Soil pH influences the toxicity of Basamid® eluates to non-target species of primary consumers.

Basamid® is a fumigant nematicide and fungicide known to break down in several volatile compounds, mainly methyl isothiocyanate (MITC), when in contact with water. Soil abiotic parameters, such as pH, influences this breakdown process, and thus, the toxic effects of Basamid® to aquatic biota. This work studied the influence of soil pH (5.5, 6.5 and 7.5) on the toxicity of eluates (1:4, m:v), obtained from Basamid®-contaminated soils (with the recommended dose of 145 mg of dazomet/Kg of soil), on two primary consumers: Daphnia magna and Brachionus calyciflorus. For this, lethal and sublethal toxicity of eluates originated from soils at pH 5.5, 6.5 and 7.5, contaminated with Basamid® (Ba-E 5.5; 6.5 and 7.5, respectively), were assessed (dilutions between 0.096 - 100%). The LD50,24h of Basamid® eluates for D. magna varied from 3.07% to 7.82% (Ba-E 6.5 and Ba-E 5.5 respectively), while for B. calyciflorus varied from 18.1% to 84.7% (Ba-E 6.5 and Ba-E 7.5, respectively). Both species were less sensitive to Basamid® eluates originated from soils with pH 7.5 and more sensitive to those obtained from soils with pH 6.5. Regarding the sublethal effects, a lower soil pH was associated with a higher toxicity of Basamid® to D. magna reproduction (LOED: 0.125% Ba-E 5.5), while for B. calyciflorus such a higher toxicity was observed at the highest soil pH (ED20: 7.42% [5.10-9.74] at Ba-E 7.5). These results show a negative association between soil pH and the lethal toxicity of Basamid® contaminated eluates. However, such a pattern was not observed at sublethal level, at which a species dependency was observed regarding the influence of soil pH in the observed toxicity. Nevertheless, it is to highlight that very low concentrations of eluates (as 3.07%) caused significant mortality, indicating a high risk for freshwater biota. Considering that Basamid® is likely to reach the aquatic systems is real, for which reason the recommended dose must be reviewed at environmentally-relevant scenarios.

Ecotoxicity of eluates obtained from Basamid® contaminated soils is pH dependent: A study with Hydra viridissima, Xenopus laevis and Danio rerio.

Agrochemicals are mostly used to deplete pests and treat diseases in terrestrial agro-ecosystems. However, their transport through the soil, by leaching and/or runoff, may cause them to reach aquatic systems. Environmental parameters, such as soil pH, can affect this transport, by influencing the magnitude of agrochemicals degradation and chemical reaction. This work aimed at investigating the influence of soil pH on the toxicity of eluates obtained from Basamid® contaminated soils to Hydra viridissima, Xenopus laevis and Danio rerio. For this, a natural soil with pH amended to 5.5, 6.5 and 7.5, was spiked with the recommended dose (RD) of Basamid® (145 mg dazomet/kg soil) and eluates (Ba-E) were prepared with the respective species culture medium. Dilutions of the eluates (0.14-100%), obtained from the three soils (Ba-E 5.5, Ba-E 6.5 and Ba-E 7.5, corresponding to soil spiked with Basamid® RD at soil pH of 5.5, 6.5 and 7.5, respectively), were used to expose the organisms. Results showed that for H. viridissima increased soil alkalinity provoked less mortality comparatively to lower soil pH [LD50,96h of Ba-E 5.5: 10.6% and LD50,96h of Ba-E 7.5: 21.2%]. As for X. laevis and D. rerio Ba-E lethal ecotoxicity was similar across soil pH (LD50,96h varied from 5.7 to 6.9% and from 2.1 to 4.3%, respectively). For malformations, 20% effect dilution (ED) in H. viridissima was significantly higher at Ba-E 7.5 (ED20,96h: 17.4%), comparatively to Ba-E 5.5 and Ba-E 6.5 (ED20,96h: 7.9% and 7.7%, respectively). From the three tested organisms and based on both lethal and sublethal effects, H. viridissima presented the highest tolerance to Basamid® eluates and soil pH was a major factor determining the fumigant toxicity, with higher soil pH levels inducing, lower toxicity. The eluates obtained from soils contaminated with RD of Basamid® induced severe effects to the three aquatic species.

Soil pH matters in the ecotoxicity of Basamid® to freshwater microalgae and macrophytes.

Intensive agriculture along with the use of agrochemicals has been associated with low soil fertility, soil erosion, and soil acidity. Management of soil pH through liming is a common practice in agriculture to increase soil fertility and nutrient availability. When altering soil pH, different chemical reactions occur depending on soil composition and agrochemicals presence. Basamid® is a fumigant used worldwide targeting soil nematodes, fungi, and weeds in diverse crops, that can reach freshwater ecosystems by leaching through the soil layers. The major goal of this work was to assess the influence of soil pH in the toxicity of Basamid® eluates to the microalgae Raphidocelis subcapitata and the duckweed Lemna minor. For this, eluates were prepared from soils with different pH (5.5, 6.5 and 7.5), contaminated with the recommended dose of Basamid® corresponding to 145.7 mg of dazomet/Kg soil. Soil was amended with calcium carbonate (CaCO3). Raphidocelis subcapitata and L. minor were exposed to the eluates during 72 h and 7 days respectively, and multiple endpoints were assessed: growth rate, biomass, pigment as chlorophyl content and cell damage. Results showed that soil pH can influence the performance of the tested species and also be a major factor in influencing Basamid®'s toxicity. However, a clear pattern of the influence of soil pH on Basamid®'s toxicity was not observed and was species dependent. For R. subcapitata lower soil pHs induced higher toxicity of Basamid®'s to the algae [ED50 for growth rate: 30 % (confidence limits-CL: 22.8-37.2) for soil pH 5.5; >100 % for soil pH 6.5 and pH 7.5], while for L. minor the opposite was observed [ED50 for number of fronds: 27.2 % (CL: 22.8-31.6) for pH 5.5; 20.3 % (CL: 10.0-30.6) for pH 6.5 and 10.7 % (CL: 6.3-15.1)]. Overall, these results showed that leachates of Basamid® through soils, at recommended doses, can have a severe impact on aquatic systems, with or without the influence of abiotic factors.

Representativeness of Folsomia candida to assess toxicity of a new generation insecticide in different temperature scenarios.

Climate change may interfere with the behavior of pesticides and organisms, influencing pesticides toxicity to non-target organisms like collembolans. Aiming to assess the representativeness of the standardized species F. candida to the Collembola group under different temperatures, four species of collembolans - Folsomia candida, Folsomia fimetaria, Proisotoma minuta and Sinella curviseta - were exposed to a new generation insecticide of Chlorantraniliprole, under the standardized temperature of 20 °C, and a temperature foreseeing a global warming scenario of 25 °C. Results showed that F. candida, F. fimetaria and P. minuta were sensitive to Chlorantraniliprole at both temperatures, while S. curviseta was insensitive to the insecticide concentrations up to 457 mg a.i./kg of soil, regardless of the temperature. The sensitivity of F. candida and P. minuta was significantly higher at 25 °C than at 20 °C, while F. fimetaria and S. curviseta remained equally sensitive/insensitive to both temperatures. Results suggest that F. candida can be representative of F. fimetaria under standard conditions but not for F. fimetaria under 25 °C nor for P. minuta and S. curviseta under both temperatures due to the higher sensitivity of F. candida. On the other hand, due to its higher sensitivity, F. candida can be used to define environmentally protective measures (at both test temperatures) but the use of additional Collembola species is recommended to avoid the definition of over-protective goals.

Feeding exposure and feeding behaviour as relevant approaches in the assessment of the effects of micro(nano)plastics to early life stages of amphibians.

The sedimentation of micro and nanosized plastics is of considerable environmental relevance and the need to assess its sublethal effects to biota increasingly recognized. In their majority, as bottom, non-selective grazers, independent-feeding young life stages of amphibians, an already severely endangered worldwide group, may be particularly vulnerable to sedimented plastics. Alongside, they may be good model organisms for the assessment of the effects of micro(nano)plastics (MNPs) through ingestion. However, to our knowledge, few studies have assessed amphibians' exposure to MNPs through contaminated food or its effects in feeding behaviour assays. The available studies reveal a lack of consistent methodology: organisms, food type, media of exposure, or exposure conditions (temperature and light) in the assessment of effects. This perspective article, will address major differences found in the available studies, identifying type, size and concentrations of the polymers tested, species, and observed effects, aiming to highlight the importance of feeding exposure assays when attempting to evaluate the effect of MNPs in amphibians.

Effects of Long-Term Exposure to Increased Salinity on the Amphibian Skin Bacterium Erwinia toletana.

Amphibian's skin bacterial community may help them to cope with several types of environmental perturbations, including osmotic stress caused by increased salinity. This work assessed whether an amphibian skin bacterium could increase its tolerance to NaCl after a long-term exposure to this salt. A strain of Erwinia toletana, isolated from the skin of Pelophylax perezi, was exposed to two salinity scenarios (with 18 g/L of NaCl): (1) long-term exposure (for 46 days; Et-NaCl), and (2) long-term exposure followed by a recovery period (exposure for 30 days to NaCl and then to LB medium for 16 days; Et-R). After exposure, the sensitivity of E. toletana clonal populations to NaCl was assessed by exposing them to 6 NaCl concentrations (LB medium spiked with NaCl) plus a control (LB medium). Genotypic alterations were assessed by PCR-based molecular typing method (BOX-PCR). The results showed that tolerance of E. toletana to NaCl slightly increased after the long-term exposure, EC50 for growth were: 22.5 g/L (8.64-36.4) for Et-LB; 30.3 g/L (23.2-37.4) for Et-NaCl; and 26.1 g/L (19.332.9) for Et-R. Differences in metabolic activity were observed between Et-LB and Et-R and between Et-NaCl and Et-R, suggesting the use of different substrates by this bacterium when exposed to salinized environments. NaCl-induced genotypic alterations were not detected. This work suggests that E. toletana exposed to low levels of salinity, activate different metabolic pathways to cope with osmotic stress. These findings may be further explored to be used in bioaugmentation procedures through the supplementation with this bacterium of the skin microbiome of natural populations of amphibians exposed to salinization.

An integrated view of redox and catalytic properties of B-type PpDyP from Pseudomonas putida MET94 and its distal variants.

PpDyP from Pseudomonas putida MET94 is an extremely versatile B-type dye-decolourising peroxidase (DyP) capable of efficient oxidation of a wide range of anthraquinonic and azo dyes, phenolic substrates, the non-phenolic veratryl alcohol and even manganese and ferrous ions. In reaction with H2O2 it forms a stable Compound I at a rate of (1.4±0.3)×10(6)M(-1)s(-1), comparable to those of classical peroxidases and other DyPs. We provide the first report of standard redox potential (E(0')) of the Compound I/Native redox couple in a DyP-type peroxidase. The value of E(0')Cpd I/N=1.10±0.04 (V) is similar to those found in peroxidases from the mammalian superfamily but higher than in peroxidases from the plant superfamily. Site-directed mutagenesis has been used to investigate the role of conserved distal residues, i.e. to replace aspartate 132 by asparagine, and arginine 214 and asparagine 136 by leucine. The structural, redox and catalytic properties of variants are addressed by spectroscopic, electrochemical and kinetic measurements. Our data point to the importance of the distal arginine in the catalytic mechanism of PpDyP, as also observed in DyPB from Rhodococcus jostii RHA1 but not in DyPs from the A and D subfamilies. This work reinforces the idea of existence of mechanistic variations among members of the different sub-families of DyPs with direct implications for their enzymatic properties and potential for biotechnological applications.