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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).)

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 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.)

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.)

A field study was established to assess the effects of a sewage sludge (SS), a mixed municipal solid waste compost (MMSWC) and a compost produced from agricultural wastes (AWC), in a Vertisol, using Lolium multiflorum L. The amendments were applied for two consecutive years: 6, 12 and 24t dry matter ha -1 for SS, and the amendment doses for MMSWC and AWC were calculated to deliver the same amount of organic matter (OM) per unit area. The amendments had significant beneficial effects on some soil properties (e.g. soil OM, N Kjeldahl , extractable P and K), and on plant productivity parameters (e.g. biomass yield, chlorophyll, foliar area). For instance, soil OM increased from 0.78% to 1.71, 2.48 and 2.51%, after two consecutive years of application of 24t dry matter ha -1 of SS, MMSWC and AWC, respectively, while the plant biomass obtained increased from 7.75tha -1 to 152.41, 78.14 and 29.26tha -1 , for the same amendments. On the plant, effects were more pronounced for SS than for both compost applications, a consequence of its higher capacity to provide N to the plant in a readily available form. However, after two years of application, the effects on soil properties were more noticeable for both composts, as their OM is more resistant to mineralization, which endures their beneficial effects on soil. Cadmium, Cr, Ni and Pb pseudo-total concentrations, were not affected significantly by the application of the organic wastes to soil, in all tested doses, neither their extractability by 0.01M CaCl 2 . On the contrary, Cu and Zn pseudo-total concentrations increased significantly in the second year of the experiment, following the application of the higher rate of MMSWC and AWC, although their extractability remained very low (<0.5% of their pseudo-total fraction). Trace elements concentrations in the aboveground plant material were lower than their maximum tolerable levels for cattle, used as an indicator of risk of their entry into the human food chain. Despite these results, it is interesting to note that the SS promoted a significant increase in the foliar concentrations of Cu, Ni and Zn that did not happen in composts application, which can be explained by the reduction of the soil pH, as a consequence of SS degradation in soil. Concluding, if this type of organic wastes were to be used in a single application, the rate could be as high as 12 or even 24tha -1 , however, if they are to be applied in an annual basis, the application rates should be lowered to assure their safe application (e.g. to 6tha -1 ). Moreover, it is advisable to use more stable and mature organic wastes, which have longer lasting positive effects on soil characteristics., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.)

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.

Sewage sludge application to soils is regulated by its total metal content. However, the real risk of metals is determined by the fraction that is biologically available. The available fraction is highly related to the strength of metal binding by the matrix, which is a dynamic process. The evaluation of the fate of metals in time can contribute increased accuracy of ecological risk assessment. Aiming to evaluate short-term changes in metal availability when metals were applied to soil directly (metal-spiked) or by way of an organic matrix (sludge-amended), a laboratory experiment was performed using open microcosms filled with agricultural soil. A concentration gradient of industrial sludge (11, 15, 55, and 75 t/ha) that was contaminated predominantly with chromium, copper, nickel, and zinc, or soil freshly spiked with the same concentrations of these metals, were applied on top of the agricultural soil. After 0, 3, 6, and 12 weeks, total (HNO(3) 69 %) and 0.01 M CaCl(2)-extractable metal concentrations in soil and metal content in the percolates were measured. Results demonstrated that comparison between sludge-amended and metal-spiked soils may give important information about the role of sludge matrix on metal mobility and availability in soil. In sludge-amended soils, extractable-metal concentrations were independent of the sludge concentration and did not change over time. In metal-spiked soils, metal extractability decreased with time due to ageing and transport of metals to deeper layers. In general, the sludge matrix increased the adsorption of metals, thus decreasing their mobility in soils.

Nineteen Mediterranean natural soils with a wide range of properties and the Organisation for Economic Co-operation and Development (OECD) artificial soil were used to assess the influence of soil properties on the results of avoidance and reproduction tests carried out with the soil collembolan species Folsomia candida. Compared to natural soils, the OECD soil was mostly rejected by individuals when a natural soil was offered in avoidance tests, and the number of offspring produced was generally lower than the one obtained in natural soils. None of the soil properties assessed showed a significant influence on the avoidance behavior. More precisely, only soil moisture was included in the model explaining the avoidance response (avoidance increased with increasing differences in moisture), but its contribution was marginally not significant. The model derived explained only 16% of the variance in avoidance response. On the contrary, several soil properties significantly influenced reproduction (number of offspring increased with increasing moisture content, increasing coarse texture, and decreasing nitrogen content). In this case, the model explained 45% of the variance in reproduction. These results, together with the fact that most of the selected soils fulfilled the validity criteria in both avoidance and reproduction tests, confirm the literature experience showing that this species is relatively insensitive to soil properties and hence highly suitable to be used in ecotoxicological tests with natural soils. In addition, our study highlights the need for accuracy in soil moisture adjustment in soil ecotoxicological tests with this species. Otherwise, results of both avoidance and reproduction tests might be biased., (Copyright © 2011 SETAC.)

A current challenge in soil ecotoxicology is the use of natural soils as test substrates to increase ecological relevance of data. Despite the existence of six natural reference soils (the Euro-soils), some parallel projects showed that these soils do not accurately represent the diversity of European soils. Particularly, Mediterranean soils are not properly represented. To fill this gap, 12 natural soils from the Mediterranean regions of Alentejo, Portugal; Cataluña, Spain; and Liguria, Italy, were selected and used in reproduction and avoidance tests to evaluate the soil habitat function for earthworms (Eisenia andrei) and enchytraeids (Enchytraeus crypticus). Predictive models on the influence of soil properties on the responses of these organisms were developed using generalized linear models. Results indicate that the selected soils can impact reproduction and avoidance behavior of both Oligochaete species. Reproduction of enchytraeids was affected by different soil properties, but the test validity criteria were fulfilled. The avoidance response of enchytraeids was highly variable, but significant effects of texture and pH were found. Earthworms were more sensitive to soil properties. They did not reproduce successfully in three of the 10 soils, and a positive influence of moisture, fine sand, pH, and organic matter and a negative influence of clay were found. Moreover, they strongly avoided soils with extreme textures. Despite these limitations, most of the selected soils are suitable substrates for ecotoxicological evaluations., (Copyright © 2011 SETAC.)

The ecotoxicological characterization of sewage sludge takes into account the additive, antagonistic and synergistic effects that occur as a result of multi-chemical interactions. Such an evaluation therefore is essential to complement the chemical analysis that, although required by law, is clearly insufficient. Using a tiered approach in the toxic evaluation of sewage sludge allows for characterization of toxicity in a timely manner. According to the literature, reproduction tests with Folsomia candida are suitable tools for the toxic assessment of organic sludges. Therefore, the inclusion of Collembola avoidance tests at a screening level (low tier), and acting as a trigger for longer-period tests (high tier; e.g. reproduction test), may provide a successful strategy, and may complement the currently proposed test battery. To evaluate the use of both avoidance and reproduction tests with collembolans in such a tiered approach, three sewage sludges (urban, olive and electroplating industries) were mixed in with a field-collected soil at different concentrations. Avoidance and reproduction tests were performed with the soil-sludge mixtures after 0, 4 and 12 weeks of incubation. The tests detected no toxicity in soil-sludge mixtures of urban and olive sludges at any incubation period. Mixtures with sludge from the electroplating industry induced toxicity only in the avoidance tests with freshly prepared and 4-week incubated samples. These results demonstrate the ability of Collembola avoidance tests to assess sewage sludge toxicity over time and its potential for hazardous sludge characterization at low tier levels.

The ability of organisms to avoid contaminated soils can act as an indicator of toxic potential in a particular soil. Based on the escape response of earthworms and Collembola, avoidance tests with these soil organisms have great potential as early screening tools in site-specific assessment. These tests are becoming more common in soil ecotoxicology, because they are ecologically relevant and have a shorter duration time compared with standardized soil toxicity tests. The avoidance response of soil invertebrates, however, can be influenced by the soil properties (e.g., organic matter content and texture) that affect behavior of the test species in the exposure matrix. Such an influence could mask a possible effect of the contaminant. Therefore, the effects of soil properties on performance of test species in the exposure media should be considered during risk assessment of contaminated soils. Avoidance tests with earthworms (Eisenia andrei) and springtails (Folsomia candida) were performed to identify the influence of both organic matter content and texture on the avoidance response of representative soil organisms. Distinct artificial soils were prepared by modifying quantities of the standard artificial soil components described by the Organization for Economic Co-operation and Development to achieve different organic matter and texture classes. Several combinations of each factor were tested. Results showed that both properties influenced the avoidance response of organisms, which avoided soils with low organic matter content and fine texture. Springtails were less sensitive to changes in these soil constituents compared with earthworms, indicating springtails can be used for site-specific assessments of contaminated soils with a wider range of respective soil properties.