Influence of soil management on soil biodiversity and ecosystem services provisioning under climate change scenarios

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Using time-lapse omics correlations to integrate toxicological pathways of a formulated fungicide in a soil invertebrate

Publication . Simões, Tiago; Novais, Sara C.; Natal-da-Luz, Tiago; Devreese, Bart; de Boer, Tjalf; Roelofs, Dick; Sousa, José P.; Van Straalen, Nico M.; Lemos, Marco F. L.

The use of an integrative molecular approach can actively improve the evaluation of environmental health status and impact of chemicals, providing the knowledge to develop sentinel tools that can be integrated in risk assessment studies, since gene and protein expressions represent the first response barriers to anthropogenic stress. This work aimed to determine the mechanisms of toxic action of a widely applied fungicide formulation (chlorothalonil), following a time series approach and using a soil model arthropod, Folsomia candida. To link effects at different levels of biological organization, data were collected on reproduction, gene expression and protein levels, in a time series during exposure to a natural soil. Results showed a mechanistic mode of action for chlorothalonil, affecting pathways of detoxification and excretion, immune response, cellular respiration, protein metabolism and oxidative stress defense, causing irregular cell signaling (JNK and NOD ½ pathways), DNA damage and abnormal cell proliferation, leading to impairment in developmental features such as molting cycle and reproduction. The omics datasets presented highly significant positive correlations between the gene expression levels at a certain time-point and the corresponding protein products 2e3 days later. The integrated omics in this study has provided useful insights into pesticide mechanisms of toxicity, evidencing the relevance of such analyses in toxicological studies, and highlighting the importance of considering a time-series when integrating these datasets.

2019-02Journal article

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Fate and effects of two pesticide formulations in the invertebrate Folsomia candida using a natural agricultural soil

Publication . Simões, Tiago; Novais, Sara C.; Natal-da-Luz, Tiago; Leston, Sara; Rosa, João; Ramos, Fernando; Pouca, Ana Sofia Vila; Freitas, Andreia; Barbosa, Jorge; Roelofs, Dick; Sousa, José P.; Van Straalen, Nico M.; Lemos, Marco F. L.

Degradation rates of two widely used pesticides were assessed, and acute and chronic effects on a standard invertebrate species investigated. An herbicide (Montana®) and fungicide (Bravo500®) formulations were investigated and results were compared to the isolated active substances of each formulation (glyphosate and chlorothalonil, respectively). Tests were performed using the invertebrate Folsomia candida as test species and an agricultural natural soil. Degradation rate tests were determined under aerobic conditions at 20 ± 2 °C, using an ecologically relevant concentration of 5 mg (a.i.) kg−1 of soil for both chemicals. Results demonstrated degradation half-lives (DT50) of 2.2 days for Montana® and 2.8 days when pure glyphosate was tested. Values of 1.1 and 2.9 days were registered for Bravo500® and its active substance chlorothalonil, respectively. There were no effects on survival for the tested concentrations of both forms of the herbicide (up to 17.3 mg kg−1). However, reproduction was affected, but only by the herbicide formulation, with an estimated EC50 value of 4.63 mg (a.i.) kg−1. Effects were most unlikely related to glyphosate. For chlorothalonil, both tested forms affected survival and reproduction. The estimated LC50 values were 117 mg (a.i.) kg−1 and 73.5 mg (a.i.) kg−1, and the EC50 41.3 mg (a.i.) kg−1 and 14.9 mg kg−1 for the formulation and the active ingredient, respectively. The effects of the active ingredient were significantly stronger, indicating the major influence of the active substance in the effects caused also by the formulation. Overall results demonstrate the importance of evaluating the effects of the formulated chemicals, as they are applied in the field, and not only their isolated active ingredients.

2019-04Journal article

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From laboratory to the field: Validating molecular markers of effect in Folsomia candida exposed to a fungicide-based formulation

Publication . Simões, Tiago; Novais, Sara C.; Natal-da-Luz, Tiago; Renaud, Mathieu; Leston, Sara; Ramos, Fernando; Römbke, Jörg; Dick, Roelofs; Van Straalen, Nico M.; Sousa, José P.; Lemos, Marco F. L.

Under controlled laboratory conditions, toxicity data tend to be less variable than in more realistic in-field studies and responses may thus differ from those in the natural environment, creating uncertainty. The validation of data under environmental conditions is therefore a major asset in environmental risk assessment of chemicals. The present study aimed to validate the mode of action of a commercial fungicide formulation in the soil invertebrate F. candida, under more realistic exposure scenarios (in-field bioassay), by targeting specific molecular biomarkers retrieved from laboratory experiments. Organisms were exposed in soil cores under minimally controlled field conditions for 4 days to a chlorothalonil fungicide dosage causing 75% reduction of reproduction in a previous laboratory experiment (127 mg a.i. kg−1 ) and half this concentration (60 mg a.i. kg−1 ). After exposure, organisms were retrieved and RNA was extracted from each pool of organisms. According to previous laboratorial omics results with the same formulation, ten genes were selected for gene expression analysis by qRT-PCR, corresponding to key genes of affected biological pathways including glutathione metabolism, oxidation-reduction, body morphogenesis, and reproduction. Six of these genes presented a dose-response trend with higher up- or down-regulation with increasing pesticide concentrations. Highly significant correlations between their expression patterns in laboratory and in-field experiments were observed. This work shows that effects of toxicants can be clearly demonstrated in more realistic conditions using validated biomarkers. Our work outlines a set of genes that can be used to assess the early effects of pesticides in a realistic agricultural scenario.

2019-06Journal article

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