Diogo N. Cardoso

Ecotoxicity and genotoxicity of a binary combination of triclosan and carbendazim to Daphnia magna

In the environment, chemical substances appear as complex mixtures and consequently organisms are exposed to a variety of chemicals from different sources (e.g. wastewater treatment plants, agriculture runoffs). When studying chemical mixtures, there are two conceptual models usually used to predict toxicity: the Independent Action (IA) and Concentration Addition (CA) models. However, deviations from these reference models can occur as synergism or antagonism, dose ratio or dose level dependency. The aim of the present study was to investigate the effects of triclosan and carbendazim, and their binary mixture to Daphnia magna. With this purpose, immobilisation, feeding inhibition, and reproduction were assessed as main ecotoxicity endpoints. In addition, in vivo genotoxicity of both chemicals was investigated using the comet assay. In the single exposure, carbendazim was more toxic to D. magna than triclosan. When daphnids were exposed to both single compounds, DNA damage was observed. Concerning mixture exposures, different endpoints followed different patterns of response, from additivity: IA model (feeding inhibition and reproduction data), to deviations that indicate interaction between chemicals inside the organism: dose level dependency (immobilisation data) and dose ratio dependency (DNA damage). This study showed that additivity does not rule the dose-effect relation in chemical mixtures of carbendazim and triclosan and interactions between both chemicals might induce generally higher toxicity than predicted based on single chemical exposures.

Carbaryl toxicity prediction to soil organisms under high and low temperature regimes

Many studies on risk assessment of pesticides on non-target organisms have been performed based on standardized protocols that reflect conditions in temperate climates. However, the responses of organisms to chemical compounds may differ according to latitude and thus predicting the toxicity of chemicals at different temperatures is an important factor to consider in risk assessment. The toxic effects of the pesticide carbaryl were evaluated at different temperature regimes, which are indicative of temperate and tropical climates and are relevant to climate change predictions or seasonal temperature fluctuations. Four standard organisms were used (Folsomia candida, Eisenia andrei; Triticum aestivum and Brassica rapa) and the effects were assessed using synergistic ratios, calculated from EC/LC50 values. When possible, the MIXTOX tool was used based on the reference model of independent action (IA) and possible deviations. A decrease on carbaryl toxicity at higher temperatures was found in F. candida reproduction, but when the mixtox tool was used no interactions between these stressors (Independent Action) was observed, so an additive response was suggested. Synergistic ratios showed a tendency to synergism at high temperatures for E. andrei and B. rapa and antagonism at low temperatures for both species. T. aestivum showed to be less affected than expected (antagonism), when exposed to both low and high temperatures. The results showed that temperature may increase the deleterious effects of carbaryl to non-target organisms, which is important considering both seasonal and latitude related differences, as well as the global climate change context.

Long-term exposure of the isopod Porcellionides pruinosus to nickel: Costs in the energy budget and detoxification enzymes

Terrestrial isopods from the species Porcellionides pruinosus were exposed to the maximum allowed nickel concentration in the Canadian framework guideline (50 mg Ni/kg soil) and to 5× this concentration (250 mg Ni/kg soil). The exposure lasted for 28 days and was followed by a recovery period of 14 days where organisms were changed to clean soil. Organisms were sampled after 24 h, 48 h, 96 h, 7 days, 14 days, 21 days, and 28 days of exposure, and at days 35 and 42 during the recovery period. For each sampling time the acetylcholinesterase (AChE), glutathione-S-transferases (GST), catalase (CAT), lactate dehydrogenase (LDH) activities were determined as well as lipid peroxidation rate (LPO) along with lipids, carbohydrates, proteins content, energy available (Ea), energy consumption (Ec) and cellular energy allocation (CEA). The integrated biomarker response (IBR) was calculated for each sampling time as well as for each one of the above parameters. In addition, mortality was also recorded throughout the assay. The results obtained showed that nickel induced oxidative stress, evidenced by results on GST, GPx, CAT or LPO, but also on changes in the energy reserves content of these organisms. In addition, this study showed that these organisms possess a specific strategy to handle nickel toxicity. In this case, biomarkers were associated with costs in the energy budget, and the increase of energy reserves has a compensation for that cost.

Effects of short-term exposure to fluoxetine and carbamazepine to the collembolan Folsomia candida

Pharmaceuticals, emerging environmental contaminants, have their ecotoxicological effects to non-target organisms in soil largely unknown. This study assessed short-term effects of two human pharmaceuticals, carbamazepine and fluoxetine, to Folsomia candida. Avoidance to spiked soils was assessed after 48 and 96 h exposure and biochemical changes (acetylcholinesterase and glutathione S-transferase activities, and lipid peroxidation levels) after 96 h. F. candida avoided soils spiked with 0.04, 0.4 and 4 mg carbamazepine kg(-1) after 48 h. However, higher number of organisms were found in soils with 40 mg carbamazepine kg(-1), a behavior also displayed for 40 mg fluoxetine kg(-1) spiked soils. After 96 h, F. candida showed avoidance behavior to soils with 4 and 40 mg carbamazepine kg(-1). Acetylcholinesterase activity decreased in 0.4 mg fluoxetine kg(-1) exposed organisms. Peroxidative damages were detected in organisms exposed to 4 and 40 mg kg(-1) carbamazepine and glutathione S-transferase inhibition was observed at 40 mg kg(-1). Data suggests that carbamazepine and fluoxetine may pose risk to soil collembolan.

Metabolic responses of the isopod Porcellionides pruinosus to nickel exposure assessed by 1H NMR metabolomics

UNLABELLED This work aimed at characterizing the metabolome of the isopod Porcellionides pruinosus and at assessing its variations over 14 days under laboratory culture conditions and upon exposure to the contaminant metal Nickel (Ni). The spectral profiles obtained by (1)H NMR spectroscopy were thoroughly assigned and subjected to multivariate analysis in order to highlight consistent changes. Over 50 metabolites could be identified, providing considerable new knowledge on the metabolome of these model organisms. Several metabolites changed non-linearly with Ni dose and exposure time, showing distinct variation patterns for initial (4 days) and later time points (7 and 14 days). In particular, at day 4, several amino acids were increased and sugars were decreased (compared to controls), whereas these variations were inverted for longer exposure, possibly reflecting earlier and more intensive moulting. Other variations, namely in betaines and choline-containing compounds, were suggested to relate with osmoregulation and detoxification mechanisms. Ni also had a marked effect on several nucleotides (increased upon exposure) and a moderate impact on lipids (decreased upon exposure). Overall, this study has provided new information on the Ni-induced metabolic adaptations of the P. pruinosus isopod, paving the way for improved mechanistic understanding of how these model organisms handle soil contamination. SIGNIFICANCE This study provided, for the first time to our knowledge, a detailed picture of the NMR-detectable metabolome of terrestrial isopods and of its fluctuations in time and upon exposure to the contaminant metal Nickel. Several time- and dose-dependent changes were highlighted, providing mechanistic insight into how these important model organisms handle Ni contamination.

Molluscicide baits impair the life traits of Folsomia candida (Collembola): Possible hazard to the population level and soil function

The application of molluscicides baits on the soil surface is the most common practice to control terrestrial gastropods. There seems to be a gap in the accurate evaluation of molluscicidal baits effects to soil arthropods, since their hazard to non-target organisms has been considered low after mixing baits into soil. In this work the ecotoxicological effects of two molluscicide baits (metaldehyde and methiocarb) to the collembolan Folsomia candida were evaluated using two different approaches: (1) molluscicidal baits were applied to the top soil once and only at the beginning of the exposure and avoidance behaviour and reproduction were evaluated; and (2) baits were replaced by new ones after 14-d of exposure, simulating the recommended application rate recommended by the manufacturer and reproduction was assessed (repeated/pulse exposure). A preference for the side contaminated for methiocarb was observed but the distribution of collembolans in the avoidance test with metaldehyde was random. Exposure to metaldehyde resulted in a significant increase in mortality. For methiocarb, a reduction in the juveniles produced but no acute effects were observed. In the bait pulse test, the toxic effects of each chemical was significantly increased compared with the single exposure test, for all treatments used (both reproduction and mortality). In summary, molluscicides have an adverse effect on F. candida, with severe effects on their behaviour (only for methiocarb), reproduction and survival (for both), which can lead to population collapse with time.

Toxicity interaction between chlorpyrifos, mancozeb and soil moisture to the terrestrial isopod Porcellionides pruinosus

A main source of uncertainty currently associated with environmental risk assessment of chemicals is the poor understanding of the influence of environmental factors on the toxicity of xenobiotics. Aiming to reduce this uncertainty, here we evaluate the joint-effects of two pesticides (chlorpyrifos and mancozeb) on the terrestrial isopod Porcellionides pruinosus under different soil moisture regimes. A full factorial design, including three treatments of each pesticide and an untreated control, were performed under different soil moisture regimes: 25%, 50%, and 75% WHC. Our results showed that soil moisture had no effects on isopods survival, at the levels assessed in this experiment, neither regarding single pesticides nor mixture treatments. Additivity was always the most parsimonious result when both pesticides were present. Oppositely, both feeding activity and biomass change showed a higher sensitivity to soil moisture, with isopods generally showing worse performance when exposed to pesticides and dry or moist conditions. Most of the significant differences between soil moisture regimes were found in single pesticide treatments, yet different responses to mixtures could still be distinguished depending on the soil moisture assessed. This study shows that while soil moisture has the potential to influence the effects of the pesticide mixture itself, such effects might become less important in a context of complex combinations of stressors, as the major contribution comes from its individual interaction with each pesticide. Finally, the implications of our results are discussed in light of the current state of environmental risk assessment procedures and some future perspectives are advanced.

Joint effects of chlorpyrifos and mancozeb on the terrestrial isopod Porcellionides pruinosus: A multiple biomarker approach

The exposure to pesticides by nontarget soil biota has long been regarded as a serious downside of modern agricultural regimes and the subject of heated debate. Of utmost relevance is the exposure to pesticide mixtures because their effects have been shown to not necessarily reflect the individual toxicity of their components, and even the simple addition of effects may lead to consequences not clearly anticipated. In the present study, a multiple biomarker approach was employed to identify the mechanistic and time effects underlying several single and mixture treatments of chlorpyrifos (CPF) and mancozeb (MCZ) in juveniles and adults of the terrestrial isopod Porcellionides pruinosus. The effects of the individual pesticides and the mixtures at recommended doses were mostly transitory under these controlled conditions and one-pulse exposure. Whereas imbalances were identified on detoxification and oxidative stress-related enzymes, the isopods generally showed the ability to recover through the end of the experiment. However, juveniles displayed greater vulnerability than adults. Most of the differences between life stages occurred in energy-related parameters where distinct performances and stress-handling behaviors were observed, suggesting higher metabolic costs to juveniles. Our results stress that understanding the time dependence of the underlying mechanisms governing the joint effects of the pesticides can help in assessing and anticipating the effects of the pesticide mixtures. Moreover, we emphasize the importance of taking life stage-related differences into consideration when evaluating the environmental risks of pesticides and pesticide mixtures. Environ Toxicol Chem 2018;37:1446-1457.