Search

Recently, a bioremediation tool consisting of freshly grown cells of the Arthrobacter aurescens strain TC1 proved successful in the cleanup of terbuthylazine (TBA) contaminated soil, with potential to prevent TBA dispersal via the soil-water pathway. The present work aimed at examining the feasibility of preparing and formulating bioaugmentation bacterial cells in advance which retain adequate viability and herbicide-degrading activity during storage and transport to contaminated sites. Three different types of formulation of A. aurescens TC1 cells were performed, namely cell paste, lyophilized, and adsorbed onto the mineral carrier vermiculite. Vermiculite-based cell material and cell paste offered survival rates significantly higher (≥78% of cells recovered viable, when compared to the initial numbers) than lyophilized cells (≥65%) after storage at 4 °C for up to a maximum period of 30 days. Inocula prepared from the three types of formulated and stored cells supported adequate levels of herbicide-biodegradation activity, ultimately allowing an almost complete removal of TBA from either liquid buffer or soil. In soil microcosms, the efficacy of bioremediation of TBA-contaminated soil in terms of decontamination (%) based on the ecotoxicity assessment of soil eluates towards a freshwater microalga was found to differ within formulations in the order: lyophilized cells (~100% efficacy at the end of one or two weeks of bioremediation treatment with an initial inoculum of ~2.5 × 107 viable cells/g of soil; similar to fresh cells) > vermiculite-adsorbed cells (81% with ~2.4 × 108 cells/g) > cell paste (68% with ~2.4 × 108 cells/g). The limitations and advantages of each type of bacterial formulation, together with the prediction of their applicability in the field are discussed.

Even though empirical data supporting sound ecoregion-specific ecotoxicological evaluations are still scarce, the differences of environmental (including climatic) conditions in specific ecoregions are already currently being regulated for environmental risk assessment of pesticides in Europe. To shed new light on the ecotoxicological effects of pesticides on aquatic communities across ecoregions, the model pollutant pyrimethanil (fungicide) was tested in an outdoor mesocosm study with macrophyte-dominated communities in the European “South” (Portugal) and “Centre” (Germany) regulatory zones. Phytoplankton indicators monitored over 12 months indicated a low risk of the fungicide (0.73 or 0.77 mg pyrimethanil L−1, single application) to phytoplankton functioning; as expected since exposure simulated worst-case scenarios. However, the growth of key structural macroalgae and macrophytes was affected by the fungicide and negative effects occurred, especially in the Central zone experiment. Such effects were not detected earlier than approximately nine months post single pyrimethanil application. The presence or absence of such extremely long-lasting/delayed pyrimethanil effects depended on species, competitive situation, and ecoregion-specific physico-chemical environment. The present findings suggest that a better understanding of both direct and indirect effects of fungicide pollution on aquatic flora in two European ecoregions helps to consolidate the environmental risk assessment of pesticides in specific regulatory zones.

Several pesticides were developed to be repellent to the targeted pest. However, non-target species could also move away from pesticide-contaminated habitats. These populations would face local ext...

Lines of evidence used in ecological risk assessment (ERA) are essentially three: chemistry, biology and ecotoxicology. Until now, the fundamental assumption made when measuring ecological risks is that organisms are forcedly exposed to stressors. However, when organisms can avoid disturbed habitats by escaping to less stressful areas, the assumption that exposure is mandatory to pose risk may not match field disturbance scenarios. A non-forced exposure approach using a linear free-choice multi-compartmented system has been proposed previously as a complementary tool to assess the effects on organisms' spatial avoidance/preference responses. Yet, the linearity of the latter system limits avoidance measurements to one spatial dimension. A novel, heterogeneous multi-habitat assay system (HeMHAS) consisting of 18 connected circular compartments (3 compartments on a vertical axis in each one of 6 zones on a longitudinal axis; a 2D system) is put forward here to be used in heterogeneous-habitat selection studies, as it makes it possible to assess the ability of organisms to detect contamination and other stressors and select more favorable habitats. In the present study, the avoidance to copper by zebrafish (Danio rerio) was tested after exposing organisms to a copper gradient in the HeMHAS and compared with that in the linear system. Avoidance occurred for all copper concentrations: 43% in the lowest (21 μg·L-1) to 72% in the highest (221 μg·L-1). Results obtained within the HeMHAS (AC50: 60 μg·L-1) were statistically (p = 0.72) similar to avoidance of copper by D. rerio in the linear non-forced system (AC50: 89 μg·L-1). In summary, the simulation of a copper gradient in the HeMHAS (2D system) allowed to assess the potential repellency of copper to zebrafish and to corroborate the ability of organisms to detect and avoid potentially toxic concentrations.

Information on how atrazine can affect the spatial distribution of organisms is non-existent. As this effect has been observed for some other contaminants, we hypothesized that atrazine-containing leachates/discharges could trigger spatial avoidance by the fish Poecilia reticulata and form a chemical barrier isolating upstream and downstream populations. Firstly, guppies were exposed to an atrazine gradient in a non-forced exposure system, in which organisms moved freely among the concentrations, to assess their ability to avoid atrazine. Secondly, a chemical barrier formed by atrazine, separating two clean habitats (extremities of the non-forced system), was simulated to assess whether the presence of the contaminant could prevent guppies from migrating to the other side of the system. Fish were able to avoid atrazine contamination at environmentally relevant concentrations (0.02 μg L−1), below those described to cause sub-lethal effects. The AC50 (atrazine concentration causing avoidance to 50% of the population) was 0.065 μg L−1. The chemical barrier formed by atrazine at 150 μg L−1 (concentration that should produce an avoidance around 82%) caused a reduction in the migratory potential of the fish by 47%; while the chemical barrier at 1058 μg L−1 (concentration that produces torpidity) caused a reduction in the migratory potential of the fish by 91%. Contamination by atrazine, besides driving the spatial distribution of fish populations, has potential to act as a chemical barrier by isolating fish populations. This study includes a novel approach to be integrated in environmental risk assessment schemes to assess high-tier contamination effects such as habitat fragmentation and population displacement and isolation.

At present, the European Union legislation facilitates the use of similar pesticides among European Member States, thereby assuming that biodiversity and ecosystems have equal sensitivities to contaminants throughout the whole of Europe. However, with this assumption, fundamental environmental and biological differences between climatic zones are being ignored in Environmental Risk Assessment. Such differences may strongly influence the behaviour of contaminants, their effects on biodiversity and on the natural functioning of ecosystems. Furthermore, toxicity testing in European ecoregions other than cold-temperate has largely depended on standardized tests using cold-temperate species and conditions, which may lead to a false estimation of risks to organisms from other ecoregions. The present study aim was to determine the response of freshwater macroinvertebrate communities to the fungicide pyrimethanil by conducting aquatic mesocosm experiments in two different ecoregions with different climates: cold-temperate (Frankfurt, Germany) and warm-temperate (Coimbra, Portugal). The results indicate that the community in the cold-temperate climate was more sensitive to the fungicide in comparison to the warm-temperate community. This difference was most likely related to a different rate of fungicide disappearance, which was slower in the colder climate. Based upon our results we discuss important implications for improving Environmental Risk Assessment across climate zones and under present-day global climate change scenarios.

The preparation of covalently linked manganese(III) meso-tetrakis(2,6-dichlorophenyl)porphyrin acetate to aminopropyl functionalized silica gel and its full characterization by standard spectroscopic methods including Fourier transform infrared spectroscopy (FTIR), thermogravimetry (TG) and scanning electronic microscopy (SEM) is described. The supported metalloporphyrin was evaluated as catalyst in the degradation of trimethoprim (TMP), using hydrogen peroxide as green oxidant. Catalyst was reused for five consecutive cycles without significant loss of activity or catalyst degradation/leaching. Degradation products were identified by UHPLC-MS, allowing to propose a plausible degradation pathway. Furthermore, ecotoxicity (EC50 and EC20) of TMP-based solutions (before and after degradation) were evaluated towards the bacterium V. fischeri, the microalgae R. subcapitata and the invertebrate B. calyciflorus.

Habitat selection by fish is the outcome of a choice between different stimuli. Typically, the presence of food tends to attract organisms, while contamination triggers an avoidance response to prevent toxic effects. Given that both food and contaminants are not homogeneously distributed in the environment and that food can be available in contaminated zones, a key question has been put forward in the present study: does a higher availability of food in contaminated areas interfere in the avoidance response to contaminants regardless of the contamination level? Tilapia fry (Oreochromis sp.; 2.5-3.0 cm and 0.5-0.8 g) were exposed to two different effluent samples, diluted along a free-choice, non-forced exposure system simulating a contamination gradient. Initially, avoidance to the effluents was checked during a one hour exposure. Afterwards, food was added to the system so that the availability of food increased with the increase in the level of contamination, and the avoidance response to contamination was checked during another hour. Results clearly showed a concentration-dependent avoidance response for both effluents during the first hour (i.e., with no food). However, in presence of the food, the avoidance pattern was altered: organisms were propelled to intermittently move towards contaminated areas where food availability was higher. The incursions were taken regardless of the potential risk linked to the toxic effects. In conclusion, even when the risk of toxicity was imminent, tilapia fry were more intensively stimulated by the attractiveness of the food than by repulsion to the contamination.

Spatial avoidance is a mechanism by which many organisms prevent their exposure to environmental stressors, namely chemical contaminants. Numerous studies on active avoidance and drift by aquatic organisms, as well as the main approaches used to measure both responses, were reviewed. We put forward a particular recommendation regarding methodological approaches: active avoidance should preferably be evaluated under a dilution gradient in a multi-compartmented system instead of in a bi-compartmented system. Available data on spatial avoidance from contamination indicate that emigration can occur at even lower contaminant concentrations than sub-individual noxious effects (assessed with the traditional forced-exposure assays), challenging the widely accepted paradigm in ecotoxicology that contaminant-driven adverse consequences at the population level result from a time delayed cascade of sequentially linked biochemical, cellular, physiological, and finally whole organism deleterious effects. Therefore, contaminants should not be viewed solely as potential toxicants at the individual level, but also as potential disturbers of habitats, by making the latter, at least partially, unsuited to accommodate life. Also, exposure to contamination is needed to trigger avoidance, but uptake is not mandatory, which demands the concept of exposure to be expanded, to include also the mere perception of the stressor. Since emigration eventually leads to local population extinction, and thus to severe implications for ecosystem structure and functioning, we then recommend that avoidance data be incorporated in environmental risk assessment schemes. Keywords: Avoidance, Drift, Ecological relevance, Habitat disturbance

Although essential to conservation, little is known about how stress intensity can provoke emigration from disturbed habitats and allow recolonisation of those same environments. To demonstrate the applicability of laboratory experiments, we tested two hypotheses empirically using zebrafish response to artificially polluted environments that exhibited a linear gradient of stressor (acid mine drainage) levels. We hypothesized that emigration is distance-independent but time-correlated (spacelessness hypothesis). Additionally, we hypothesized that stressor-driven emigration could predict the extent of population growth in recovering habitats (avoidance-recolonisation hypothesis). For example, if half the organisms emigrate at a given stressor level, then the remainder should be able to recolonise a habitat experiencing the same stressor intensity. Comparisons of the small-scale experiment with a larger-scale simulation suggested that controlled laboratory results can be extrapolated to field populations (although time to perceive the contamination gradient may pose differential individual effects) because AC50 (median avoidance concentration) values of an acid mine drainage sample (AMD) were not statistically different when fish were exposed to the same gradient in 3-m long [0.50% (0.43-0.57)] or 30-m long [0.73% (0.30-2.2)] systems. Regarding the avoidance-recolonisation hypothesis, the number of recolonisers was inversely proportional to the number of avoiders (ACx = RC100-x). In particular, the similar distribution of fish along the 0-3% AMD gradient in both avoidance and recolonisation experiments resulted in identical AC50 and RC50 values: 0.55% (0.34-0.87) and 0.55% (0.45-0.67) AMD, respectively. The inclusion of avoidance and recolonisation responses in the environmental risk assessments provides a novel perspective of risk based on the emigration of organisms and contributes to the understanding and prediction of biological invasions and ecosystem recovery after restoration.

Traditional ecotoxicity testing neglects possible contaminant-driven emigration, thus potentially underestimating ecological risks. A literature survey indicates several aquatic organisms may move away from contaminant-disturbed habitats, even before suffering toxicological effects. If spatial avoidance only occurs at partially lethal contaminant concentrations, coupled effects increase the population immediate extinction probability. Methodologies and a conceptual framework are already available to easily estimate contaminant-driven emigration and recolonisation patterns. We recommend contaminants be regarded not only as poisons, but also as repellents, and that avoidance and recolonisation testing be included in ecological risk assessments.

The effect of the fungicide pyrimethanil (0.7 mg L(−1)) on biofilm development and alder leaf litter decomposition in aquatic ecosystems was assessed in outdoor lentic mesocosms immediately and 274 days after pyrimethanil application. Pyrimethanil decreased ergosterol concentrations (an indicator of fungal biomass) and the abundance and richness of the macroinvertebrate community associated with decomposing leaves. However, because neither fungi nor macroinvertebrates were main factors contributing to decomposition in this particular system, organic matter processing rates were not affected. After 274 days, pyrimethanil concentration in the water column was ≤0.004 mg L(−1) but richness, biomass and composition of the invertebrate community associated with decomposing leaf-litter still showed the effect. The comparison of ergosterol (a molecule existing on both algae and fungal cell membranes), with chlorophyll (an indicator of algal biomass) associated with biofilm suggests that pyrimethanil may decrease fungal biomass and alter the relative abundance of algae and fungi on biofilm developing in control- and treated-mesocosms.

Aquatic ecotoxicity assays used to assess ecological risk assume that organisms living in a contaminated habitat are forcedly exposed to the contamination. This assumption neglects the ability of organisms to detect and avoid contamination by moving towards less disturbed habitats, as long as connectivity exists. In fluvial systems, many environmental parameters vary spatially and thus condition organisms' habitat selection. We assessed the preference of zebra fish (Danio rerio) when exposed to water samples from two western Ecuadorian rivers with apparently distinct disturbance levels: Pescadillo River (highly disturbed) and Oro River (moderately disturbed). Using a non-forced exposure system in which water samples from each river were arranged according to their spatial sequence in the field and connected to allow individuals to move freely among samples, we assayed habitat selection by D. rerio to assess environmental disturbance in the two rivers. Fish exposed to Pescadillo River samples preferred downstream samples near the confluence zone with the Oro River. Fish exposed to Oro River samples preferred upstream waters. When exposed to samples from both rivers simultaneously, fish exhibited the same pattern of habitat selection by preferring the Oro River samples. Given that the rivers are connected, preference for the Oro River enabled us to predict a depression in fish populations in the Pescadillo River. Although these findings indicate higher disturbance levels in the Pescadillo River, none of the physical-chemical variables measured was significantly correlated with the preference pattern towards the Oro River. Non-linear spatial patterns of habitat preference suggest that other environmental parameters like urban or agricultural contaminants play an important role in the model organism's habitat selection in these rivers. The non-forced exposure system represents a habitat selection-based approach that can serve as a valuable tool to unravel the factors that dictate organisms' spatial distribution in connected ecosystems.

In situ assays based on feeding depression have been proposed as sublethal assays able to assess immediate contaminant effects on key ecosystem functions, long before effects on life-history traits can be detected. The in situ peculiarities provide more realistic exposure scenarios than laboratory-controlled conditions, which is particularly relevant for estuarine ecosystems where environmental conditions are highly variable. In this context, we developed a short-term cost-effective in situ assay based on the postexposure feeding of the estuarine species, Cyathura carinata, through the following steps: i) develop a methodology to quantify feeding under laboratory conditions; ii) quantify the sensitivity of the postexposure feeding response under laboratory conditions; iii) deploy the in situ assay at various field sites covering different environmental variables in a reference estuary and degrees of contamination in a contaminated estuary to, respectively, derive a model to predict postexposure feeding rates across sites varying in environmental variables and evaluate the potential of this in situ tool to assess sediment contamination in estuaries. A quantity of 100 defrosted nauplii of Artemia franciscana per isopod given during a short period of 30 min in darkness was adequate to allow estimating precise postexposure feeding rates. Laboratory toxicity tests showed the high sensitivity of postexposure feeding to Cd as a sublethal endpoint for C. carinata (7-fold lower than the corresponding 48-h LC50). However, this species appears to be able to face high concentrations of non-essential metals as Cd, with 96-h LC50 values (37 mg Cd/L) higher than values reported for other isopods, and among the highest concentrations reported for saltwater invertebrates. Results of in situ deployments at reference sites indicated that sediment with size fraction between 63-125 µm (very fine sand) influences feeding rates. The in situ assay across contaminated sites was able to discriminate between unpolluted and polluted estuaries, and also to detect degrees of toxicity among sampling sites within an estuary, after statistically adjusting feeding rates on the basis of the proportion of the sediment very fine sand influencing baseline feeding rates. The in situ postexposure feeding assay with C. carinata was found to be, therefore, a potential useful cost-effective tool for estuarine sediment toxicity assessments.

Contaminants can behave as toxicants, when toxic effects are observed in organisms, as well as habitat disturbers and fragmentors, by triggering avoidance responses and generating less- or uninhabited zones. Drift by stream insects has long been considered a mechanism to avoid contamination by moving to most favorable habitats. Given that exploration and transportation of crude oil represent a threat for surrounding ecosystems, the key goal of the present study was to assess the ability of autochthonous groups of aquatic insects from the Ecuadorian paramo streams to avoid by drift different concentrations of polycyclic aromatic hydrocarbons (PAH) contained in the soluble fraction of locally transported crude oil. In the laboratory, different groups of insects were exposed to PAH for 12h. Three different assays, which varied in taxa and origin of the organisms, concentrations of PAH (0.6-38.8µgL(-1)), and environment settings (different levels of refuge and flow) were performed. For Anomalocosmoecus palugillensis (Limnephilidae), drift was a major cause of population decline in low concentration treatments but at higher concentrations mortality dominated. PAH was highly lethal, even at lower concentrations, for Chironomidae, Grypopterygidae (Claudioperla sp.) and Hydrobiosidae (Atopsyche sp.), and, therefore, no conclusion about drift can be drawn for these insects. Contamination by PAH showed to be a threat for benthic aquatic insects from Ecuadorian paramo streams as it can cause a population decline due to avoidance by drift and mortality.

Contaminant-driven feeding inhibition has direct and immediate consequences at higher levels of biological organization, by depressing the population consumption and thus hampering ecosystem functioning (e.g., grazing, organic matter decomposition). The present study aimed at developing a short-term laboratory and in situ assay based on the postexposure feeding of the freshwater snail Theodoxus fluviatilis. A method to precisely quantify feeding rates was first developed, consisting of a 3-h feeding period, in darkness, on 150 defrosted nauplii of the brine shrimp Artemia franciscana. Postexposure feeding after a 48-h exposure to cadmium was approximately as sensitive as survival, with the median effective concentration (EC50) and median lethal concentration (LC50) being 85 µg/L and 102 µg/L, respectively, and the 20% effective concentration (EC20) and 20% lethal concentration (LC20) being 41 µg/L and 77 µg/L, respectively. Together, both effects at the LC20 reduced population consumption by 56%. In situ experiments at reference sites covering broad ranges of current velocity, hardness, conductivity, sediment organic matter content, and sediment particle size distribution revealed the influence of these abiotic conditions on postexposure feeding, in the absence of contamination, to be negligible. The effectiveness of the in situ assay was evaluated at 5 sites contaminated with acid mine drainage. Surviving organisms at the single partially lethal site (37% mortality) presented a 54% feeding inhibition relative to the reference, whereas the population consumption would be inhibited by 71%, confirming the integration of survival and feeding to be pertinent for estimating contaminant effects at higher levels of biological organization. Environ Toxicol Chem 2013;32:2144–2152. © 2013 SETAC

In summer, streams in Portuguese eucalyptus forests frequently experience drought resulting in isolated pools, frequently saturated with leaf litter, in which the leaf leachates may generate toxic and hypoxic conditions. We assessed the ecological effects of Eucalyptus globulus leachate with and without aeration (i.e., low flow vs pool scenarios) on microbial decomposition of eucalyptus leaves and on toxicity to and survival, avoidance, and feeding behavior of Chironomus riparius Meigen (Diptera:Chironomidae), Echinogammarus meridionalis Pinkster (Amphipoda:Gammaridae), and Sericostoma vittatum Rambur (Trichoptera:Sericostomatidae). Eucalyptus globulus leaves immersed in a gradient of aerated or nonaerated eucalyptus leachate (100, 40.0, 16.0, 6.40, 2.50% volume/volume) were colonized by species-poor fungal assemblages. Leaf mass loss did not differ among treatments, but hypoxia suppressed conidia production and negatively affected fungal biomass. A concentration-dependent effect on fungal biomass...

IRET The impact of diazinon spraying in an agricultural tropical soil through the evaluation of both the habitat and retention functions of the soil system was never reported. To fill this gap, five times the recommended dose of a commercial diazinon formulation was sprayed in an agricultural area of Costa Rica, and dilution gradients of the sprayed soil were prepared in the laboratory. Avoidance and reproduction tests with soil organisms (Eisenia andrei, Enchytraeus crypticus and Folsomia candida) to evaluate losses in terrestrial habitat function, and growth and reproduction tests with aquatic organisms (Chlorella vulgaris and Daphnia magna, respectively) to evaluate the retention function of soil were performed. Results demonstrated that regarding habitat function, F. candida reproduction was the most sensitive endpoint (EC50 = 0.288 mg a.i./kg), followed by avoidance behaviour of E. andrei (EC20 = 1.75 mg a.i./kg). F. candida avoidance and the reproduction of E. andrei and E. crypticus were not affected by diazinon. The toxicity tests with aquatic organisms showed that the soil retention function was insufficient to prevent effects of diazinon either on microalgae growth (EC50 B ≤.742 mg/L and EC20 B ≤.223 mg/L) and on the reproduction of the cladoceran (EC50 B ≤.00771 mg/L and EC 20 B ≤.00646 mg/L). Results suggested that diazinon exerted toxic effects even at the dilution corresponding to the recommended dose, fact which makes its misuse an issue of environmental concern. Furthermore, the present study highlighted the importance and complementary nature of the assessment of both habitat and retention functions to an ecological risk assessment in tropical systems. Nunca se informó sobre el impacto de la fumigación con diazinón en un suelo agrícola tropical a través de la evaluación tanto del hábitat como de las funciones de retención del sistema del suelo. Para llenar este vacío, se roció cinco veces la dosis recomendada de una formulación comercial de diazinón en un área agrícola de Costa Rica y se prepararon gradientes de dilución del suelo rociado en el laboratorio. Se realizaron pruebas de evitación y reproducción con organismos del suelo (Eisenia andrei, Enchytraeus crypticus y Folsomia candida) para evaluar pérdidas en la función del hábitat terrestre, y pruebas de crecimiento y reproducción con organismos acuáticos (Chlorella vulgaris y Daphnia magna, respectivamente) para evaluar la función de retención del suelo. realizado. Los resultados demostraron que con respecto a la función del hábitat, la reproducción de F. candida fue el criterio de valoración más sensible (CE50 = 0,288 mg a.i./kg), seguido del comportamiento de evitación de E. andrei (CE20 = 1,75 mg a.i./kg). F. La evitación de cándida y la reproducción de E. andrei y E. crypticus no se vieron afectadas por el diazinón. Las pruebas de toxicidad con organismos acuáticos mostraron que la función de retención del suelo era insuficiente para prevenir los efectos del diazinón en el crecimiento de microalgas (EC50 B ≤.742 mg / L y EC20 B ≤.223 mg / L) y en la reproducción del cladocerano ( EC50 B ≤.00771 mg / L y EC 20 B ≤.00646 mg / L). Los resultados sugirieron que el diazinón ejercía efectos tóxicos incluso a la dilución correspondiente a la dosis recomendada, hecho que hace que su mal uso sea un problema ambiental. Además, el presente estudio destacó la importancia y la naturaleza complementaria de la evaluación de las funciones de hábitat y retención a una evaluación de riesgo ecológico en los sistemas tropicales. O impacto da pulverização de diazinon em um solo agrícola tropical por meio da avaliação do habitat e das funções de retenção do sistema de solo nunca foi relatado. Para preencher esta lacuna, cinco vezes a dose recomendada de uma formulação comercial de diazinon foi pulverizada em uma área agrícola da Costa Rica, e gradientes de diluição do solo pulverizado foram preparados em laboratório. Os testes de evitação e reprodução com organismos do solo (Eisenia andrei, Enchytraeus crypticus e Folsomia candida) para avaliar as perdas na função do habitat terrestre, e os testes de crescimento e reprodução com organismos aquáticos (Chlorella vulgaris e Daphnia magna, respectivamente) para avaliar a função de retenção do solo foram realizada. Os resultados demonstraram que em relação à função do habitat, a reprodução de F. candida foi o desfecho mais sensível (EC50 = 0,288 mg ia./kg), seguido pelo comportamento de evitação de E. andrei (EC20 = 1,75 mg ia./kg). A evitação de F. candida e a reprodução de E. andrei e E. crypticus não foram afetadas pelo diazinon. Os testes de toxicidade com organismos aquáticos mostraram que a função de retenção do solo foi insuficiente para prevenir os efeitos do diazinon no crescimento de microalgas (EC50 B ≤.742 mg / L e EC20 B ≤.223 mg / L) e na reprodução do cladócero ( EC50 B ≤,00771 mg / L e EC 20 B ≤,00646 mg / L). Os resultados sugeriram que o diazinon exerceu efeitos tóxicos mesmo na diluição correspondente à dose recomendada, o que torna seu uso indevido uma questão de preocupação ambiental. Além disso, o presente estudo destacou a importância e a natureza complementar da avaliação das funções de habitat e retenção para uma avaliação de risco ecológico em sistemas tropicais. University of Coimbra, Portugal Universidad Nacional, Costa Rica Instituto Regional de Estudios en Sustancias Tóxicas

The present study evaluated the bioremediation efficacy of a cleanup tool for atrazine-contaminated soils (Pseudomonas sp. ADP plus citrate [P. ADP + CIT]) at a semifield scale, combining chemical and ecotoxicological information. Three experiments representing worst-case scenarios of atrazine contamination for soil, surface water (due to runoff), and groundwater (due to leaching) were performed in laboratory simulators (100 × 40 × 20 cm). For each experiment, three treatments were set up: bioremediated, nonbioremediated, and a control. In the first, the soil was sprayed with 10 times the recommended dose (RD) for corn of Atrazerba and with P. ADP + CIT at day 0 and a similar amount of P. ADP at day 2. The nonbioremediated treatment consisted of soil spraying with 10 times the RD of Atrazerba (day 0). After 7 d of treatment, samples of soil (and eluates), runoff, and leachate were collected for ecotoxicological tests with plants (Avena sativa and Brassica napus) and microalgae (Pseudokirchneriella subcapitata) species. In the nonbioremediated soils, atrazine was very toxic to both plants, with more pronounced effects on plant growth than on seed emergence. The bioremediation tool annulled atrazine toxicity to A. sativa (86 and 100% efficacy, respectively, for seed emergence and plant growth). For B. napus, results point to incomplete bioremediation. For the microalgae, eluate and runoff samples from the nonbioremediated soils were extremely toxic; a slight toxicity was registered for leachates. After only 7 d, the ecotoxicological risk for the aquatic compartments seemed to be diminished with the application of P. ADP + CIT. In aqueous samples obtained from the bioremediated soils, the microalgal growth was similar to the control for runoff samples and slightly lower than control (by 11%) for eluates. Environ. Toxicol. Chem. 2012; 31: 1564–1572. © 2012 SETAC

Most laboratory tests may underestimate adverse effects in real scenarios of contamination because they imply the forced exposure of organisms to contaminants, thus overlooking the possibility of emigration. Avoidance from contaminants has been observed in several aquatic organisms, and avoidance-based tests have been recommended to be included in risk assessment studies. To reduce uncertainty in the extrapolation of laboratory derived results, the first aim of the present study was to compare both the median avoidance concentration and the lowest-observed-effect gradient (LOEG) values of atrazine for the cladoceran Daphnia magna, between an already developed 1.1-m-long system and a scaled-up system, three times longer. Second, the present study aimed at evaluating the population immediate decline--the proportion of the population that disappears (avoids and, if not, dies)--through the integration of the relationships between lethality and avoidance versus contaminant concentration. Daphnia magna significantly avoided atrazine, during 12-h exposures, with similar results in the original and scaled-up systems. The population immediate decline at the 48-h median lethal concentration would be 94%. Even at a concentration eliciting only 5% mortality, the population immediate decline would be over 50%. Achieving a higher pertinence of avoidance results and a better understanding of the LOEG values and their time dependence, scaling up the system further both spatially and temporally, and modeling explicit spatial dynamics in exposure and organism movement in space and time are needed.

Consumption inhibition in natural populations, namely due to contaminants, may have direct and immediate effects on ecosystems, by hampering ecosystem key functions (e.g., organic matter decomposition, grazing), long before effects at the individual level (e.g., reproduction, growth, emergence) have time-delayed consequences at successively higher levels of biological organization. The present study aimed at developing a cost-effective (short and easy) toxicity test based on the postexposure feeding of a freshwater amphipod present in the Iberian Peninsula and at evaluating the immediate impact of contamination on the population-level consumption rate. First, methodologies to quantify postexposure feeding were developed and optimized, the most important criterion being a feeding period as short as possible to minimize physiological recovery from the contaminant exposure. Second, the sensitivities of 48-h postexposure feeding and 48-h lethality tests were compared, using a reference chemical – copper. Third, the latter responses were integrated in a single parameter, the median population consumption inhibitory concentration. When Echinogammarus meridionalis males were fed on 100 defrosted Artemia franciscana nauplii during 30 min in darkness at 19–21 °C, the eaten proportion was approximately 80%, without truncated data distributions. The 48-h median effective copper concentration for postexposure feeding was approximately two times lower than the 48-h LC50 – 91 versus 198 μg L−1, respectively. Two techniques were used to quantify the median population consumption inhibitory copper concentration, both leading to similar values (75 and 68 μg L−1). In conclusion, when contaminant concentrations elicit both lethality and feeding depression, the integration of both responses can provide an ecologically relevant evaluation on the potency of a contaminant to immediately disrupt ecosystem functioning.

In the last years the chloro-s-triazine active substance terbuthylazine has been increasingly used as an herbicide and may leave residues in the environment which can be of concern. The present study aimed at developing a bioaugmentation tool based on the soil bacterium Arthrobacter aurescens strain TC1 for the remediation of terbuthylazine contaminated soils and at examining its efficacy for both soil and aquatic compartments. First, the feasibility of growing the bioaugmentation bacterium inocula on simple sole nitrogen sources (ammonium and nitrate) instead of atrazine, while still maintaining its efficiency to biodegrade terbuthylazine was shown. In sequence, the successful and quick (3 days) bioremediation efficacy of ammonium-grown A. aurescens TC1 cells was proven in a natural soil freshly spiked or four-months aged with commercial terbuthylazine at a dose 10× higher than the recommended in corn cultivation, to mimic spill situations. Ecotoxicity assessment of the soil eluates towards a freshwater microalga supported the effectiveness of the bioaugmentation tool. Obtained results highlight the potential to decontaminate soil while minimizing terbuthylazine from reaching aquatic compartments via the soil-water pathway. The usefulness of this bioaugmentation tool to provide rapid environment decontamination is particularly relevant in the event of accidental high herbicide contamination. Its limitations and advantages are discussed.

Purpose The present study presents data on the screening phase (tier 1) of a site-specific ecological risk assessment in a former smelter area heavily contaminated with metals (Santo Amaro, Bahia, Brazil). Joining information from three lines of evidence (LoE), chemical, ecotoxicological, and ecological, integrated risk values were calculated to rank sites within the area and identify those that may need further investigation in tier 2.

The seasonal diet and size of fish prey of the otterLutra lutrawere studied in an almost pristine river of North Portugal (Paiva river) through the analysis of spraints collected during 1 year. To identify Iberian freshwater fish species (Salmo trutta,Micropterus salmoides,Lepomis gibbosus,Luciobarbus bocagei,Cyprinus carpio,Carassius auratus,Pseudochondrostoma polylepis,Achondrostoma arcasii, andIberocypris alburnoides) consumed by the otter keys based on scales and vertebrae were first developed and tested. Both keys allowed identification of all fish species, although three Cyprinidae species could not be discriminated (P. polylepis,A. arcasii, andI. alburnoides). Regression equations were calculated to estimate the dimensions of preyed fish. Highly significant linear relationships were obtained for the standard length versus vertebrae measurements, for all fish species, except forC. carpio. Fish was the predominant prey category in autumn and winter, but the otter also preyed on amphibians, reptiles, mammals, and birds, particularly during spring and summer. In general, the otter consumed high proportions of small size fish (3–12 cm for the three indiscriminate Cyprinidae, 3–9 cm forC. carpio, and 6–12 cm forS. trutta).

7 páginas, 3 figuras, 3 tablas., This work presents the results of an interlaboratory proficiency exercise for whole-sedimenttoxicityassays with the benthicmarinediatomCylindrotheca closterium. An assay protocol was established and followed by all participating laboratories. Cell growth after 72 h exposure was the endpoint used. Four sediment samples of unknown toxicity were assayed. The main problem encountered during this exercise was the differences in the cell growth of algae exposed to reference sediment. Those differences may be associated with changes in the physiological status of the initial culture due to temperature changes during transport to the other laboratories. In general, the method proposed presented good replicability (precision between replicates) and reproducibility (interlaboratory precision). Around 80% (17 out of 21) of results obtained were classified as satisfactory (Z-scores < 2). The whole-sedimentassay with C. closterium presented here can be considered sufficiently successful for possible use as a standard toxicitytest. The assay is simple to perform, the proposed species is ecologically relevant as an integral component of microphytobenthos, and is widely distributed around the world. These positive factors suggest that the whole-sedimentassay with the benthicmarinediatomC. closterium can be used as a reliable tool in marinesediment quality assessment., This work has been funded by the Spanish Ministry of Science and Education (project MECASEC: CTM2006-01437/MAR).

Studies on avoidance of contaminants by aquatic organisms verified that such behavior may have crucial ecological implications. Yet, avoidance tests have not been considered in ecological risk assessments. This study aimed at developing a short-term test with Daphnia magna Straus based on avoidance of pulp mill effluents and at comparing its sensitivity to the standard 21 d D. magna reproduction test. The avoidance effective dilution values (12 h EDil20 and EDil50) were as sensitive as the 21 d EDil20 and EDil50 values for reproduction. Therefore, this easily standardizable short-term test can be recommended as a valuable complementary tool in ecological risk assessments. http://dx.doi.org/10.1007/s00128-008-9538-y

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.

The feeding behaviour of Peringia ulvae was studied with the aim of assessing its preferential feeding niche (habit) as well as its potential role in controlling or preventing the occurrence of macroalgal blooms. Both these aspects were studied in the Mondego estuary because of the clear long-lasting eutrophication process that favours the growth of two opportunistic macroalgae, Gracilaria spp. and Ulva spp., over the local beds of the macrophyte Zostera nolteii, which is another potential food source. All three species, both with and without periphyton, were used as a single food source in feeding experiments to assess different feeding parameters of the gastropod. Sediment with microphytobenthos was used together with the latter three species in feeding niche preference experiments. None of the food types provided were resistant to grazing. Higher levels of grazing occurred on Ulva spp. (apical parts) and Z. nolteii, both with periphyton. The presence of periphyton did not change grazing rates. When a choice between each food type was offered, the sediment was clearly the feeding niche preferred by the snails. As P. ulvae spent more time on the sediment (preferred feeding niche), the hypothesis that it has a role in preventing macroalgal blooms was not supported., This research was partially funded by the Fundacao para a Ciencia e a Tecnologia (FCT, Portugal) through Ciencia 2007/2008 – Human Potential Operational Program (POPH) and Quadro de Referencia Estrategico Nacional (QREN) through European Social Fund (FSE) and Ministry of Education and Science (MEC) funds, and by the Institute for Interdisciplinary Research (University of Coimbra) through the project EXTREMIS (contract number III/36/2008). CVMA is grateful to FCT for postdoctoral fellowships (SFRH/BPD/74044/2010

The European Water Framework Directive (WFD) represents a transformation of the guidelines for water quality assessment and monitoring across all EU Member States. At present, it is widely accepted that the WFD requires holistic and multidisciplinary ecological approaches by integrating multiple lines of evidence. Within the scope of the WFD, the scientific community identified clear opportunities to take advantage of an ecotoxicological line of evidence. In this context, ecotoxicological tools, namely biomarkers and bioassays, were proposed to contribute to the integration of the chemical and biological indicators, and thus to provide an overall insight into the quality of a water body. More than one decade after the publication of the WFD, we reviewed the studies that have attempted to integrate ecotoxicological tools in the assessment of surface water bodies. For this purpose, we reviewed studies providing an ecological water status assessment through more conventional community based approaches, in which biomarkers and/or bioassays were also applied to complement the evaluation. Overall, from our review emerges that studies at community level appear suitable for assessing the ecological quality of water bodies, whereas the bioassays/biomarkers are especially useful as early warning systems and to investigate the causes of ecological impairment, allowing a better understanding of the cause–effect-relationships. In this sense, community level responses and biomarkers/bioassays seem to be clearly complementary, reinforcing the need of combining the approaches of different disciplines to achieve the best evaluation of ecosystem communities’ health., SI

This study presents data on the detailed evaluation (tier 2) of a site-specific ecological risk assessment (ssERA) in a former smelter area contaminated with metals (Santo Amaro, Bahia, Brazil). Combining information from three lines of evidence (LoE), chemical (ChemLoE), ecotoxicological (EcotoxLoE) and ecological (EcoLoE), in the Triad approach, integrated risk values were calculated to rank sites and confirm the potential risk disclosed with tier 1. Risk values were calculated for the habitat and for the retention functions in each sampling point. Habitat function included the ChemLoE calculated from total metal concentrations. The EcotoxLoE was based on reproduction tests with terrestrial invertebrates (Folsomia candida, Enchytraeus crypticus, Eisenia andrei), shoot length and plant biomass (Avena sativa, Brassica rapa). For the EcoLoE, ecological parameters (microbial parameters, soil invertebrate community, litter breakdown) were used to derive risk values. Retention function included the ChemLoE, calculated from extractable metal concentrations, and the EcotoxLoE based on eluate tests with aquatic organisms (Daphnia magna reproduction and Pseudokirchneriella subcapitata growth). Results related to the habitat function indicated that the metal residues are sufficient to cause risk to biota, while the low metal levels in extracts and the general lack of toxicity in aquatic tests indicated a high soil retention capacity in most sampling points. Integrated risk of tier 2 showed the same trend of tier 1, suggesting the need to proceed with remediation actions. The high risk levels were related to direct toxicity to organisms and indirect effects, such as failure in the establishment of vegetation and the consequent loss of habitat quality for microorganisms and soil fauna. This study shed some light on the selection of tools for the tier 2 of an ssERA in tropical metal-contaminated sites, focusing on ecological receptors at risk and using available chemical methods, ecological surveys and ecotoxicity tests.

Microalgae immobilization in alginate matrixes has been recently used to perform in situ experiments. However, the susceptibility of alginate matrixes to cation chelating agents and to antigelling cations, which can cause bead disruption or dissolution, is a major limitation for in situ exposures in estuarine and marine systems. The ultimate goal of this study was to produce alginate beads stable in seawater and suited for Phaeodactylum tricornutum growth. For this, different concentrations of alginate isolated from Macrocystis pyrifera (1.5, 1.9 and 2.3% [w/v]) and Laminaria hyperborea (4.0, 4.9 and 5.8% [w/v]), two concentrations of the hardening cations calcium and strontium (2.0 and 4.0% [w/v]), and the use of the polycation chitosan were investigated. Only beads found to be more stable after 16 days of exposure in seawater were inoculated with the microalga. P. tricornutum immobilized in beads prepared from 5.8% L. hyperborea alginate and in all beads in which a chitosan hardening treatment was applied showed a weak microalgal growth. Beads prepared using 4.9% of L. hyperborea alginate and a 4% (w/v) strontium solution were found to be the most stable and the most suitable for microalgal growth, and were exposed in the field, under natural fluctuating conditions of light and temperature. In situ growth rates of immobilized P. tricornutum cells demonstrated the potential of these beads for future use in in situ experiments in estuarine and marine systems.

Submitted by Amadeu Mortágua Velho da Maia Soares (asoares@ua.pt) on 2012-01-10T23:52:16Z No. of bitstreams: 1 97_BECT_2005_74_48.pdf: 1194359 bytes, checksum: 55ede64d68c0b8afd432ee8ebb735f7f (MD5) Made available in DSpace on 2012-02-14T12:13:31Z (GMT). No. of bitstreams: 1 97_BECT_2005_74_48.pdf: 1194359 bytes, checksum: 55ede64d68c0b8afd432ee8ebb735f7f (MD5) Previous issue date: 2005

In December 2000, the ship ‘Coral Bulker’ ran aground at the entrance of the port of Viana do Castelo (North–west coast of Portugal). A large amount of fuel oil was spilled and part of it reached the shore. To evaluate the spatial and temporal impact of this oil spill, a field study, and several laboratory toxicity tests were performed using Mytilus galloprovincialis as biological indicator of environmental contamination and the biomarkers glutathione S-transferases (GSTs) and acetylcholinesterase (AChE) as indicative criteria. Fifteen days after the oil spill, mussels collected at stations located near the ship presented higher and lower values of GSTs and AChE activity, respectively. These results, and those obtained in the laboratory toxicity tests, evidence that these biomarkers were sensitive indicators of exposure to this kind of pollution and were able to monitor a spatial impact of the oil spill of at least 10 km, confirming the higher level of contamination near the ship and a contamination gradient along the sampling stations. One year after the accident, such a contamination gradient was no longer evident. This study highlight the potential suitability of a biomarker approach for assessing spatial and temporal impacts of marine pollution accidents, such as fuel oil spills, suggesting the inclusion of these biomarkers in risk assessment studies, as cost-effective and early warning recognized tools. Major advantages and limitations of the biomarker approach used in this study are further discussed. http://dx.doi.org/10.1007/s10646-003-4422-3

The present study is aimed at evaluating whether a gene expression assay with the microbial eukaryotic model Saccharomyces cerevisiae could be used as a suitable warning tool for the rapid preliminary screening of potential toxic effects on organisms due to scenarios of soil and water contamination with pyrimethanil. The assay consisted of measuring changes in the expression of the selected pyrimethanil-responsive genes ARG3 and ARG5,6 in a standardized yeast population. Evaluation was held by assessing the toxicity of surface runoff, a major route of pesticide exposure in aquatic systems due to non-point-source pollution, which was simulated with a pyrimethanil formulation at a semifield scale mimicking worst-case scenarios of soil contamination (e.g. accident or improper disposal). Yeast cells 2-h exposure to the runoff samples led to a significant 2-fold increase in the expression of both indicator genes. These results were compared with those from assays with organisms relevant for the aquatic and soil compartments, namely the nematode Caenorhabditis elegans (reproduction), the freshwater cladoceran Daphnia magna (survival and reproduction), the benthic midge Chironomus riparius (growth), and the soil invertebrates Folsomia candida and Enchytraeus crypticus (survival and reproduction). Under the experimental conditions used to simulate accidental discharges into soil, runoff waters were highly toxic to the standard test organisms, except for C. elegans. Overall, results point out the usefulness of the yeast assay to provide a rapid preview of the toxicity level in preliminary screenings of environmental samples in situations of inadvertent high pesticide contamination. Advantages and limitations of this novel method are discussed.

Contaminant-induced feeding inhibition has direct and immediate consequences at higher levels of biological organization, by depressing the population consumption and thus hampering ecosystem functioning (e.g. grazing, organic matter decomposition). Thus, similarly to lethality and avoidance, feeding is mechanistically linked to ecosystem processes and is therefore an unequivocal ecologically meaningful response. The objective of the present study was to develop a short-term assay with the small freshwater fish Danio rerio, based on feeding. For this, a methodology to easily and precisely quantify feeding was first optimized: each fish was allowed to prey on ten live Daphnia magna juveniles, for 1h, just before the end of a 48-h exposure test period. Secondly, copper sensitivity of feeding relatively to survival and growth was evaluated. At the growth EC20 (40 μg L(-1)), feeding was inhibited by 53%, and at the feeding EC50 (36 μg L(-1)), mortality was negligible (1.3%). Integrating feeding and survival revealed a 97% depression in the population consumption at the LC50 (61 μg L(-1)). Thirdly, the influence of pH, conductivity and hardness on the feeding background variability was assessed by assaying waters collected at eight reference sites and was found to be negligible, within tested ranges. Fourthly, feeding assays with natural waters contaminated with acid mine drainage confirmed the integration of lethality and feeding to be pertinent at estimating contaminant effects at higher levels of biological organization.

Several oil spills due to ruptures in the pipeline oil systems have occurred at the Andean paramo. A sample of this crude oil was mixed with water from a nearby Andean lagoon and the toxicity of the soluble fraction was assessed through lethal and avoidance assays with a locally occurring copepod (Boeckella occidentalis intermedia). The integration of mortality and avoidance aimed at predicting the immediate decline of copepod populations facing an oil leakage. The 24-h median lethal PAH concentration was 42.7 (26.4-91.6) µg L(-1). In the 12-h avoidance assay, 30% avoidance was recorded at the highest PAH concentration (19.4 µg L(-1)). The mortality at this PAH concentration would be of 25% and, thus, the population immediate decline would be of 55%. The inclusion of non-forced exposure testing with the quantification of the avoidance response in environmental risk assessments is, therefore, supported due to underestimation of the lethal assays.

There is growing concern that modifications to the global environment such as ocean acidification and increased ultraviolet radiation may interact with anthropogenic pollutants to adversely affect the future marine environment. Despite this, little is known about the nature of the potential risks posed by such interactions. Here, we performed a multifactorial microcosm experiment to assess the impact of ocean acidification, ultraviolet B (UV-B) radiation and oil hydrocarbon contamination on sediment chemistry, the microbial community (composition and function) and biochemical marker response of selected indicator species. We found that increased ocean acidification and oil contamination in the absence of UV-B will significantly alter bacterial composition by, among other things, greatly reducing the relative abundance of Desulfobacterales, known to be important oil hydrocarbon degraders. Along with changes in bacterial composition, we identified concomitant shifts in the composition of oil hydrocarbons in the sediment and an increase in oxidative stress effects on our indicator species. Interestingly, our study identifies UV-B as a critical component in the interaction between these factors, as its presence alleviates harmful effects caused by the combination of reduced pH and oil pollution. The model system used here shows that the interactive effect of reduced pH and oil contamination can adversely affect the structure and functioning of sediment benthic communities, with the potential to exacerbate the toxicity of oil hydrocarbons in marine ecosystems.

Amphibians have experienced an accentuated population decline in the whole world due to many factors, one of them being anthropogenic contamination. The present study aimed to assess the potential effect of copper, as a worldwide and reference contaminant, on the immediate decline of exposed population due to avoidance and mortality responses in tadpoles of three species of amphibians across climatic zones: a South American species, Leptodactylus latrans , a North American species, Lithobates catesbeianus , and a European species, Pelophylax perezi . A non-forced exposure system with a copper gradient along seven compartments through which organisms could freely move was used to assess the ability of tadpoles to detect and avoid copper contamination. All species were able to avoid copper at a concentration as low as 100 μg L −1 . At the lowest (sublethal) concentrations (up to 200 μg L −1 ) avoidance played an exclusive role for the population decline, whereas at the highest concentrations (>450 μg L −1 ) mortality was the response determining population decline. The median concentrations causing exposed population immediate decline were 93, 106 and 180 μg L −1 for Le . latrans , Li . catesbeianus and P . perezi , respectively. Contaminants might, therefore, act as environmental disruptors both by generating low-quality habitats and by triggering avoidance of tadpoles, which could be an important response contributing to dispersion patterns, susceptibility to future stressors and decline of amphibian populations (together with mortality).