Assessment of the reproduction of six collembolan species in tropical soils naturally rich in potentially toxic elements.

Laboratory ecotoxicological tests are important tools for the management of environmental changes derived from anthropogenic activities. Folsomia candida is usually the model species used in some procedures. However, this species may not be sufficiently representative of the sensitivity of the other collembolan species. This study aimed to evaluate (i) the effects of soils naturally rich in potentially toxic elements (PTE) and soil characteristics on the reproduction and survival of different collembolan species, (ii) whether the habitat function of these soils is compromised, and (iii) to what extent F. candida is representative of the other collembolan species. For this, reproduction tests with six collembolan species were conducted in 14 different samples of soils. In general, collembolan reproduction was not completely inhibited in none of the natural tested soils. Even soils with high pollution load index values did not negatively affect collembolan reproduction for most of the species. In contrast, the lowest collembolan reproduction rates were found in a visually dense soil (lowest volume/weight ratio), highlighting that soil attributes other than total PTE concentration also interfere in the reproduction of collembolans. Our results support the idea that the F. candida species might not be representative of other collembolan species and that laboratory tests to assess soil contaminations should be conducted using diverse collembolan species.

Intermediate-tier options in the environmental risk assessment of plant protection products for soil invertebrates-Synthesis of a workshop.

The European environmental risk assessment (ERA) of plant protection products follows a tiered approach. The approach for soil invertebrates currently consists of two steps, starting with a Tier 1 assessment based on reproduction toxicity tests with earthworms, springtails, and predatory mites. In case an unacceptable risk is identified at Tier 1, field studies can be conducted as a higher-tier option. For soil invertebrates, intermediate tiers are not implemented. Hence, there is limited possibility to include additional information for the ERA to address specific concerns when the Tier 1 fails, as an alternative to, for example, a field study. Calibrated intermediate-tier approaches could help to address risks for soil invertebrates with less time and resources but also with sufficient certainty. A multistakeholder workshop was held on 2-4 March 2022 to discuss potential intermediate-tier options, focusing on four possible areas: (1) natural soil testing, (2) single-species tests (other than standard species), (3) assessing recovery in laboratory tests, and (4) the use of assembled soil multispecies test systems. The participants acknowledged a large potential in the intermediate-tier options but concluded that some issues need to be clarified before routine application of these approaches in the ERA is possible, that is, sensitivity, reproducibility, reliability, and standardization of potential new test systems. The definition of suitable assessment factors needed to calibrate the approaches to the protection goals was acknowledged. The aims of the workshop were to foster scientific exchange and a data-driven dialog, to discuss how the different approaches could be used in the risk assessment, and to identify research priorities for future work to address uncertainties and strengthen the tiered approach in the ERA for soil invertebrates. This article outlines the background, proposed methods, technical challenges, difficulties and opportunities in the ERA, and conclusions of the workshop. Integr Environ Assess Manag 2024;20:780-793. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Amendment of soils with metal-rich sludge: Potential water toxicity due to metal release via over-time slaking.

Industrial metal-rich sludge can improve soil properties, but it is potentially toxic to soils and adjacent aquatic systems. The soil-sludge-water interactions influence metals bioavailability over time, a phenomenon mostly regulated by the still debatable "sludge physical protection" or "sludge delayed release" hypotheses. The present study aimed to investigate: (1) whether sludge increases soil aggregate stability against slaking, (2) which hypothesis mostly regulates metal release from soils to water and (3) the ecotoxicity of the metals released during soil slaking for aquatic organisms. Under a realistic field scale, soils amended with an industrial sludge or spiked with equivalent metal solutions (of Cr, Cu, Ni, Zn) were collected over three months to test soil aggregate stability, the ecotoxicity of the slaking water and metal contents in soil and water. The "sludge physical protection" was verified for all metals, though for Cu the "sludge delayed release" hypothesis appears plausible after three months. Soil amendment with sludge did not lead to effects on the growth of the microalga Raphidocelis subcapitata, contrarily to the observed for the metal-spiked soil. Criteria regulating soils sludge-amendment management should thus include doses not hazardous to biota, and not only metal threshold levels.

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.

Are mulch biofilms used in agriculture an environmentally friendly solution? - An insight into their biodegradability and ecotoxicity using key organisms in soil ecosystems.

Biobased and biodegradable plastic mulch films (aka, mulch biofilm) have emerged as a sustainable alternative to conventional plastic mulch films in agriculture, promising to reduce soil contamination with plastic residues through in situ biodegradation. However, current standards certifying biodegradable plastics cannot predict biodegradability in natural settings. The scarce studies considering the possible biodegradation and ecotoxicity of mulch biofilms in soil systems question the environmental friendliness of these alternative options. This study assessed the biodegradation of a commercially available mulch biofilm by the soil-dwelling fungus Penicillium brevicompactum (in solid culture media and soil for 15 and 28 days, respectively), and the ecotoxicological effects of mulch biofilm microplastics on the earthworm Eisenia andrei (pristine or UV-weathered, at 0.125-0.250-0.500 g/kg). Results (from microplastics' mass loss, microscopy, and FTIR spectroscopy) suggest that the presence of P. brevicompactum promotes mulch biofilm's biodegradation. Exposure to environmental concentrations of pristine biofilm microplastics (and its ingestion) increased earthworms' sensitivity to touch, induced physiological alterations, decreased energy reserves, and decreased their reproduction (>30%). Conversely, exposure to weathered biofilm microplastics slightly increased earthworms' sensitivity, as well as carbohydrate reserves,without affecting their reproduction. The tested mulch biofilm seems to be, at first sight, an environmentally friendly alternative as it presented susceptibility for biodegradation by a widespread fungus, and the absence of ecotoxicological chronic effects on a key macroinvertebrate species in soil ecosystems when considering environmental relevant concentrations and plastics weathered conditions. Notwithstanding, the obtained results highlight the need to revise current standards, as they often neglect the role of, and their chronic effects on, naturally occurring organisms.

The Recolonization Concentration Concept: Using Avoidance Assays with Soil Organisms to Predict the Recolonization Potential of Contaminated Sites.

In this study the recolonization concentration concept for soil organisms is presented and validated. This concept is based on the empirically deduced avoidance-recolonization hypothesis, which shows a negative correlation between avoidance (ACx) and recolonization (RCx) (ACx = RC100-x) responses. The concept was validated in a two-step approach composed by (i) individual placement tests, to demonstrate the non-influence of individual placement in a dual chamber avoidance test and (ii) small scale gradient tests to demonstrate that the number of colonizers reaching a soil patch with a certain concentration is independent on their previous exposure to lower concentrations. Overall, data show that avoidance data can be used, when framed under the recolonization concentration concept, to evaluate the recolonization potential of contaminated sites. The recolonization concept is an important theoretical concept that when coupled with spatial modelling tools could be used to tackle the spatial and temporal recovery dynamics of contaminated soil.

In situ and ex situ bioassays with Cantareus aspersus for environmental risk assessment of metal(loid) and PAH-contaminated soils.

Environmental risk assessment of contaminated soils requires bioindicators that allow the assessment of bioavailability and toxicity of chemicals. Although many bioassays can determine the ecotoxicity of soil samples in the laboratory, few are available and standardized for on-site application. Bioassays based on specific threshold values that assess the in situ and ex situ bioavailability and risk of metal(loid)s and polycyclic aromatic hydrocarbons (PAHs) in soils to the land snail Cantareus aspersus have never been simultaneously applied to the same soils. The aims of this study were to compare the results provided by in situ and ex situ bioassays and to determine their respective importance for environmental risk assessment. The feasibility and reproducibility of the in situ bioassay were assessed using an international ring test. This study used five plots located at a former industrial site and six laboratories participated in the ring test. The results revealed the impact of environmental parameters on the bioavailability of metal(loid)s and PAHs to snails exposed in the field to structured soils and vegetation compared to those exposed under laboratory conditions to soil collected from the same field site (excavated soils). The risk coefficients were generally higher ex situ than in situ, with some exceptions (mainly due to Cd and Mo), which might be explained by the in situ contribution of plants and humus layer as sources of exposure of snails to contaminants and by climatic parameters. The ring test showed good agreement among laboratories, which determined the same levels of risk in most of the plots. Comparison of the bioavailability to land snails and the subsequent risk estimated in situ or ex situ highlighted the complementarity between both approaches in the environmental risk assessment of contaminated soils, namely, to guide decisions on the fate and future use of the sites (e.g., excavation, embankments, and land restoration). Integr Environ Assess Manag 2022;18:539-554. © 2021 SETAC.

Are structural and functional endpoints of soil communities similarly affected by metal mixtures? - A terrestrial model ecosystem approach.

Soils are habitat to a variety of flora and fauna in a linked ecosystem which provides essential ecosystem services. In soil, metals can accumulate at high concentrations, because of anthropogenic activities, leading to toxic effects, threatening the ecosystem and the services it provides. In most real-world contamination scenarios, metals occur as complex mixtures which can interact and produce different toxicity than predicted from individual metal data. Current regulatory guidelines are based on single species responses to individual metals and ignore indirect effects inherent to the inter-linked nature of ecosystems. Also, the evaluation of anthropogenic impacts to the soil communities is usually measured through structural endpoints (e.g. abundance) disregarding functional measurements (e.g. organic matter decomposition rates), which are often seen as tightly related, and thus, similarly affected. In this study we tested three mixture ratios of five metal oxides (lead, copper, nickel, zinc, cobalt) at three dose levels (Low, Med, High) in a terrestrial model ecosystem experiment and measured structural and functional endpoints. Exposure to metal mixtures for 16 weeks did not affect the microarthropod community, but produced severe effects on soil microbial activity (PNR and DHA) reducing activity below 50% compared to control levels, in all dosed treatments. Metal contamination also significantly affected feeding activity and organic matter decomposition, but effects were not as pronounced as on microbial activity. Data suggest that, in the risk assessment of metals and their mixtures, effects on ecosystem structure and functions must be considered to provide adequate environmental protection.

Community effect concentrations as a new concept to easily incorporate community data in environmental effect assessment of complex metal mixtures.

The goal of this study was to incorporate community data into the effect assessment of environmental and regulatory relevant metal mixtures. In this experiment three fixed mixture ratios (Canadian soil quality guideline ratio - CSQG; Agricultural, residential and Loamy ratio - ARL; and Sudbury ratio - SUD) were tested in a natural community microcosm with 11 doses for each mixture ratio. The effect of metal mixtures on the community was measured using the community effect concentration (EC) concept which assumes that as contamination increases, the community similarity between test and control treatments decreases producing a dose response curve allowing the calculation of community effect concentrations. In regulatory mixture ratios (CSQG and ARL) community EC10s were four times higher than regulatory thresholds and current regulation might be overprotective of the microarthropod communities in some soils. For the contaminated site ratio (SUD), the field dose in the contaminated site corresponded to a community EC20 and if metal concentrations were reduced by 1TU, (from 3.1TU to 2.1TU) effects would be below a community EC10. Overall, the community EC concept was successfully applied and has the potential for inclusion in risk assessment schemes as a measure of community response.

Effect of temperature on the toxicity of imidacloprid to Eisenia andrei and Folsomia candida in tropical soils.

The influence of temperature on the chronic toxicity and risk of imidacloprid to soil non-target species was assessed in tropical soils. Earthworms Eisenia andrei and collembolans Folsomia candida were exposed to a tropical artificial soil (TAS) and two natural tropical soils from Brazil (Entisol and Oxisol) with increasing concentrations of imidacloprid under atmospheric temperatures of 20, 25 and 28 °C. The effect of temperature on the reproduction of both species was assessed through the number of juveniles and earthworm's growth, and the risk associated was estimated through the Toxicity-Exposure Ratio (TER). Toxicity of imidacloprid increased with temperature in all tested soils, being generally lower in TAS soil (EC50s of 1.48, 0.66 and 0.40 mg kg-1 for E. andrei and 0.3, 0.2 and 0.06 mg kg-1 for F. candida at 20, 25 and 28 °C, respectively) compared to Entisol (EC50s of 0.19, 0.03 and 0.14 mg kg-1 for E. andrei and 0.04, 0.02, 0.01 mg kg-1 for F. candida at 20, 25 and 28 °C, respectively) and Oxisol (EC50s of 0.21, 0.07, 0.06 mg kg-1 for E. andrei and 0.16, 0.09, 0.06 mg kg-1 for F. candida at 20, 25 and 28 °C, respectively) within each temperature for both species. These values indicate that properties of TAS may not be representative of natural/local soils to adequately estimate the toxicity of pesticides to non-target soil species. At higher temperatures, the variability of imidacloprid toxicity between soils was lower, which suggests that the influence of soil properties on imidacloprid toxicity was overshadowed by temperature. TER values revealed that risk is also greater at higher temperatures. Data reported enforce the need for the inclusion of more realistic conditions in single-species tests in prospective risk assessment of pesticides to avoid underestimation of risk to non-target species.

Assessing the most sensitive and reliable endpoints in plant growth tests to improve arsenic risk assessment.

Arsenic (As) is toxic to humans and the environment. Its toxicity has been assessed in many ways, including plant growth tests integrated in As risk assessments (RA). The tiered approach used in RA schemes assumes that lower tiers are more conservative than higher tiers. Plant growth tests may comprise lower tier of a RA and include the measurement of several endpoints. However, only few of these endpoints are highly sensitive and reliable, which makes them more appropriate to comprise lower tiers. Therefore, the selection of those endpoints is needed. The present study aimed to evaluate the most appropriate endpoints of plant growth tests to use in lower tier As RA schemes. This selection of endpoints was based on their sensitivity and reliability, using different tropical soils and plant species. In order to achieve this objective, six plant species were exposed to eight levels of As contamination (0; 8; 14.5; 26; 46.5; 84; 150; 270 mg kg-1), in three different tropical soils (Oxisol, Inceptisol, and tropical artificial soil). The endpoints measured were: first germination count (FrC), plant height (PH), relative leaf area (RLA), stem diameter (SD), total germination (TG), germination speed index (GSI), dry mass (DM), number of completely expanded leaves (CEL), plant survival (PS), soil plant analysis development chlorophyll level (SPAD), and the final germination count (FnC). Toxic values for 50% of effect were estimated for each endpoint within each species and test soil, to rank them according to their sensitivity and reliability. The most sensitive endpoints were: FrC, RLA, DM, GSI, PH, and FnC, while the most reliable endpoints were: FrC, DM, GSI, DM, PH, FnC, and TG. Our findings suggest that FrC, DM, GSI, PH, and FnC are the most adequate endpoints to be used in plant growth tests as lower tiers of As RA in tropical regions.

Assessing the Brazilian prevention value for soil arsenic: Effects on emergence and growth of plant species relevant to tropical agroecosystems.

One of the entry routes of arsenic (As) into the food chain is through the consumption of edible parts of crops contaminated by this element. Different plant species present distinctive As accumulation and tolerance capacities. These differences are also influenced by As availability and speciation in soils. This study assessed the effect of As contamination on plant emergence and initial growth, as well as on accumulated As contents in different crops grown in tropical soils. In addition, it was intended to verify the protection level of the current soil As prevention value adopted in Brazil, which should be applicable for conceivably other tropical soils in Latin America. Plants of maize, rice, sorghum, common bean, sunflower, and radish were cultivated in two different tropical soils (Oxisol and Inceptisol) and in a standard substrate (tropical artificial soil - TAS) dosed with As (0; 8; 14.5; 26; 46.5; 84; 150; 270 mg kg-1). Early germination, total dry mass, As content, and bioconcentration factor were evaluated. The EC20 and EC50 values (the As concentration for 20% or 50% of effect relative to control treatment) based on total As concentration were more variable among different soils than the corresponding EC20 and EC50 values based on extractable (phytoavailable) As concentration. From the studied species, common bean was the most sensitive and maize was the least sensitive to As. Those species were the ones that accumulated the lowest As levels in shoot tissues. Arsenic concentrations measured in plant tissues and estimated bioaccumulation factors were not related to relative As toxicity among species. Data obtained suggest that the current Brazilian prevention value for arsenic is adequate for soils with high arsenic adsorption capacity.

Wing membrane and fur samples as reliable biological matrices to measure bioaccumulation of metals and metalloids in bats.

There is a growing conservation concern about the possible consequences of environmental contamination in the health of bat communities. Most studies on the effects of contaminants in bats have been focused on organic contaminants, and the consequences of bat exposure to metals and metalloids remain largely unknown. The aim of this study was to evaluate the suitability of external biological matrices (fur and wing membrane) for the assessment of exposure and bioaccumulation of metals in bats. The concentration of arsenic, cadmium, cobalt, chromium, copper, manganese, nickel, lead, selenium and zinc was measured in internal organs (liver, heart, brain), internal (bone) and external tissues (wing membrane, fur) collected from bat carcasses of four species (Hypsugo savii, Nyctalus leisleri, Pipistrellus pipistrellus, Pipistrellus pygmaeus) obtained in windfarm mortality searches. With the exception of zinc (P = 0.223), the results showed significant differences between the concentrations of metals in the analyzed tissues for all metals (P < 0.05). Significant differences were also found between organs/tissues (P < 0.001), metals (P < 0.001) and a significant interaction between organs/tissues and metals was found (P < 0.001). Despite these results, the patterns in terms of metal accumulation were similar for all samples. Depending on the metal, the organ/tissue that showed the highest concentrations varied, but fur and wing had the highest concentrations for most metals. The variability obtained in terms of metal concentrations in different tissues highlights the need to define standardized methods capable of being applied in monitoring bat populations worldwide. The results indicate that wing membrane and fur, biological matrices that may be collected from living bats, yield reliable results and may be useful for studies on bats ecotoxicology, coupled to a standardized protocol for large-scale investigation of metal accumulation.

Exploring the Use of Species Sensitivity Distributions to Define Protective Limits for the Use of Organic Wastes as Soil Amendments.

The use of organic wastes as soil amendments can be an important measure to improve soil quality and reduce waste accumulation and landfilling. However, the potential contaminant loads of such wastes, can be a source of environmental concern. Consequently, legislation has been developed to regulate the use of these wastes in agricultural soils. However, the regulations only consider chemical parameters, which are insufficient to establish the level of environmental risk. A possible solution is the use of species sensitivity distributions (SSDs), employing ecotoxicological data from test batteries that could be incorporated into legislation. In the present study, 2 different hazardous concentrations affecting 5 and 50% of the soil community (HC5 and HC50, respectively) were determined using ecotoxicological data (effect concentrations, 10 and 50% [EC10 and EC50, respectively]) for 5 different wastes. The results demonstrate that, as expected, current legislative thresholds do not translate to environmental risk/protection and that SSDs may be an important tool allowing the simple inclusion and interpretation of ecotoxicological data from test batteries in legislation. On the other hand, SSDs must be used with caution because there are still doubts about their actual value in risk prediction and about which estimates provide adequate protection. For instance, the use of HC50EC10 values is not recommended; these values overlap with the more conservative HC5EC50 data, highlighting the fact that the use of lower effect concentrations may not always provide the most protective approach. Also, hazardous concentrations need to be calibrated at the field or semifield level, to verify environmental protection in different soils/environments and the adequacy of standard test organisms. Environ Toxicol Chem 2019;38:1569-1576. © 2019 SETAC.

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

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 ±â€¯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.

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

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.

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

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 2-3 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.

Should oral exposure in Hypoaspis aculeifer tests be considered in order to keep them in Tier I test battery for ecological risk assessment of PPPs?

The laboratory reproduction test with the predatory mite Hypoaspis aculeifer is currently a mandatory test in the new EU data requirements for prospective environmental risk assessment of Plant Protection Products (PPPs). However, the low sensitivity often shown by this mite towards PPPs, when compared to other invertebrates (namely Folsomia candida and Eisenia fetida), makes the test with this species not very useful in the lower tier test battery. However, the current test protocol only considers exposure to contaminants via contaminated soil, disregarding exposure via contaminated food and does not take into account the fact that H. aculeifer is a predatory species. Therefore, through this protocol, the toxicity of contaminants to soil mites might be underestimated and, thus, an adaptation of the test performance, by including exposure via contaminated food, may be necessary. With this aim, two reproduction tests with H. aculeifer were performed using copper chloride as model substance, artificial soil as test substrate and cheese mites as food. The OECD guideline was followed but, while in one test cheese mites from normal laboratory breeding cultures (clean prey mites) were provided, in the other test, cheese mites previously exposed to copper (Cu pre-exposed prey mites) were provided. Predatory mites were affected at lower concentrations in tests using Cu pre-exposed prey compared to test with clean-prey (NOEC = 1225 and 1508 mg kg-1 and EC10 = 1204 and 1903 mg kg-1 using Cu pre-exposed and clean prey, respectively). However, this higher sensitivity was not detected by EC50 values (EC50 = 2634 and 2814 mg kg-1 using Cu pre-exposed and clean prey, respectively). Further tests are needed in order to (i) investigate the relevance of oral exposure to different PPPs, (ii) optimize the contamination of prey mites according to the chemical properties of each substance and (iii) substantiate a proposal to adapt the standard protocol.

An integrative omics approach to unravel toxicity mechanisms of environmental chemicals: effects of a formulated herbicide.

The use of integrative molecular approaches can aid in a comprehensive understanding of the effects of toxicants at different levels of biological organization, also supporting risk assessment. The present study aims to unravel the toxicity mechanisms of a widely used herbicide to the arthropod Folsomia candida exposed in a natural soil, by linking effects on reproduction, proteomics and genome-wide gene expression. The EC50 effects on reproduction over 4 weeks was 4.63 mg glyphosate/kg of soil. The formulation included a polyethoxylated tallowamine as an adjuvant, which at 50% effect on reproduction had an estimated concentration of 0.87-1.49 mg/kg of soil. No effects were observed on survival and reproduction when using the isolated active substance, pointing the toxicity of the formulated product to the co-formulant instead of the active ingredient, glyphosate. RNA sequencing and shotgun proteomics were applied to assess differential transcript and protein expressions between exposed and control organisms in time, respectively. Specific functional categories at protein and transcriptome levels were concordant with each other, despite overall limited correlations between datasets. The exposure to this formulation affected normal cellular respiration and lipid metabolism, inducing oxidative stress and leading to impairment in biological life cycle mechanisms such as molting and reproduction.

Bacterial communities in soil become sensitive to drought under intensive grazing.

Increasing climatic and anthropogenic pressures on soil ecosystems are expected to create a global patchwork of disturbance scenarios. Some regions will be strongly impacted by climate change, others by agricultural intensification, and others by both. Soil microbial communities are integral components of terrestrial ecosystems, but their responses to multiple perturbations are poorly understood. Here, we exposed soils from sustainably- or intensively-managed grasslands in an agro-silvo-pastoral oak woodland to month-long intensified drought and flood simulation treatments in a controlled mesocosm setting. We monitored the response of the bacterial communities at the end of one month as well as during the following month of recovery. The communities in sustainably-managed plots under all precipitation regimes were richer and more diverse than those in intensively-managed plots, and contained a lower proportion of rapidly-growing taxa. Soils from both land managements exhibited changes in bacterial community composition in response to flooding, but only intensively-managed soils were affected by drought. The ecologies of bacteria favored by both drought and flood point to both opportunism and stress tolerance as key traits shaping the community following disturbance. Finally, the response of several taxa (i.e. Chloracidobacteria RB41, Janthinobacterium sp.) to precipitation depended on land management, suggesting that the community itself affected individual disturbance responses. Our findings provide an in-depth view of the complexity of soil bacterial community responses to climatic and anthropogenic pressures in time, and highlight the potential of these stressors to have multiplicative effects on the soil biota.