Amadeu M.V.M. Soares

Protein differential expression induced by endocrine disrupting compounds in a terrestrial isopod.

Endocrine disrupting compounds (EDCs) have been studied due to their impact on human health and increasing awareness of their impact on wildlife species. Studies concerning the organ-specific molecular effects of EDC in invertebrates are important to understand the mechanisms of action of this class of toxicants but are scarce in the literature. We have used a dose/response approach to unravel the protein expression in different organs of isopods exposed to bisphenol A (BPA) and vinclozolin (Vz) and assess their potential use as surrogate species. Male isopods were exposed to a range of Vz or of BPA concentrations. After animal dissection, proteins were extracted from gut, hepatopancreas and testes. Protein profiles were analysed by electrophoresis and differentially expressed proteins were identified by MALDI mass spectrometry. EDCs affected proteins involved in the energy metabolism (arginine kinase), proteins of the heat shock protein family (Hsp70 and GRP78) and most likely microtubule dynamics (tubulin). Different proteins expressed at different concentrations in different organs are indicative of the organ-specific effects of BPA and Vz. Additionally, several proteins were up-regulated at lower but not higher BPA or Vz concentrations, bringing new data to the non-monotonic response curve controversy. Furthermore, our findings suggest that some common responses to EDCs in both vertebrates and invertebrates may exist.

The Daphnia bioassay: a critique

Daphnia magna is used widely as a standard ecotoxicological indicator organism, and protocols exist for its use in assessing the toxicity of substances under acute and chronic experimental conditions. Problems exist in repeatability of such bioassays between laboratories. Sources of variation are identified using a simple quantitative genetics model. Presenting specific examples, we conclude that these problems are tractable, but only if the genotype and culture conditions prior to and during tests are strictly controlled.

Should the use of inhibition of cholinesterases as a specific biomarker for organophosphate and carbamate pesticides be questioned

Recently there has been evidence that contaminants other than organophosphate and carbamate pesticides may inhibit the activity of the enzyme acetylcholinesterase (AChE) both under in vitro and in vivo conditions. In this study we investigated the in vitro effect of three detergents \[dodecyl benzyl sulphonate (DBS), sodium dodecyl sulphate (SDS) and a mixture commonly used as domestic detergent (X)] and three metals \[molybdenum, barium and chromium (VI)] on AChE activity of Mytilus galloprovincialis haemolymph. All the detergents tested significantly inhibited the activity of the enzyme. The lowest observed effect concentrations were equal to 12 5 for DBS and 50 mg l-1 for SDS and X. Among the metals, molybdenum and barium had no effect on AChE activity, whereas chromium (VI) significantly depressed the activity of the enzyme at concentrations equal to or higher than 25 mg l-1. These results suggest that the use of AChE as a specific biomarker for organophosphate and carbamate pesticides should be quest...Recently there has been evidence that contaminants other than organophosphate and carbamate pesticides may inhibit the activity of the enzyme acetylcholinesterase (AChE) both under in vitro and in vivo conditions. In this study we investigated the in vitro effect of three detergents \[dodecyl benzyl sulphonate (DBS), sodium dodecyl sulphate (SDS) and a mixture commonly used as domestic detergent (X)] and three metals \[molybdenum, barium and chromium (VI)] on AChE activity of Mytilus galloprovincialis haemolymph. All the detergents tested significantly inhibited the activity of the enzyme. The lowest observed effect concentrations were equal to 12 5 for DBS and 50 mg l-1 for SDS and X. Among the metals, molybdenum and barium had no effect on AChE activity, whereas chromium (VI) significantly depressed the activity of the enzyme at concentrations equal to or higher than 25 mg l-1. These results suggest that the use of AChE as a specific biomarker for organophosphate and carbamate pesticides should be questioned and that the use of this enzyme as a biomarker could be extended.

Acute effects of copper and mercury on the estuarine fish Pomatoschistus microps: Linking biomarkers to behaviour ☆

The main objective of the present study was to investigate possible links between biomarkers and swimming performance in the estuarine fish Pomatoschistus microps acutely exposed to metals (copper and mercury). In independent bioassays, P. microps juveniles were individually exposed for 96 h to sub-lethal concentrations of copper or mercury. At the end of the assays, swimming performance of fish was measured using a device specially developed for epibenthic fish (SPEDE). Furthermore, the following biomarkers were measured: lipid peroxidation (LPO) and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), glutathione S-transferases (GST), 7-ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx). LC(50)s of copper and mercury (dissolved throughout metal concentrations) at 96h were 568 microg L(-1), and 62 microg L(-1), respectively. Significant and concentration-dependent effects of both metals on swimming resistance and covered distance against water flow were found at concentrations equal or higher than 50 microg L(-1) for copper and 3 microg L(-1) for mercury (dissolved throughout metal concentrations). These results indicate that SPEDE was efficacious to quantify behavioural alterations in the epibenthic fish P. microps at ecologically relevant concentrations. Significant alterations by both metals on biomarkers were found including: inhibition of AChE and EROD activities, induction of LDH, GST and anti-oxidant enzymes, and increased LPO levels, with LOEC values ranging from 25 to 200 microg L(-1), for copper and from 3 to 25 microg L(-1) for mercury (dissolved throughout metal concentrations). Furthermore, significant and positive correlations were found between some biomarkers (AChE and EROD) and behavioural parameters, while negative correlations were found for others (LPO, anti-oxidant enzymes and LDH) suggesting that disruption of cholinergic function through AChE inhibition, decreased detoxification capability due to EROD inhibition, additional energetic demands to face chemical stress, and oxidative stress and damage may contribute to decrease the swimming performance of fish. Since a reduced swimming capability of fish may reduce their ability to capture preys, avoid predators, and interfere with social and reproductive behaviour, the exposure of P. microps to copper and/or mercury concentrations similar to those tested here may decrease the fitness of wild populations of this species.

Lactate dehydrogenase activity as an effect criterion in toxicity tests with Daphnia magna straus

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

METAL-BASED NANOPARTICLES IN SOIL: FATE, BEHAVIOR, AND EFFECTS ON SOIL INVERTEBRATES

Metal-based nanoparticles (NPs) (e.g., silver, zinc oxide, titanium dioxide, iron oxide) are being widely used in the nanotechnology industry. Because of the release of particles from NP-containing products, it is likely that NPs will enter the soil compartment, especially through land application of sewage sludge derived from wastewater treatment. This review presents an overview of the literature dealing with the fate and effects of metal-based NPs in soil. In the environment, the characteristics of NPs (e.g., size, shape, surface charge) and soil (e.g., pH, ionic strength, organic matter, and clay content) will affect physical and chemical processes, resulting in NP dissolution, agglomeration, and aggregation. The behavior of NPs in soil will control their mobility and their bioavailability to soil organisms. Consequently, exposure characterization in ecotoxicological studies should obtain as much information as possible about dissolution, agglomeration, and aggregation processes. Comparing existing studies is a challenging task, because no standards exist for toxicity tests with NPs. In many cases, the reporting of associated characterization data is sparse, or missing, making it impossible to interpret and explain observed differences in results among studies.

A comparative study of genotype sensitivity to acute toxic stress using clones of Daphnia magna Straus

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

In vitro and in vivo inhibition of Daphnia magna acetylcholinesterase by surfactant agents: possible implications for contamination biomonitoring

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

Silver nanoparticles and silver nitrate induce high toxicity to Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio

Silver nanoparticles (AgNP) have gained attention over the years due to the antimicrobial function of silver, which has been exploited industrially to produce consumer goods that vary in type and application. Undoubtedly the increase of production and consumption of these silver-containing products will lead to the entry of silver compounds into the environment. In this study we have used Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio as model organisms to investigate the toxicity of AgNP and AgNO₃ by assessing different biological endpoints and exposure periods. Organisms were exposed following specific and standardized protocols for each species/endpoints, with modifications when necessary. AgNP were characterized in each test-media by Transmission Electron Microscopy (TEM) and experiments were performed by Dynamic Light Scattering (DLS) to investigate the aggregation and agglomeration behavior of AgNP under different media chemical composition and test-period. TEM images of AgNP in the different test-media showed dissimilar patterns of agglomeration, with some agglomerates inside an organic layer, some loosely associated particles and also the presence of some individual particles. The toxicity of both AgNO₃ and AgNP differ significantly based on the test species: we found no differences in toxicity for algae, a small difference for zebrafish and a major difference in toxicity for Daphnia magna.

Proteins in ecotoxicology - how, why and why not?

The growing interest in the application of proteomic technologies to solve toxicology issues and its relevance in ecotoxicology research has resulted in the emergence of “ecotoxicoproteomics”. There is a general consensus that ecotoxicoproteomics is a powerful tool to spot early molecular events involved in toxicant responses, which are responsible for the adverse effects observed at higher levels of biological organization, thus contributing to elucidate the mode of action of stressors and to identify specific biomarkers. Ultimately, early‐warning indicators can then be developed and deployed in “in situ” bioassays and in environmental risk assessment. The number of field experiments or laboratory trials using ecologically relevant test‐species and involving proteomics has been, until recently, insufficient to allow a critical analysis of the real benefits of the application of this approach to ecotoxicology. This article intends to present an overview on the applications of proteomics in the context of ecotoxicology, focusing mainly on the prospective research to be done in invertebrates. Although these represent around 95% of all animal species and in spite of the key structural and functional roles they play in ecosystems, proteomic research in invertebrates is still in an incipient stage. We will review applications of ecotoxicoproteomics by evaluating the technical methods employed, the organisms and the contexts studied, the advances achieved until now and lastly the limitations yet to overcome will be discussed.