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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)., (© 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Natal-da-Luz, Tiago reports financial support was provided by H2020 Food Security Sustainable Agriculture and Forestry Marine Maritime and Inland Water Research and the Bioeconomy. Has patent pending to., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 (AC x ) and recolonization (RC x ) (AC x = RC 100-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.

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., (© 2021 SETAC.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 (EC 50 s 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 (EC 50 s 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 (EC 50 s 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 EC 20 and EC 50 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 EC 20 and EC 50 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., (Copyright © 2019. Published by Elsevier B.V.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 HC50 EC10 values is not recommended; these values overlap with the more conservative HC5 EC50 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., (© 2019 SETAC.)

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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 EC 10  = 1204 and 1903 mg kg -1 using Cu pre-exposed and clean prey, respectively). However, this higher sensitivity was not detected by EC 50 values (EC 50  = 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Neonicotinoids are the most prominent group of insecticides in the world and are commercialized in over 120 countries for the control of agricultural pests mainly due to their broad-spectrum activity and versatility in application. Though non-target soil organisms are likely to be exposed during application, there is paucity of information in scientific literature regarding their sensitivity to neonicotinoids. This study attempts to fill this gap by evaluating, under laboratory conditions, the chronic toxicity of the neonicotinoids thiacloprid and acetamiprid, through their commercial formulations (CF), to the soil invertebrates Folsomia candida, Eisenia andrei and Enchytraeus crypticus. Results obtained indicate that the relative reproductive sensitivity of the test organisms can be expressed as: F. candida = E. andrei > E. crypticus (for acetamiprid CF) and E. andrei > F. candida > E. crypticus (for thiacloprid CF). To extrapolate from laboratory test results to field conditions, predicted environmental concentrations (PECs) and predicted no-effect concentrations were derived. Calculated toxicity-exposure ratios (TER = EC10/PEC) were below trigger values for acetamiprid and thiacloprid, when estimated with initial PEC. While estimated hazard quotients (HQ = PEC/PNEC), were greater than the European Commission trigger value. Therefore, with the current data under standard environmental risk assessment schemes it can be considered that the risk of thiacloprid and acetamiprid to the soil compartment is unacceptable. However, further research into the effects of these substances on different organisms is required to increase the confidence in the risk assessment estimates for instance, by calculating hazardous concentrations using species sensitivity distribution curves., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Using organic wastes, as soil amendments, is an important alternative to landfilling with benefits to soil structure, water retention, soil nutrient and organic matter concentrations. However, this practice should be monitored for its environmental risk due to the frequent presence, of noxious substances to soil organisms. To evaluate the potential of eight organic wastes with different origins, as soil amendments, reproduction tests with four soil invertebrate species (Folsomia candida, Enchytraeus crypticus, Hypoaspis aculeifer, Eisenia fetida) were performed using gradients of soil-waste mixtures. Results obtained demonstrated that contaminant concentrations required by current legislation might not be a protective measure for the soil ecosystem, as they do not properly translate the potential toxicity of wastes to soil invertebrates. Some wastes with contaminant loadings below thresholds showed higher toxicity than wastes with contaminants concentrations above legal limits. Also, test organism reproduction was differently sensitive to the selected wastes, which highlights the need to account for different organism sensitivities and routes of exposure when evaluating the toxicity of such complex mixtures. Finally this study shows that when combining chemical and ecotoxicological data, it is possible to postulate on potential sources of toxicity, contributing to better waste management practices and safer soil organic amendment products., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Field-based studies are an essential component of research addressing the behavior of organic chemicals, and a unique line of evidence that can be used to assess bioaccumulation potential in chemical registration programs and aid in development of associated laboratory and modeling efforts. To aid scientific and regulatory discourse on the application of terrestrial field data in this manner, this article provides practical recommendations regarding the generation and interpretation of terrestrial field data. Currently, biota-to-soil-accumulation factors (BSAFs), biomagnification factors (BMFs), and bioaccumulation factors (BAFs) are the most suitable bioaccumulation metrics that are applicable to bioaccumulation assessment evaluations and able to be generated from terrestrial field studies with relatively low uncertainty. Biomagnification factors calculated from field-collected samples of terrestrial carnivores and their prey appear to be particularly robust indicators of bioaccumulation potential. The use of stable isotope ratios for quantification of trophic relationships in terrestrial ecosystems needs to be further developed to resolve uncertainties associated with the calculation of terrestrial trophic magnification factors (TMFs). Sampling efforts for terrestrial field studies should strive for efficiency, and advice on optimization of study sample sizes, practical considerations for obtaining samples, selection of tissues for analysis, and data interpretation is provided. Although there is still much to be learned regarding terrestrial bioaccumulation, these recommendations provide some initial guidance to the present application of terrestrial field data as a line of evidence in the assessment of chemical bioaccumulation potential and a resource to inform laboratory and modeling efforts., (© 2015 SETAC.)

The impact of sugarcane vinasse on soil invertebrates was assessed through ecotoxicological assays. Increasing concentrations of two vinasses from different distillery plants (VA and VB), and a vinasse from a laboratory production (VC), were amended on two natural tropical Oxisols (LV and LVA) and a tropical artificial soil (TAS) to characterize the effects of the vinasses on earthworms (Eisenia andrei), enchytraeids (Enchytraeus crypticus), mites (Hypoaspis aculeifer) and collembolans (Folsomia candida). The highest concentrations of VA and VB were avoided by earthworms in all soils and by collembolans especially in the natural soils. The presence of VC in all of the tested soils did not cause avoidance behavior in these species. The reproduction of earthworms, enchytraeids and collembolans was decreased in the highest concentrations of VA and VB in the natural soils. In TAS, VB reduced the reproduction of all test species, whereas VA was toxic exclusively to E. andrei and E. crypticus. The vinasse VC only reduced the number of earthworms in TAS and enchytraeids in LVA. The reproduction of mites was reduced by VB in TAS. Vinasses from distillery plants were more toxic than the vinasse produced in laboratory. The vinasse toxicities were influenced by soil type, although this result was most likely because of the way the organisms are exposed to the contaminants in the soils. Toxicity was attributed to the vinasses' high salt content and especially the high potassium concentrations. Data obtained in this study highlights the potential risk of vinasse disposal on tropical soils to soil biota. The toxic values estimated are even more relevant when considering the usual continuous use of vinasses in crop productions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Chromate can be considered a potent environmental contaminant and consequently, an understanding of chromate availability and toxicity to soil biology is essential for effective ecological assessment of metal impact in soils. This study shows the response of two bacterial bioreporters, pCHRGFP1 Escherichiacoli and pCHRGFP2 Ochrobactrumtritici, to increasing concentrations of chromate in two different soils. The bioreporters, carrying the regulatory gene chrB transcriptionally fused to the gfp reporter system, exhibited different features. In both, the fluorescence signal and the chromate concentration could be linearly correlated but E. coli biosensor functioned within the range of 0.5-2 μM and O. tritici biosensor within 2-10 μM chromate. The bioreporters were validated through comparative measurements using the chemical chromate methods of diphenylcarbazide and ionic chromatography. The bacterial sensors were used for the estimation of bioavailable fraction of chromate in a natural soil and OECD artificial soil, both spiked with chromate in increasing concentrations of 0-120 mg Cr(VI) kg(-1) of soil. OECD soil showed a faster chromate decrease comparing to the natural soil. The toxicity of soils amended with chromate was also evaluated by ecotoxicological tests through collembolan reproduction tests using Folsomia candida as test organism. Significant correlations were found between collembolans reproduction and chromate concentration in soil (lower at high chromate concentrations) measured by biosensors. Data obtained showed that the biosensors tested are sensitive to chromate presence in soil and may constitute a rapid and efficient method to measure chromate availability in soils., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The ecotoxicity of a dredged sediment from the Guanabara Bay (Rio de Janeiro, RJ, Brazil) was evaluated using reproduction tests with Eisenia andrei, Folsomia candida and Enchytraeus crypticus, and avoidance and feeding inhibition tests with Folsomia candida. The sediment was mixed with artificial soil to obtain the following doses: 1.25, 2.5, 5.0, 10.0, 20.0 and 40.0 %. Lead, nickel, chromium, copper and zinc concentrations were determined in the test mixtures. In reproduction tests, E. andrei was the most sensitive species (EC50 = 2.94 %), followed by F. candida (EC50 = 7.72 %) and E. crypticus (EC50 = 10.10 %). The percentage of initial weight of earthworms was significantly higher in all test concentrations compared to the control except at the highest one where earthworms biomass significantly decreased. No feeding inhibition of F. candida was observed for any test mixture and the number of organisms with a dark gut (the fed collembolans) generally increased with the increasing dose of sediment. Significant avoidance responses of F. candida were observed towards all test mixtures, however, the avoidance behaviour was the less sensitive endpoint after feeding inhibition. The results showed that chemical analysis is not sufficient to foresee toxic effects in terrestrial systems resulting from sediment disposal in soil if not complemented with an ecotoxicological evaluation.

Metal reference values established in Brazilian legislation for terrestrial disposal of dredged sediments and soil quality were derived for temperate regions. To evaluate the adequacy of such metal reference values to tropical soils, the ecotoxicity of a dredged sediment (from the Guanabara bay, Rio de Janeiro, Brazil) was investigated in two local soils (ferralsol and chernosol) by performing avoidance and reproduction tests using Folsomia candida and Enchytraeus crypticus. Test doses consisted of 0 %, 1.25 %, 2.5 %, 5, 10 %, and 20 %. Total and potentially bioavailable metal concentrations were determined in the test mixtures. Although the chernosol mixtures had the highest total metal concentrations, the influence of the expansive clay minerals (with high ability to adsorb metals) and the high contents of nutrients typical from this type of soils, seem to reduce the ecotoxicity. Collembolan avoidance behavior was the least sensitive endpoint. The lowest sediment doses increased the reproduction of F. candida in ferralsol mixtures. E. crypticus reproduction in the ferralsol mixtures were more pronounced at lower concentrations than in chernosol mixtures. Possibly the low nutrient content of the ferralsols, in connection with the addition of small amounts of sediment, created particular conditions that promoted reproduction of the test species. Data obtained in the ecotoxicological tests may support the establishment of a "safe" ecological dose of dredged sediments to be applied in tropical soils, supporting decision-makers in programs of environmental management.

The upper limit concentrations of metals established by international legislations for dredged sediment disposal and soil quality do not take into consideration the properties of tropical soils (generally submitted to more intense weathering processes) on metal availability and ecotoxicity. Aiming to perform an evaluation on the suitability of these threshold values in tropical regions, the ecotoxicity of metal-contaminated dredged sediment from the Guanabara Bay (Rio de Janeiro, Brazil) was investigated. Acute and avoidance tests with Eisenia andrei were performed with mixtures of dredged sediment with a ferralsol (0.00, 6.66, 13.12, 19.98, and 33.30 %) and a chernosol (0.00, 6.58, 13.16, 19.74, and 32.90 %). Mercury, lead, nickel, chromium, copper, and zinc concentrations were measured in test mixtures and in tissues of surviving earthworms from the acute tests. While ferralsol test mixtures provoked significant earthworm avoidance response at concentrations ≥13.31 %, the chernosol mixtures showed significant avoidance behavior only at the 19.74 % concentration. The acute tests showed higher toxicity in ferralsol mixtures (LC50 = 9.9 %) compared to chernosol mixtures (LC50 = 16.5 %), and biomass increased at the lowest sediment doses in treatments of both test soils. Most probably, the expansive clay minerals present in chernosol contributed to reduce metal availability in chernosol mixtures, and consequently, the ecotoxicity of these treatments. The bioconcentration factors (BCF) for zinc and copper were lower with increasing concentrations of the dredged sediment, indicating the existence of internal regulating processes. Although the BCF for mercury also decreased with the increasing test concentrations, the known no biological function of this metal in the earthworms metabolism lead to suppose that Hg measured was not present in bioaccumulable forms. BCFs estimated for the other metals were generally higher in the highest dredged sediment doses.

The replacement of native Portuguese forests by Eucalyptus globulus is often associated with deleterious effects on terrestrial and aquatic communities. Several studies have suggested that such a phenomenon is linked with the leaf essential oils released into the environment during the Eucalyptus leaf degradation process. However, to date, the way these compounds affect leaf degradation in terrestrial systems i.e. by direct toxic effects to soil invertebrates or indirectly by affecting food of soil fauna, is still unknown. In order to explore this question, the effect of essential oils extracted from E. globulus leaves on terrestrial systems was investigated. Fungal growth tests with species known as leaf colonizers (Mucor hiemalis, Alternaria alternata, Penicillium sp., Penicillium glabrum and Fusarium roseum) were performed to evaluate the antifungal effect of essential oils. In addition, a reproduction test with the collembolans Folsomia candida was done using a gradient of eucalyptus essential oils in artificial soil. The influence of essential oils on feeding behaviour of F. candida and the isopods Porcellio dilatatus was also investigated through food avoidance and consumption tests. Eucalyptus essential oils were lethal at concentrations between 2.5-20 µL/mL and inhibited growth of all fungal species between 1.25-5 µL/mL. The collembolan reproduction EC50 value was 35.0 (28.6-41.2) mg/kg and both collembola and isopods preferred leaves without oils. Results suggested that the effect of essential oils in leaf processing is related to direct toxic effects on fungi and soil fauna and to indirect effects on the quality and availability of food to soil invertebrates.

The impact of diazinon spraying in an agricultural tropical soil through the evaluation of both the habitat and retention functions of the soil system was never reported. To fill this gap, five times the recommended dose of a commercial diazinon formulation was sprayed in an agricultural area of Costa Rica, and dilution gradients of the sprayed soil were prepared in the laboratory. Avoidance and reproduction tests with soil organisms (Eisenia andrei, Enchytraeus crypticus and Folsomia candida) to evaluate losses in terrestrial habitat function, and growth and reproduction tests with aquatic organisms (Chlorella vulgaris and Daphnia magna, respectively) to evaluate the retention function of soil were performed. Results demonstrated that regarding habitat function, F. candida reproduction was the most sensitive endpoint (EC(50) = 0.288 mg a.i./kg), followed by avoidance behaviour of E. andrei (EC(20) = 1.75 mg a.i./kg). F. candida avoidance and the reproduction of E. andrei and E. crypticus were not affected by diazinon. The toxicity tests with aquatic organisms showed that the soil retention function was insufficient to prevent effects of diazinon either on microalgae growth (EC(50) ≤ 0.742 mg/L and EC(20) ≤ 0.223 mg/L) and on the reproduction of the cladoceran (EC(50) ≤ 0.00771 mg/L and EC(20) ≤ 0.00646 mg/L). Results suggested that diazinon exerted toxic effects even at the dilution corresponding to the recommended dose, fact which makes its misuse an issue of environmental concern. Furthermore, the present study highlighted the importance and complementary nature of the assessment of both habitat and retention functions to an ecological risk assessment in tropical systems.

Earthworms may promote the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in soil, but the mechanism through which they exert such influence is still unknown. To determine if the stimulation of PAH degradation by earthworms is related to changes in microbial communities, a microcosm experiment was conducted consisting of columns with natural uncontaminated soil covered with PAH-contaminated dredge sediment. Columns without and with low and high Eisenia andrei densities were prepared. Organic matter and PAH content, microbial biomass, and dehydrogenase activity (DHA) were measured in soil and sediment over time. Biolog Ecoplate™ and polymerase chain reaction using denaturing gradient gel electrophoresis were used to evaluate changes in metabolic and structural diversity of the microbial community, respectively. Earthworm activity promoted PAH degradation in soil, which was significant for biphenyl, benzo[a]pyrene, and benzo[e]pyrene. Microbial biomass and DHA activity generally did not change over the experiment. Earthworm activity did change microbial community structure, but this did not affect its functioning in terms of carbon substrate consumption. Results suggest no relationship between changes in the microbial community by earthworm activity and increased PAH disappearance. The role of shifts in soil microbial community structure induced by earthworms in PAH removal needs further investigation., (Copyright © 2011 SETAC.)