Bruno B. Castro

Toxicity evaluation of three pesticides on non-target aquatic and soil organisms: commercial formulation versus active ingredient

The Ecological Risk Assessment of pesticides requires data regarding their toxicity to aquatic and terrestrial non-target species. Such requirements concern active ingredient(s), generally not considering the noxious potential of commercial formulations. This work intends to contribute with novel information on the effects of short-term exposures to two herbicides, with different modes of action (Spasor®, Stam Novel Flo 480®), and an insecticide (Lannate®), as well as to corresponding active ingredients (Glyphosate, Propanil and Methomyl, respectively). The microalga Pseudokirchneriella subcapitata (growth inhibition), the cladoceran Daphnia magna (immobilisation), and the earthworm Eisenia andrei (avoidance behaviour) were used as test species. Both herbicides were innocuous to all test organisms at environmentally realistic concentrations, except for Stam and Propanil (highly toxic for Pseudokirchneriella; moderately toxic to Daphnia). Lannate and Methomyl were highly toxic to Daphnia and caused Eisenia to significantly avoid the spiked soil at realistic application rates. The toxicity of formulations either overestimated (e.g. Stam/Propanil for P. subcapitata) or underestimated (e.g. Stam/Propanil for D. magna) that of the active ingredient.

Chronic responses of different clones of Daphnia longispina (field and ephippia) to different food levels

Abstract Resting eggs are a fundamental reproductive strategy among freshwater cladocerans. Under adverse environmental conditions, whole Daphnia populations can disappear from a lake and a new community will arise from ephippial eggs. Since these new populations are subjected to genetic variation, their responses to environmental stress or contaminants can be different from the “original” population. In the present study, life history responses (reproduction and growth) of Daphnia longispina to different food concentrations was studied. Two Daphnia populations were tested: (a) field clones and (b) ephippial clones. Food ( Selenastrum capricornutum ) concentration was the stressor tested (absence of food, and low to high food concentrations). The results showed that reproductive responses of D. longispina to the tested food concentrations varied among clones, independently from their origin.

In situ bioassay chambers and procedures for assessment of sediment toxicity with Chironomus riparius

The purpose of this study was to develop an in situ sediment bioassay chamber and respective procedures, suitable for performing toxicity bioassays with benthic invertebrates, using the midge Chironomus riparius. It was also our objective to compare the responses obtained under controlled conditions (laboratory 10-day larval growth and survival test) with those obtained in situ. Clean sand and a formulated sediment were incorporated in the in situ bioassay, along with local sediments, as a way of minimizing natural variability due to physicochemical differences among sediments or due to interactions with indigenous organisms. Recovery of organisms was good (80-100% in the control and reference site), indicating that the developed chamber and protocol were suitable for exposing and retrieving C. riparius in situ. Results also showed differences between responses obtained with formulated and natural sediment in situ, as well as between laboratory and in situ.

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.

Rotifer community structure in three shallow lakes: seasonal fluctuations and explanatory factors

The present work aimed at studying the rotifer communities of three shallow eutrophic lakes in Portugal (lakes Mira, Vela and Linhos). At the time of the study, Mira and Vela faced large inputs of allochthonous nutrients, while Linhos was facing terrestrialisation, with cycles of dominance-senescence of macrophytes. The three lakes differed in terms of their abiotic features, with Linhos presenting very high nutrient levels and low pH, while Vela and Mira shared most of the characteristics. The rotifer communities of these two lakes were poorly diversified but highly abundant (max. > 2000 ind l−1), with a clear dominance of eurytopic euplanktonic species (mainly Keratella cochlearis). On the other hand, Linhos presented lower abundances (<1000 ind l−1) but higher species richness, mainly due to macrophyte-associated taxa, such as the littoral genera Lepadella, Testudinella and Squatinella. In all lakes, summertime represented a peak in terms of abundance and diversity. Canonical correspondence analysis (CCA) identified two main environmental gradients that shape up the rotifer assemblages: a temporal gradient, mainly related to temperature, and a eutrophy gradient, associated with nitrogenous nutrients. The latter gradient is clearly dependent on between-lake variation, due to the high nutrient levels observed in lake Linhos. Variance partitioning using CCA revealed that the largest portion (27.5%) of the total variation explained (52.1%) was attributed to the interaction between lake and environmental variables.

Impaired microbial activity caused by metal pollution: A field study in a deactivated uranium mining area.

European frameworks for the ecological risk assessment (ERA) of contaminated sites integrate information from three lines of evidence: chemical, ecotoxicological, and ecological. Regarding the last one, field observations at the contaminated sites are compared to reference site(s) and the differences recorded are analysed at the light of a cause-effect relationship, taking into account the site-specific contamination. Thus, included in the tier 2 of a site-specific risk assessment that is being carried out in an deactivated uranium mining area, a battery of soil enzyme activities (dehydrogenases, urease, arysulphatase, cellulase, acid phosphate) and potential nitrification were assessed in seven sampling sites (A-D-E-F-G-H-I) at different distances from the mine pit. These parameters have been considered good indicators of impacts on soil microbial communities and, subsequently, on soil functions. Soil enzyme activities were impaired in the most contaminated site (A, near the mine pit), for which a higher degree of risk was determined in the tier 1 of ERA. Three other sites within the mining area (F, G, and D) were discriminated on the basis of their low microbial activity, using uni- and multivariate approaches, and validating what had been previously found with chemical and ecotoxicological lines of evidence. We observed considerable among-site heterogeneity in terms of soil physical and chemical properties, combined with seasonal differences in enzyme activities. Still, the correlation between microbial parameters and soil general physical and chemical parameters was weak. In opposition, significant and negative correlations were found between soil enzyme activities and several metallic elements (Al, Be, Cu, U). These findings suggest a clear correlation between compromised soil function (nutrient recycling) and metal contamination. Such information reinforces the evidence of risks for some sites within the mining area and is an important contribution for the usefulness of soil enzyme activities for evaluating changes in soil health.

Toxic potential of paracetamol to freshwater organisms: a headache to environmental regulators?

Paracetamol is one of the most prescribed drugs globally, due to its antipyretic and analgesic properties. However, it is highly toxic at elevated doses, with involvement of an already described oxidative stress pathway. Despite this, the number of ecotoxicological studies on potential effects of paracetamol in wild organisms is still scarce. The present article presents a comprehensive series of standardized assays for the assessment of paracetamol effects in freshwater organisms. The results show that paracetamol toxicity is widely variable among species, even when these species are phylogenetically related. Furthermore, comparisons between data from the literature and our results reinforce this conclusion, providing evidence of the inadequacy of standardized toxicity testing guidelines for pharmaceutical compounds in wild organisms. Paracetamol toxicity can be modulated by unpredictable physiological conditions that might compromise extrapolations and comparisons of responsiveness among species. The ecological relevance of data obtained from classical tests for this compound is further discussed.

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.

Performance of standard media in toxicological assessments with Daphnia magna: chelators and ionic composition versus metal toxicity

Fully artificial test media can increase reproducibility and standardization in ecotoxicological assessments, but there is still a lack of convergence among ecotoxicology laboratories in aquatic test media with respect to ionic composition, chelators, and organic supplements. We compared the performance of Daphnia magna in three widely-used reconstituted media. The tested media differed in composition: (a) ADaM, an artificial medium based in a synthetic sea salt, with no a priori known chelating properties; (b) ASTM hard water supplemented with algal extract, a semi-artificial medium with unknown chelating properties; and (c) M7, a complex artificial medium containing EDTA as a chelator. All three media were suitable for rearing D. magna (although performance in M7 was suboptimal) and acute EC50 values for reference substances (3,4-DCA, K2Cr2O7) were similar between media. In acute exposures to Cu and Cd, daphniids were least sensitive when reared in M7, as expected due to metal chelation by EDTA. Daphnia sensitivity to Cd was low in ADaM. Thus, these two media were suboptimal for assessing the toxicity of some metals to D. magna in acute tests. We suggest that both the ionic composition of the medium and the presence of chelators should be taken into account when metal toxicity is concerned. Chronic toxicity profiles for Cu suggested a mild chelating effect of the algal extract in ASTM medium. Still, ASTM hard water persists as one of the most suitable media for acute toxicity assessments of metals and metal-contaminated samples.

Life history responses of Daphnia longispina to mosquitofish (Gambusia holbrooki) and pumpkinseed (Lepomis gibbosus) kairomones

In the present study, the effect of chemical cues from two fish species (mosquitofish and pumpkinseed), at different concentrations, was tested in life history experiments with Daphnia longispina. The two fish species used represent the most abundant planktivores of many Mediterranean shallow lakes (SW Europe), where the indigenous fish communities have been replaced by such exotic assemblages. Results have shown a similar response of D. longispina to both fish species: kairomones stimulated daphnids to produce more offspring, which resulted in higher fitness (r), relatively to a fishless control. Fish presence also induced an earlier first reproduction, a smaller size at maturity of daphnids, and the production of smaller-sized neonates. Significant correlations with fish concentration (indirect measure of fish kairomone concentration) were found for size at maturity and neonate size, for both fish species. These results are in accordance to the “positive response” observed by other authors, which represents a defence mechanism to face losses caused by fish predators. The chemically mediated size reduction of mature females and neonates is an adaptive response to the size-selective predation exerted by fish. Pumpkinseed introduction is very recent in the lake of origin of the daphnids used in the experiments and its kairomone produced similar effects to mosquitofish in the life history of D. longispina. These results are contrary to the existence of a species-specific kairomone and support the hypothesis of a general fish kairomone.