at increasing zinc concentrations. A quantitative genetic analysis revealed that genotype and genotype x environmental factors governed population responses to mixtures of both metals. More specifically, genetic variation in tolerance to sublethal levels of Cd decreased at those Zn concentrations where there were no effects on feeding, and increased again at Zn concentrations that affected feeding. The existence of genotype x environmental interactions indicated that the genetic consequences of exposing D. magna to mixtures of Cd and Zn cannot be predicted from the animals' response to single metals alone. Therefore, current ecological risk assessment methodologies for predicting the effects of chemical mixtures may wish to incorporate the concept of genetic variability. Furthermore, exposure to low and moderate concentrations of Zn increased the sublethal tolerance to Cd. This induction of tolerance to Cd by Zn was also observed for D. magna fed algae pre-loaded with both metals. Furthermore, in only one clone, physiological acclimatization to zinc also induced tolerance to cadmium. These results suggest that the feeding responses of D. magna may be related to gut poisoning induced by the release of metals from algae under low pH conditions. In particular, both induction of metallothionein synthesis by Zn and competition between Zn and Cd ions for uptake at target sites on the gut wall may be involved in determining sublethal responses to mixtures of both metals.

Abstract: Enchytraeids are important members of the soil fauna living in the true soil layer instead of the humus like most earthworms, resulting in a different interaction with chemicals in soil. It is well known that the detectable concentration of contaminants in the soil cannot fully predict a biological effect; therefore bioaccumulation studies are of great importance. In this study the bioaccumulation pattern of the pesticide lindane ([(14)C]gamma-HCH) in Enchytraeus albidus was analyzed in two different soil types, an artificial Organisation for Economic Co-operation and Development (OECD) soil and a natural agricultural soil. The effect of aging on bioaccumulation and bioavailability was also analyzed. Aging experiments consisted of having the same procedures at different times after soil contamination: Immediately after contamination and 1, 2, 4, 6, and 10 months after that. Major differences occurred within the first month. Considering the overall data (10 months), it is possible to observe that in OECD soil there is a correlation between concentration in soil and in the organisms, but that was not observed for the natural soil. The decrease patterns for concentration in soil and in watery soil extracts were different: monophasic behavior occurred in OECD soil, and a biphasic pattern was measured in natural soil. It was observed that the bioavailable ( i.e., water-extractable) fraction of lindane in the natural soil decreased faster than the total soil concentration. In natural soil the decrease of concentration appeared faster than in OECD soil; this may be related to the lower amount of organic matter content.

Abstract: To extrapolate credibly from individuals in the laboratory to field populations, it is essential to account for genetic differences in susceptibility to toxic stress and thus incorporate genetic variability into ecological risk estimates. In this study, the distribution of sensitivities across two toxic chemicals among and within field populations of Daphnia magna were used to quantify genetic variability. The study employed 30 D. magna clones from three geographically separate European populations. The sensitivity of each population studied and its constituent clones was estimated in terms of the concentrations of lambda-cyhalothrin and cadmium impairing individual fitness by 10 and 50% (EC10-50). Results revealed that differences in tolerance among clones within populations were large when compared with differences between populations and that the genetic range of sensitivities to toxic stress within populations was log-normally distributed. Furthermore, reported variation in sensitivity values to toxic stress among different laboratory species, populations, and clones was similar to that observed among and within field populations of Daphnia. These results suggest that it is possible to estimate genetic variability by estimating the tolerance distribution of laboratory populations and clones and that extrapolation approaches currently used in ecological risk assessment should explicitly incorporate genetic variability in tolerance into risk estimates.

Abstract: Previous attempts to test the hypothesis that laboratory selection of isogenetic populations can produce test organisms with a significantly increased mean tolerance to toxic substances have failed. One possible explanation for such failure is that the tolerance of laboratory populations is largely constrained by their origins (were the source populations composed of tolerant genotypes?). To address this question, among- and within-population variability in stress tolerance was assessed by calculating the variance in individual fitness and longevity across a cadmium gradient (0-10 microg/L). The study employed Daphnia magna clones from four geographically separate European populations. Results revealed significant differences in tolerance to lethal levels of toxic stress among populations. The distribution of tolerances within two of the studied populations showed high amounts of genetic variation in tolerance. Genetic relationships between tolerance traits and life history performance under nonstressful environments differed among the studied populations. One population showed significant but low costs associated with tolerance, whereas no costs were associated with tolerance in the other population. These results suggest that laboratory selection will favor individuals with high fitness or reproductive performance under optimal laboratory conditions resulting in laboratory populations with similar or lower tolerance to toxic stress than their original field populations. Given that populations can exhibit high levels of genetic variability in tolerance to toxic stress, minimizing genetic diversity in toxicity tests will increase the uncertainty attendant in extrapolating from the lab to the field.

Abstract: The in vivo effects of parathion and endosulfan on the isopod Porcellio dilatatus were investigated. Feeding parameters (food consumption and assimilation rates), growth, and energy reserves (glycogen, lipid, and protein contents) of pesticide-exposed isopods were compared with those of control animals. Isopods were exposed to a wide range of concentrations of parathion or endosulfan (0.1, 1, 10, 25, 50, 100, 250, and 500 microg/g of food) for 21 days. The route of uptake of the pesticides was through the diet (alder leaves). Results revealed that parathion induces a significant depression of glycogen, lipid, and protein contents. However, no significant effect was observed on either feeding parameters or growth. Animals fed endosulfan-contaminated food had smaller amounts of glycogen and lipid than control animals, while protein levels were similar in all treatments. Endosulfan also induced a significant decrease in food consumption and assimilation rates at the highest concentrations tested. Growth rate was also significantly affected by endosulfan. These results suggest that the isopod P. dilatatus is a suitable species for use in toxicity tests and that energy reserves could be used as effect criteria in both laboratory and field studies.

Abstract: Biochemical factors contributing to response variation in five clones of Daphnia magna exposed to ethyl parathion were studied. Differences in sensitivities to ethyl parathion between sensitive and resistant clones varied between four- and ninefold. Acute toxicity and in vivo acetylcholinesterase (AChE) inhibition responses to ethyl parathion were similar, whereas in vitro AChE sensitivities to paraoxon were higher. Thus indicating that, in vivo, not all ethyl parathion present is converted by Daphnia juveniles to active metabolite, or if so, only a proportion of the paraoxon produced is in direct contact with its target. Comparison of AChE activities obtained during in vitro and in vivo exposures with acute responses revealed clonal concordance only between in vivo AChE sensitivities and acute toxicity. This latter finding indicates that clonal differences in ethyl parathion metabolism rather than differences in AChE sensitivities are the most likely explanations of the observed genetic differences in tolerance.

Abstract: Activity of lactate dehydrogenase (LDH) was used as an effect criterion in toxicity tests with Daphnia magna. In the first part of the work, the conditions for the use of LDH activity in toxicity tests with juveniles and adults of D. magna, were optimized. The influence of parameters such as the number of animals per sample, nutritional status, age and the presence of eggs in the brood chamber were investigated. In the second part of the study, both in vivo and in vitro tests based on the alteration of LDH activity of D. magna were developed and tested using zinc chloride as test substance. The results obtained indicate that LDH activity of D. magna may be used as an indicative parameter in aquatic toxicity tests.

Abstract: In the present study, the authors investigated and defined development stages of Daphnia magna eggs that could be potential endpoints for sublethal toxicity tests with in vitro parthenogenetic egg cultures. Such an "egg test" could be a cost-effective alternative to the classic 21-day test with D. magna. Three main stages of embryonic development were considered: release of the egg external membrane, release of the internal membrane, and separation of the caudal spine. The first embryonic stage was attained approximately 30 h after transfer of eggs from ovaries to the brood chamber (considered as time zero), the second stage at 48 h, and the third stage at 68 h. Embryonic development was considered completed with the caudal spine separation. Thereafter, juveniles were able to swim in the water column. Egg mortality, duration of each egg stage, egg diameter, and egg abnormalities were investigated as potential endpoints. In vitro tests were carried out with several toxicants (DBS, 3,4-DCA, cadmium, and copper) and with acid mine drainage, sensitivity generally being higher than with the accepted chronic 21-day test with D. magna.

Abstract: This paper describes the experimental results for optimizing isopod culture conditions for terrestrial ecotoxicity testing. The influence of animal density and food quality on growth and reproduction of Porcellio dilatatus was investigated. Results indicate that density influences isopod performance in a significant way, with low-density cultures having a higher growth rate and better reproductive output than medium- or high-density cultures. Alder leaves, as a soft nitrogen-rich species, were found to be the best-quality diet; when compared with two other food mixtures, alder leaves induced the best results, particularly in terms of breeding success. Guidance regarding culture conditions of isopod species, especially in terms of culture density and diet, is given.

Abstract: In the present work the sensitivity of yeast strains of Kluyveromyces marxianus, Pichia anomala, Candida utilis, Schizosaccharomyces pombe and Saccharomyces cerevisiae, to the fungicides cymoxanil, penconazol, and dichlofluanid, was evaluated. Dichlofluanid induced the most negative effects, whereas penconazol in general was not very toxic. Overall, our results show that the parameters IC50 for specific respiration rates of C. utilis and S. cerevisiae and C(D) for cell viability of S. cerevisiae can be applied to quantify the toxicity level of the above compounds in yeast. Hence, could be explored as an alternative or at least as a complementary test in toxicity studies and, therefore, its potential for inclusion in a tier testing toxicity test battery merits further research.

Abstract: Growth inhibition tests for copper were carried out on four marine microalgal species: Chlorella autotrophyca, Nannochloris atomus (Chlorophyceae), Phaeodactylum tricornutum (Bacillariophyceae), and Isochrysis aff. galbana (Primnesiophyceae). The test initial cellular densities were reduced to 50 and 10% from the recommended initial cellular density in most of standardized assays. OECD test protocol (originally described for freshwater) was adapted for seawater. The EC(50) values were reduced when initial cellular density decreased. The green algae used in this study exhibited lower sensitivity than P. tricornutum and quite lower than I. aff. galbana. The latter species was found to be very sensitive to copper. The concept of cellular toxic quote (amount of toxic per cell) is defined in order to improve the results of toxicity tests.

Abstract: This study was designed to investigate the effect of two surfactants, dodecyl benzyl sulfonate (DBS), sodium dodecyl sulfate (SDS), and of a domestic detergent (Y) on the AChE activity of the crustacean cladoceran Daphnia magna. All the chemicals significantly inhibit the activity of the enzyme, both in vitro and in vivo conditions. In vitro lowest observed effect concentration (LOEC) values ranged from 12.5 to 100 mg/l and correspondent IC50 (50% inhibition concentration) values ranged from 6.6 to 58.5 mg/l. In vivo LOEC values ranged from 2 to 11.9 mg/l, while EC50 (50% effect concentration) values ranged from 11.4 to 56.7 mg/l. AChE inhibition by environmental contaminants such as surfactants, detergents and metals may lead to false diagnostics and even wrong conclusions in biomonitoring studies based on the use of AChE as a specific biomarker for organophosphorous and carbamate compounds.

Abstract: As a result of a widespread application in numerous industrial processes, chromium is a contaminant of many environmental systems. Chromium and their compounds are toxic to both invertebrates and vertebrates and, for this reason, there has been a search for suitable and less toxic alternatives. Molybdenum compounds have been studied as alternative to chromium compounds for some industrial applications. The toxicity of chromium is well known but the effects of molybdenum and molybdenum mining on natural populations and communities of freshwater invertebrates have not often been studied. However, chromium, and molybdenum (and their compounds) are included in the same list (List II) of European Union dangerous substances. In this study, the acute and chronic effects of sodium molybdate and sodium dichromate to Daphnia magna Straus were evaluated. Furthermore, in vitro and in vivo effects of these two metals on acetylcholinesterase (AChE) activity of D. magna Straus were investigated. LC(50) values determined at 48 h were 0.29 and 2847.5 mg L(-1) for chromium (as sodium dichromate) and molybdenum (as sodium molybdate), respectively. No significant in vitro effects of both metals on AChE were found. However, both toxicants inhibited AChE in vivo at concentrations under the respective 48-h LC(50) values. Both sodium dichromate and sodium molybdate inhibited the reproduction and growth of D. magna, but the concentrations inducing significant effects were different for the two chemicals. Sodium molybdate had significant lower toxicity to D. magna Straus than sodium dichromate.

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Abstract: In situ bioassays are becoming very popular for laboratory test validation and field extrapolation. Natural conditions, including environmental variables, affect the "behavior" of contaminants and, consequently, their toxicity. This work aimed to develop protocols for in situ chronic bioassays with cladocerans and to assess the ecological relevance of conventional protocols for laboratory testing (water-column and sediment). An aquatic system impacted with acid mine drainage was chosen for the study. At some stations that were contaminated with heavy metals, water-column bioassays did not reveal any toxicity, while sediment and, especially in situ bioassays, revealed a delay in reproduction and a reduction in fertility. Thus, both laboratory bioassays underestimated the actual toxicity of studied sites: sediment, besides being an important contaminant source, became significantly altered with sampling, transport, storage, and manipulation. Therefore, an extra caution is needed when interpreting laboratory results, since the extrapolation to natural systems could be a very problematic step.

Abstract: In this study, the association between the acute toxicity of 15 compounds to Daphnia magna, expressed as 24- and 48-h LC(50) values, and the corresponding oral LD(50) values for the rat was tested. Since there was evidence of a strong relationship between the two species, the sample was extended to 54 cases by including the values for acute toxicity to D. magna and rat of more chemicals published by other authors. Thus, a total of 54 data points were further used to ascertain the relationship between the acute toxicity of chemical compounds to D. magna and that to the rat. To summarize its validity, the D. magna test is more specific than sensitive as an indicator of toxicity to the rat. When it is used with a chemical that has a high probability of being very toxic to D. magna (LC(50)< 0.22 mg/L), the test provides considerable information if it is positive, virtually giving evidence of toxicity to the rat (with a probability of 0.83). On the other hand, a negative test (D. magna LC(50)>0.22 mg/L) has a probability of correctly assigning nontoxicity to the rat equal to 0.74. This study and results published by other authors provide good evidence of the applicability of using invertebrate tests as prescreening methods, thus considerably reducing the number of mammals required in toxicity testing.

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Abstract: Four marine microalgal species (Chlorella autotrophyca, Nannochloropsis gaditana, Tetraiselmis chuii, and Isochrysis aff. galbana) were exposed for 24 h to 1 mg L(-1) dissolved copper and then transferred to fresh medium. After that, a group of 10 neonate rotifers were fed with these four microalgal species. The levels of accumulated copper in cellular concentrations of the microalgae were checked, with the result of around 40% of original concentration, with the exception of I. aff. galbana (25% of original concentration). In all cases, cells with preaccumulated metal caused a delay of 1 or 2 days in populational development of rotifers (increase in "lag phase"). The microalgae that were not fed to rotifers (disposed in parallel series) did not significantly transfer metal to the medium after the first day.

Abstract: Several techniques have been developed to assess the ecotoxicity of contaminated watercourses. Most of these techniques involve chemical alterations of water samples, by diluting it or by adding chelating agents. These changes become particularly severe when assessing the toxicity of samples with very low pH and with high quantities of contaminants. Trying to overcome this problem, a novel toxicity test, specific for acid waters, was previously developed and field validated. The toxicity of acid samples is assessed using the survival time of Ceriodaphnia dubia. During this study, the novel test was applied to a field situation, where an aquatic system is seriously impacted with acid mine drainage. Its efficiency was tested and compared with two classical toxicity tests: the Microtox and the median lethal dilution with C. dubia. The survival time test was performed without adjusting pH and after adjusting pH to a fixed value (pH 2, 3, and 4). At pH 2 and 4 no acceptable results were obtained; at pH 3 it was possible to distinguish the toxicity due to pH from the toxicity due to other toxicants. The test conducted at local pH was able to discriminate toxicity sources only for highly contaminated samples. The toxicity evaluation of acid mine drainage samples was possible neither with the median lethal dilution test nor with Microtox.

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Abstract: In this study a comparison was made of the results obtained in Daphnia magna chronic bioassays after first-brood release and after 21 days of exposure, using inhibition of normal reproduction and growth as effect criteria and EC10, EC20, EC50, no-observed-effect concentration (NOEC), and lowest-observed-effect concentration (LOEC) as statistical parameters. Test substances were sodium bromide (NaBr), 3,4-dichloroaniline (DCA), cadmium, and parathion. For NaBr, DCA, and cadmium, toxicity evaluated after the first-brood release was similar to toxicity evaluated after 21 days, using reproduction as end point. Parathion did not affect either reproduction or growth. Thus, LC50, NOEC, and LOEC were calculated using mortality as the endpoint for parathion. Results indicate that the period until release of the first brood is sufficient to predict the toxicity of some chemicals to D. magna. Values estimated on the basis of a logistic model (EC10, EC20, and EC50) were more appropriate than NOECs and LOECs for evaluating toxicity of the test substances. Furthermore, classic endpoints used for the evaluation of chronic toxicity (inhibition of normal reproduction and growth) may not be adequate to evaluate the sublethal toxicity of compounds that induce cumulative effects leading to mortality within the test period and causing no observable effects on the reproduction and growth of the species. Endpoints indicative of biochemical stress or effects on specific targets of the test compound may be useful in sublethal toxicity evaluation.

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Abstract: This study developed a bioassay with the isopod Porcellio dilatatus based on the activity of the enzymes acetylcholinesterase (AChE) and lactate dehydrogenase (LDH). The in vivo effects of the insecticides parathion-ethyl and endosulfan-sulfate on AChE and LDH activities of P. dilatatus under laboratory conditions were investigated. The route of uptake of the pesticides was through the food (alder leaves). Isopods were exposed to a wide range of concentrations of parathion or endosulfan (0.1, 1, 10, 25, 50, 100, 250, and 500 microg/g of food) for 21 days. After this period, the activity of AChE and LDH was determined. Parathion induced significant depression of both AChE and LDH activities. Animals fed with endosulfan-contaminated food exhibited lower LDH activities than control animals, while AChE activity was similar in all treatments. The results of the present investigation suggest that the isopod. P. dilatatus is a suitable species for use in toxicity tests and indicate that the enzymes AChE and LDH could be used as effect criteria both in laboratory and in field studies with this species.

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Abstract: Organophosphate and carbamate insecticides are inhibitors of cholinesterases (ChE). The depression of blood ChE activity is frequently used as indicative of exposure to these chemicals. However, it is not known whether the inhibition of blood ChE activity reflects the inhibition of ChE in target tissues (e.g. brain and muscle). In this study we investigated the possibility of using whole blood ChE activity to predict frontal cerebral cortex ChE activity in rats treated with parathion. Twenty four hours after the intraperitoneal administration of several doses of parathion, the activity of ChE in whole blood and the activity of ChE in frontal cerebral cortex were determined in each animal. A high correlation between the two parameters was found (r = 0.96, p < 0.05) and the model of linear regression fitted to the data accounted for 93% of its variability. Thus, these results seem to indicate that 24 hours after the treatment with parathion the effects induced on whole blood ChE activity may be used to predict the effects caused on frontal cerebral cortex ChE activity.

Abstract: In this work we investigated the acute effects of 3,4-dichloroaniline (DCA) on the changes of specific biochemical and cellular blood parameters using male Wistar rats, 24 hours after the intraperitoneal administration of five different doses of DCA (0, 81, 162, 324, 486, and 568 mg/Kg of body weight). We also evaluated the dose-dependent toxicity of DCA on the ratio "organ weight:total body weight" for kidney, liver, spleen and thymus, and on the changes of morphological characteristics of the spleen. The LOEL (lowest observed effect level) value for spleen and thymus injury was 324 mg DCA/Kg. The cellular blood parameters affected by DCA include: methemoglobin concentration, the number of circulating leukocytes and the number and size of platelets. The relative sensitivity of all the analysis used to assess the acute toxicity of DCA was evaluated by comparing their ED50 values, which ranged from 224 to 837 mg DCA/Kg. We concluded that the most sensitive endpoint to measure the acute toxicity of DCA was the methemoglobin formation. Platelet counts, urea and creatinine showed ED50 values slightly higher than methemoglobin (329-367 mg/kg).

Abstract: In this work, the mitochondrial transmembrane electric potential (delta psi) of isolated mitochondria was used to evaluate the toxicity of some chemicals (endosulfan, 3,4-dichloroaniline, parathion, tributyltin and cadmium) and wastewater. Mitochondria were isolated from rat liver, and the delta psi measured in a suitable assay medium, using a sensitive tetraphenylphosphonium (TPP+) electrode. The test substance was pre-incubated in a rotenone-containing medium during 3 min with 1.0 mg of mitochondrial protein. Mitochondria were energised with succinate and after the establishment of a constant maximal potential ADP was added to induce the phosphorylative cycle. Chosen endpoints were the membrane potential from mitochondria oxidising succinate and the depolarisation induced by ADP. After the appropriate transformations the EC50 (effective concentration) was calculated for each toxicant. Even very low concentrations of a toxicant were able to affect the delta psi, thus showing its suitability as a biosensor in ecotoxicology and results were reproducible between tests. The utilisation of delta psi in screening tests of pure substances and wastewater seems to be very effective and can be carried out routinely.

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Abstract: The acute toxicity of six metals [Hg, Cd, Cu, Zn, Cr(II), and Cr(VI)] to Daphnia magna neonates was evaluated using three different test media (Elendt M7, a complex medium containing EDTA; ASTM hard water and EEC, two simple media free of chelators). The EC50 values, at both 24 and 48 h, obtained for Zn, Cr(II), and Cr(VI) were similar in all of the media tested. Hg was more toxic in ASTM than in M7 and in EEC media. The toxicity of Cd and Cu was similar in ASTM and EEC media and higher when evaluated in M7 medium. Thus, M7 should be used only carefully for the toxicity evaluation of mixtures and effluents containing metals. It is recommended, however, that it be excluded from use in tests evaluating samples of unknown composition, or those known to contain Cu and Cd. For the metals tested in this study, a factor of five applied to each 48-h EC50 would be sufficient in order to attain the respective acute NOECs for immobility.

Abstract: In this work we compared the effects of medium renewal and use of a carrier (ethanol) in the lethal toxicity of parathion to Daphnia magna Straus. Actual concentrations measured in test solutions with and without ethanol were not significantly different, indicating that there is no need for a carrier solution to assist dissolution of parathion, up to a concentration of 5 mg litre(-1). LC50 values at 48 h from bioassays with and without medium renewal were identical, indicating that in our experimental conditions, degradation of parathion was not a significant consideration. Similar 48-h LC50 values were obtained in all tests, with or without ethanol present. Thus, potential interactions between carrier and toxicant had no effect on the lethal toxicity of parathion to D. magna.

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Abstract: In this work we investigated the possibility of using the enzyme acetylcholinesterase (AChE) activity in Daphnia magna homogenates, both in vivo and in vitro conditions, as a specific method for rapid toxicity evaluations. The results from in vivo and in vitro AChE inhibition tests were compared with 48 hours EC50 values obtained in conventional acute bioassays. EC50 values from in vivo AChE inhibition tests were: 2.4 micrograms/l for parathion, 0.2 microgram/l for paraoxon; DCA and cadmium at the concentrations tested had no effects on enzyme activity. I50 values were 764 micrograms/l for parathion, 0.08 micrograms/l for paraoxon and 3367 micrograms/l for cadmium; DCA did not affect AChE activity measured in in vitro conditions. EC50 values from conventional acute tests were: 2.2 micrograms/l for parathion, 0.2 microgram/l for paraoxon, 163 micrograms/l for DCA and 9.5 micrograms/l for cadmium. Our results indicated that the in vivo AChE inhibition test is selective, being very sensitive to detect toxicity of the organophosphates tested. The in vitro AChE inhibition assay is less time consuming, requires less human effort and produces less toxic waste than conventional acute bioassays and the in vivo AChE inhibition test. However, it does not take into account the effect of the metabolization of the toxicants inside live organisms; since the organophosphate metabolism may be activative or degradative, the toxic potential of the parent compound may be under or over evaluated in in vitro conditions.

Abstract: Chronic life-cycle tests using the freshwater cladoceran Daphnia magna aim to measure the effect of toxic chemicals on adult reproduction, in terms of the number of viable offspring produced. A tacit assumption is made that the primary effect of toxicants in these tests is a sublethal effect on the parent, reducing egg production and hence fecundity. The authors' observations on two chemicals, sodium bromide and 3,4-dichloroaniline, demonstrate that this assumption is at least partly false. In these experiments, the primary toxic effect of these compounds was to kill developing eggs in the brood chamber. Total egg output was unaffected, whereas output of viable eggs was severely impaired--an acute lethal effect. A further experiment demonstrated that the effect was limited to those eggs developing in the brood chamber, with eggs in the ovary remaining unaffected. It was suggested that these observations may provide the basis for the development of a short, sensitive early life-stage test with this species.

Abstract: The genetic typing of laboratory strains of Daphnia magna revealed that although there are genetic differences between different testing laboratory strains, most laboratories culture single genotypes as clones. A comparative study was made of the performance of five of the more common clones in terms of neonate mortality under acute exposure to nine toxic chemicals. The range in clonal EC50 response was from less than one to more than two orders of magnitude in terms of concentration. Surprisingly, there was little pattern in rank order of response of clones exposed to different toxicants, thus suggesting that genotype-environment interactions were predominating. Nevertheless, it was suggested that specifying the genotype used in D. magna toxicity tests will result in increased test repeatability. Evidence indicating that culture environment may also significantly affect the performance of neonates in acute toxicity tests was also presented.