Sensitivity to salinization and acclimation potential of amphibian (Pelophylax perezi) and fish (Lepomis gibbosus) models.

Predictions of the International Panel for Climate Changes on sea level rise foresee that the number of coastal regions impacted with salinization will increase in a near future. The present work intended to evaluate the sensitivity to salinization of two freshwater vertebrate species (the frog Pelophylax perezi and the fish Lepomis gibbosus) and their ability to acclimate to this stressor. For this, three specific objectives were targeted: (i) to assess if NaCl may be used as a safe surrogate for risk assessment of seawater (SW) intrusion for freshwater vertebrates; (ii) to evaluate the sensitivity of two freshwater vertebrate models to increased salinity (both due to NaCl or SW); (iii) to determine the capacity of the studied species to acclimate to low levels of salinization. To assess specific objectives (i) and (ii), organisms were exposed to serial concentrations of NaCl or SW dilutions. To assess the capacity of acclimation of both species to salinization, organisms were exposed to low serial concentrations of NaCl during the embryonic development or for a period of two months, respectively, and their sensitivity to NaCl was re-evaluated after this period. Results showed that fish juveniles were more tolerant (96-h LC50 of 21.3 mS cm-1 for NaCl and 23.6 mS cm-1 for SW) than frog embryos (96-h LC50 of 10.7 mS cm-1 for NaCl and 10.7 mS cm-1 for SW) and tadpoles (96-h LC50 of 19.4 mS cm-1 for NaCl and 8.72 mS cm-1 for SW). The fish was able to cope with conductivities of almost one third of SW conductivity, while effect conductivities computed for the amphibian were much lower than SW conductivity (≈ 52 mS cm-1). The two-fold difference between the sensitivity of the two tested species reinforces the idea that ecological risk assessment for amphibians based on fish toxicity data may underestimate the risk to the former. Acclimation to low levels of salinity caused an increase in tolerance to salinization in P. perezi tadpoles but not in fish.

Sensitivity of freshwater species under single and multigenerational exposure to seawater intrusion.

Salinization of coastal freshwater ecosystems is already occurring in some regions of the world. This phenomenon raises serious concerns on the protection of coastal freshwater ecosystems, since many of them support and shelter a large number of species and are considered hotspots of biodiversity. This work intended to assess the adverse effects that salinization, caused by the intrusion of seawater (SW), may pose to freshwater organisms. In this study, three specific goals were addressed: (i) to assess if sodium chloride (NaCl) may be used as a surrogate of natural SW at early-stages of risk assessment; (ii) to identify the most sensitive freshwater species to salinity NaCl; and (iii) to determine if increased tolerance to salinity may be acquired after multigenerational exposure to low levels of salinization (induced with NaCl). A total of 12 standard monospecific bioassays were carried out by exposing organisms from different taxonomic groups (Cyanobacteria: one species, Tracheophyta: two species, Rotifera: one species, Arthropoda: two species and Mollusca: one species) to a series of concentrations of NaCl (ranging from 0.95 to 22.8 mS cm-1) or dilutions of SW (ranging from 1.70 to 52.3 mS cm-1). In general, NaCl exerted similar or higher toxicity than SW, both at lethal and sublethal levels, suggesting that it may be proposed as a protective surrogate of SW for first tiers of salinization risk assessment. Among all tested species, the cyanobacterium Cylindrospermopsis raciborskii, the daphnid Daphnia longispina and the rotifer Brachionus plicatilis were the most sensitive taxa to salinization (EC50 ≤ 4.38 mS cm-1). Given their position at the basis of the food web, it is suggested that small increments of salinity may be enough to induce structural changes in freshwater communities or induce changes in trophic relations. No clear evidences of increased tolerance after multigenerational exposure to low levels of salinity were found.This article is part of the theme issue 'Salt in freshwaters: causes, ecological consequences and future prospects'.

Transgenerational Effects of Toxicants: An Extension of the Daphnia 21-day Chronic Assay?

The assessment of transgenerational effects should be incorporated in standard chronic toxicity protocols for the sake of a realistic extrapolation of contaminant effects to the population level. We propose a simple add-on to the standard 21-day chronic Daphnia magna assay, allowing the assessment of the reproductive performance of the offspring (F1 generation) born from the first clutch of the parental (F0) generation. The extended generational assay was performed simultaneously with the standard reproduction assay. With this design, we evaluated the lethal, reproductive, and transgenerational effects of four widespread and extensively used substances: a biocide/anti-fouling (copper sulphate), an industrial oxidizing agent (potassium dichromate), a pharmaceutical (paracetamol), and a quaternary ammonium compound (benzalkonium chloride). Benzalkonium chloride was the most toxic in terms of lethality, whereas paracetamol, copper sulphate, and potassium dichromate caused deleterious effects in the reproductive performance of exposed D. magna. Adverse effects in the fitness of the daughter (F1) generation were observed in the case of maternal exposure to paracetamol and copper sulphate, although they were not very pronounced. These findings highlight the usefulness of our approach and reinforce the view-shared by other authors-of the need for a generalised formal assessment of the transgenerational effects of pollutants.

Whole-organism and biomarker endpoints in Daphnia magna show uncoupling of oxidative stress and endocrine disruption in phenolic derivatives.

During the past century, the amount of chemicals released into water bodies has increased, with particular emphasis being attributed to xenobiotics with endocrine disruption properties and/or pro-oxidant effects. Among these, it is possible to identify a specific chemical class, alkylphenols, which are of widespread use, and include a variety of chemicals with multiple uses. Bisphenol A is an important chemical used in industrial production of plastics, and has been extensively described as an endocrine disruptor. Paracetamol is a pharmaceutical compound used in human medicine, known for its therapeutic action but also for its evident pro-oxidant features. Additionally, previous studies have suggested that paracetamol may also exert endocrine disruption. The main goal of this study was to assess the effects of both paracetamol and bisphenol A as endocrine disruptors, and as promoters of oxidative stress and damage, on the freshwater microcrustacean Daphnia magna. The obtained results showed that bisphenol A was capable of altering population traits of exposed organisms, by impairing molting. On the contrary, paracetamol was not causative of any significant change in this parameter, despite having caused extensive oxidative stress.

Short-term effects of neuroactive pharmaceutical drugs on a fish species: biochemical and behavioural effects.

The presence of pharmaceutical residues in the aquatic environment is receiving great attention since significant levels of contamination have been found, not only in sewage treatment plant effluents, but also in open waters. In our study, the toxicity of three anticonvulsant drugs commonly found in the environment (diazepam, carbamazepine, and phenytoin) was evaluated in Lepomis gibbosus (pumpkinseed sunfish). This study focused on oxidative stress parameters, namely: glutathione reductase (GRed), glutathione S-transferases (GSTs), catalase (CAT), and lipid peroxidation (thiobarbituric acid reactive substances, TBARS) in the hepatic, digestive, and gill tissues of exposed animals. Simultaneously, we assessed the effects of these drugs in terms of behavioural parameters, such as scototaxis and activity. Exposure to diazepam caused an increase in GST activities in the gills and an inhibition of GRed in the digestive tract, relative to control, suggesting an antioxidant response. It also caused fish to spend more time swimming and less time in a refuge area (black compartment of an aquarium). Exposure to carbamazepine caused an increase in GSTs and GRed activity in the digestive tract, which is not always consistent with the literature. A significant positive correlation was found between carbamazepine concentration and time spent in motion and a negative correlation with time spent in black compartment. Exposure to phenytoin was responsible for adaptive responses in the activities of CAT and GSTs (in the liver), but it did not elicit any behavioural alterations. Although all three drugs seemed to induce oxidative stress in some organs, peroxidative damage (measured as TBARS concentrations) was not found at the selected range of concentrations. Our results enlighten the need for more research on the ecological consequences of pharmaceuticals in the aquatic environment, especially drugs that interfere with the CNS and behaviour, because the net outcome of these effects may be difficult to predict.

Genetic, biochemical, and individual responses of the teleost fish Carassius auratus to uranium.

Carassius auratus were exposed for 96 h to different concentrations of uranyl nitrate (corresponding to 0, 100, 450, and 2,025 microg U L(-1)) and killed after different postexposure periods (0, 48, and 96 h) to assess uranium bioaccumulation, peroxisome proliferation (catalase [CAT]), lipid peroxidation (thiobarbituric acid reactive substances [TBARS]), and DNA integrity in erythrocytes (comet assay). In addition, feeding behaviour was recorded as a general response to toxicant exposure. Results provided evidence of uranium bioaccumulation in muscle of C. auratus after exposure to the highest concentrations (450 and 2,025 microg U L(-1)). This tissue was able to depurate uranium to control levels 96 h after exposure ceased. However, no perturbations in feeding behaviour or cell damage were observed in the tested organisms, except for the apparent irreversible inhibition of CAT activity immediately after exposure in the highest concentration tested. Data on DNA integrity (comets) showed that waterborne uranium exposure was able to induce genotoxicity in C. auratus erythrocytes because fish exposed to all concentrations exhibited higher DNA damage than controls 96 h after exposure. No DNA damage repair was apparent throughout the postexposure period, which was contrary to a recovery scenario. This experiment provides evidence of uranium's ability to induce physiologic impairment and genotoxicity in freshwater fish at environmentally relevant concentrations.

In situ bioassay with Eisenia andrei to assess soil toxicity in an abandoned uranium mine.

The goal of this study was to develop an in situ bioassay with Eisenia andrei, deploying it in several locations of an abandoned mining area. Our objectives were two-fold: (i) we intended to validate the in situ soil bioassay procedures, while (ii) providing ecologically relevant data to complement the ongoing risk evaluation based on laboratorial assays. To promote cost- and time-effectiveness, the in situ exposure was short (48 h) and the endpoints analysed included oxidative stress biomarkers and metal content in soil and organisms. The bioassay was carried out under different experimental conditions, simulating local (natural soil) vs. control conditions (LUFA soil), and irrigation with artificial rainwater vs. irrigation with diluted acidic effluent. Variation in the data was mostly due to soil type, rather than irrigation water, and substantial spatial heterogeneity was observed. Oxidative stress biomarkers did not fully work as sensitive parameters to environmental contamination. Earthworm metal burdens suggested a potential concern in terms of bioaccumulation of some metallic elements.

Contribution for tier 1 of the ecological risk assessment of Cunha Baixa uranium mine (Central Portugal): II. soil ecotoxicological screening.

This study presents the first ecotoxicological data concerning the soils of the area surrounding the Cunha Baixa uranium mine. Our main goal was to categorise soils from the area based on their toxicity profiles using a battery of cost- and time-effective bioassays (elutriate approach - Microtox and Daphnia acute tests; whole-soil approach - Microtox and avoidance assays with Eisenia andrei), as a part of tier 1 of an ongoing Environmental Risk Assessment. No acute toxicity was found for any of the 10 sites/soils using Microtox or Daphnia. On the contrary, the behavioural response of E. andrei was found to be an extremely sensitive endpoint, allowing the discrimination of highly to moderately toxic soils based on their toxicity profiles (as a function of soil concentration). Soils exhibiting highest toxicity corresponded to areas subjected to runoffs or sludge deposition from the aquatic effluent, while non-toxic soils were farthest to the mine. Data obtained in avoidance assays strengthen the previous evaluation of risks based on chemical data and supported decisions about proceeding for tier 2.

Short-term effects of Quirlan (chlorfenvinphos) on the behavior and acetylcholinesterase activity of Gambusia holbrooki.

Chlorfenvinphos is a widespread organophosphorous (OP) insecticide and it is a reported hazardous chemical for aquatic nontarget organisms. This study intended to evaluate the effects of sublethal concentrations of Quirlan(R) (commercial formulation of chlorfenvinphos) on several behavioral parameters of the mosquitofish, Gambusia holbrooki. The insecticide showed high toxicity to G. holbrooki by significantly impairing all the behavioral responses (location in the test vessel, activity/excitability, swimming, and feeding), exhibiting a time-dependent pattern. Behavioral EC50s, after a 96-h exposure, ranged from 5.2 to 9.0 microg L(-1). As OP pesticides are acutely neurotoxic, acetylcholinesterase (AChE) activity was also selected for use as a biomarker in this study for the establishment of a relationship with the observed behavior abnormalities. A strong inhibition of AChE was observed in fish exposed to chlorfenvinphos (IC50 = 3.55 microg L(-1)). Behavioral impairment was registered in fish with >40% AChE inhibition levels, while mortality was only observable in fish exhibiting AChE inhibition levels >80%. Additionally, significant correlations were found between behavioral impairment and AChE inhibition, suggesting a mechanistic link. These results show the usefulness of integrating biochemical and individual endpoints in a small-sized model species, and confirm a potential hazard of chlorfenvinphos to nontarget aquatic organisms.

Evaluation of water column and sediment toxicity from an abandoned uranium mine using a battery of bioassays.

Uranium mining activities in Cunha Baixa, Mangualde (Portugal), were extensive between 1967 and 1993, with high production of poor ore. Ore exploitation left millions of tons of tailings in the surrounding area, close to human houses. Contamination of the area (water and soil compartment) presently represents a serious hazard to humans and wildlife. The aim of this work was to evaluate the acute toxicity of water and sediments from a pond that floods a uranium mine pit, in two periods (spring and autumn). High contents of metals were found in water samples (chiefly Mn, Fe, Al, U, Sr). A battery of assays was applied to screen the acute toxicity of the different compartments using algae, crustaceans and dipterans. Results showed that the sediments were non-toxic, unlike the superficial water. Water toxicity was higher in the autumn, when the effluent was more acidic, compared to spring. In the water toxicity assays, the relative sensitivity of the test species used was Daphnia longispina>Pseudokirchneriella subcapitata>Daphnia magna. The present study is part of the chemical and ecotoxicological characterisation of the aquatic compartment performed in the Tier 1 of the Ecological Risk Assessment of the Cunha Baixa mining area.