An ecotoxicological approach can complement the assessment of natural waters from Portuguese reservoirs?

Within the scope of the European Water Framework Directive (WFD), the scientific community recognized clear opportunities to take advantage of the use of ecotoxicological tools in water quality assessments. In this perspective, bioassays and biomarkers were suggested to contribute to the integration of the chemical and biological conditions, and thus to provide an overall insight into the quality of a water body. This study aimed to assess whether current bioassays as feeding rate assays with Daphnia longispina and growth inhibition assays with Lemna minor are suitable to detect potential ecotoxicity, using waters from Portuguese reservoirs. Several sampling sites were defined in reservoirs (Miranda, Pocinho, and Alqueva). The samplings were conducted in autumn of 2018 and spring of 2019. Total chlorophyll, lipid peroxidation, and proline content were also evaluated in L. minor. Results demonstrated that D. longispina showed some sensitivity to water treatments; however, the results were difficult to interpret since no reason or trend can be accurate. All parameters of L. minor did not show sensitivity to detect potential ecotoxicological risks associated with natural water understudy, since no discrimination among the water treatments was recorded. However, biomarkers/bioassays proved to be concordant to each other. Under the conditions evaluated here (reservoirs and sampling periods), the biological responses observed were not consistent, clear, and coherent with the physical-chemical parameters and chemical analyses performed, suggesting that the ecotoxicological tools selected were not sensitive to assess water quality in this type of ecosystems. In this sense, species of different trophic levels are recommended for ecotoxicological analyses due to differences in species sensitivities.

Assessment of 17α-ethinylestradiol effects in Daphnia magna: life-history traits, biochemical and genotoxic parameters.

The occurrence of pharmaceuticals in aquatic ecosystems and the need to study them have increased over the years since they enter continuously the environment. Besides, these compounds are not intended for applications with environmental purposes, and therefore, little is known about their ecological effects, particularly in non-target organisms, as invertebrate species. Inside these substances, endocrine disrupting compounds (EDCs) have recently come into the limelight, due to environmental concentrations and consequently their detrimental effects on different organisms. 17α-ethinylestradiol (EE2) has been detected in the aquatic environment in various locations around the globe since it is the main synthetic hormone used as a female oral contraceptive and is also applied in veterinary medicine and animal production. The present study was intended to assess the chronic effects of EE2, in the non-target organism as Daphnia magna. Thus, to analyze the individual and subindividual impact, this aquatic organism was chronically exposed (21 days) to 0.00 (control group), 0.10, 1.00, 10.0, and 100 µg/L of EE2. Results here obtained demonstrated that D. magna exposed to the EE2 concentrations had significant effects in individual (life-history) and sub-individual (biochemical levels) parameters. Alterations as anticipation in the age at first reproduction, a decrease of the growth rate, oxidative stress, and lipid peroxidation were detected, as well as genotoxic damage. Therefore, it was possible to infer that EE2 can disrupt several metabolic pathways and physiological functions of D. magna, since EE2 demonstrated ecotoxicity, at environmentally relevant concentrations. This work reinforces the importance of examining the effects of more relevant exposures (more prolonged and with ecologically pertinent concentrations) of potential endocrine disruptors like EE2, to the freshwater organisms and ecosystem.

Ecotoxicological evaluation of fungicides used in viticulture in non-target organisms.

The effect of fungicides, commonly used in vine cultures, on the health of terrestrial and aquatic ecosystems has been poorly studied. The objective of this study was to evaluate the toxicity of three viticulture fungicides (myclobutanil, cymoxanil, and azoxystrobin) on non-target organisms, the bacteria Rhodopirellula rubra, Escherichia coli, Pseudomonas putida, and Arthrobacter sp., the microalgae Raphidocelis subcapitata, and the macrophyte Lemna minor. Fungicide toxicity was performed in acute cell viability assay for bacteria; 72-h and 7-day growth inhibition tests for R. subcapitata and L. minor, respectively. Contents of photosynthetic pigments and lipid peroxidation in L. minor were evaluated. Arthrobacter sp. and P. putida showed resistance to these fungicides. Even though azoxystrobin affected R. rubra and E. coli cell viability, this effect was due to the solvent used, acetone. Cell viability decrease was obtained for R. rubra exposed to cymoxanil and E. coli exposed to myclobutanil (30 min of exposure at 10 mg/L and 240 min of exposure at 46 mg/L, respectively). R. subcapitata showed about 10-fold higher sensitivity to azoxystrobin (EC50-72h = 0.25 mg/L) and cymoxanil (EC50-72h = 0.36 mg/L) than L. minor to azoxystrobin and myclobutanil (EC50-72h = 1.53 mg/L and EC50-72h = 1.89 mg/L, respectively). No lipid peroxidation was observed in L. minor after fungicide exposure, while changes of total chlorophyll were induced by azoxystrobin and myclobutanil. Our results showed that non-target aquatic organisms of different trophic levels are affected by fungicides used in viticulture.

Histopathological effects of the antibiotic erythromycin on the freshwater fish species Oncorhynchus mykiss.

Pharmaceuticals are found in the aquatic compartment due to their continuous release in wastewater effluents or direct dispersal in aquaculture practices raising serious threats to human and environmental health. Erythromycin (ERY) is a macrolide antibiotic widely prescribed in human and veterinary medicine to threat a number of bacterial infections, being consequently found in the aquatic environment. The present work intends to evaluate the sub-lethal effects of ERY on juveniles of rainbow trout (Oncorhynchus mykiss) in terms of tissue damage using histochemical staining procedures. Individuals were exposed for 96 h (acute exposure: 0.001-10 mg/L) and 28 days (chronic exposure: 0.05-0.8 µg/L) to environmentally realistic concentrations of ERY. Qualitative and quantitative approaches were used to assess O. mykiss gills and liver tissue alterations after exposure to ERY. For both exposures the most common gill changes recorded were progressive (e.g. hypertrophy of mucous cells and hyperplasia of the epithelial cells). However, circulatory (e.g. aneurysms and oedemas) and regressive (e.g. epithelial lifting of lamellae and lamellar fusion) changes were also observed in the acute assay. Gill morphometric analysis revealed to be a good indicator of subtle alterations in gill architecture in agreement with the qualitative scoring system. In liver, regressive (e.g. cytoplasmic vacuolization, pyknotic nucleus and hepatocellular degeneration) and circulatory disturbances (e.g. hemorrhage and increase of sinusoidal space) were the most frequently observed alterations, but only for the acute assay. Furthermore, all histological changes observed contributed to a significant increase in the pathological index for both organs. The current data demonstrate the existence of a direct dose-effect relationship between the exposure to this specific macrolide antibiotic and the histological disorders recorded in different tissues of the exposed fish. The histopathological findings observed in this study may have been the result of several physio-metabolic dysfunctions. However, the observed tissue lesions were of minimal or moderate pathological importance, non-specific and reversible. Further investigation into the cellular mechanism of action of ERY is needed.

Effects of zinc pyrithione on biochemical parameters of the freshwater Asian clam Corbicula fluminea.

Zinc pyrithione (ZnPT) is an organometallic biocide with bactericide, algaecide, and fungicide activity. Considering this biological activity, ZnPT has been used in anti-fouling paints, and also in human therapeutics and cosmetics, in shampoos to treat dandruff and seborrhoea. Despite its potential uses and consequent presence in the aquatic environment, the ecotoxicological effects of ZnPT are poorly understood. This work aims to characterise the effects of ZnPT in biochemical parameters of the Asian clam, one of the most invasive bivalves known for its biofouling action in hydro-dependent industries, using a classical (LC50 determination) and a biomarker-based approach (quantification of the activities of catalase, GSTs, and acetylcholinesterase, and also the muscle glycogen content). The here determined LC50-96 h for zinc pyrithione was 2.17 mg/L. ZnPT caused significant increases in the activity of catalase and of cholinesterases. These findings evidence the pro-oxidative effects caused by the metabolism of ZnPT. Despite the absence of clear effects, it is important to stress that the presence of ZnPT in the wild is usually accompanied by other pyrithiones, whose co-existence can contribute to the exertion of considerable toxic effects.

Acute and chronic ecotoxicological effects of four pharmaceuticals drugs on cladoceran Daphnia magna.

The occurrence of pharmaceuticals in the aquatic environment has received increasing attention in recent years, as concerns have risen about their environmental persistence, biological activity and different effects toward nontarget organisms. Considering the magnitude of concentrations (ng L(-1) to mg L(-1)) and their often-specific modes of action, the assessment of physiological responses of exposed aquatic biota may provide significant information regarding the potential ecological consequences of exposure to these contaminants. The present study intended to assess the acute and chronic effects of four pharmaceuticals: acetaminophen, chlorpromazine, diclofenac sodium and propranolol in the cladoceran species Daphnia magna. Parameters such as immobility, total of offspring and rate of population increase were analyzed. Results of acute exposures showed a considerable variability of toxicity among pharmaceuticals, with the following ranking of toxicity: diclofenac (EC50 = 123.3 mg L(-1)) < propranolol (EC50 = 5.531 mg L(-1)) < acetaminophen (EC50 = 2.831 mg L(-1)) < chlorpromazine (EC50 = 1.805 mg L(-1)). The chronic toxicity data showed the exertion of reproductive adverse effects. The compounds chlorpromazine and propranolol caused a significant decrease in fecundity, and the rate of population increase parameter suffered a significant decrease from 0.33 mg L(-1) to 0.128 mg L(-1) onwards, respectively. The levels of exposure to which our test organism was acutely and chronically exposed were above those already reported in the wild. Nevertheless, the extensive production, prescription and release of pharmaceuticals drugs will continue to grow in the future, and consequently their loadings to the environment can result in potential long-term ecological risks to aquatic biota.

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.

Testing procedures for the determination of several biomarkers in different species, for environmental assessment of pollution.

Antioxidant defences are the primary mechanisms by which organisms compensate oxidative damage caused by contact with several types of substances prone to establish oxidative cycles. Biomarkers are one of the most useful tools to assess effects consequent to environmental exposure to a large number of xenobiotics. However, the use of biomarkers requires previous standardization of enzymatic protocols in order to guarantee accuracy and relevance of obtained results. This article describes standardized conditions for the use of several enzymatic biomarkers (oxidative stress: glutathione peroxidase, lipid peroxidation, glutathione reductase, glutathione-S-transferase and catalase; respiration: lactate dehydrogenase) for the evaluation of responses of specimens of Eisenia andrei, Pelophylax perezi (formerly Rana perezi) and Apodemus sylvaticus. Furthermore, we also present the normal values of activity for the given markers, for non-exposed organisms, that can serve as comparative indexes for subsequent studies. We conclude that these species are thus a suitable target to serve as sentinels in studies of environmental contamination by chemicals, which can lead to oxidative and physiological modifications.