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.

Bridging the Gap between Field Experiments and Machine Learning: The EC H2020 B-GOOD Project as a Case Study towards Automated Predictive Health Monitoring of Honey Bee Colonies.

Honey bee colonies have great societal and economic importance. The main challenge that beekeepers face is keeping bee colonies healthy under ever-changing environmental conditions. In the past two decades, beekeepers that manage colonies of Western honey bees (Apis mellifera) have become increasingly concerned by the presence of parasites and pathogens affecting the bees, the reduction in pollen and nectar availability, and the colonies' exposure to pesticides, among others. Hence, beekeepers need to know the health condition of their colonies and how to keep them alive and thriving, which creates a need for a new holistic data collection method to harmonize the flow of information from various sources that can be linked at the colony level for different health determinants, such as bee colony, environmental, socioeconomic, and genetic statuses. For this purpose, we have developed and implemented the B-GOOD (Giving Beekeeping Guidance by computational-assisted Decision Making) project as a case study to categorize the colony's health condition and find a Health Status Index (HSI). Using a 3-tier setup guided by work plans and standardized protocols, we have collected data from inside the colonies (amount of brood, disease load, honey harvest, etc.) and from their environment (floral resource availability). Most of the project's data was automatically collected by the BEEP Base Sensor System. This continuous stream of data served as the basis to determine and validate an algorithm to calculate the HSI using machine learning. In this article, we share our insights on this holistic methodology and also highlight the importance of using a standardized data language to increase the compatibility between different current and future studies. We argue that the combined management of big data will be an essential building block in the development of targeted guidance for beekeepers and for the future of sustainable beekeeping.

Exposure and risk assessment of acetamiprid in honey bee colonies under a real exposure scenario in Eucalyptus sp. landscapes.

Honey bee colonies have shown abnormal mortality rates over the last decades. Colonies are exposed to biotic and abiotic stressors including landscape changes caused by human pressure. Modern agriculture and even forestry, rely on pesticide inputs and these chemicals have been indicated as one of the major causes for colony losses. Neonicotinoids are a common class of pesticides used worldwide that are specific to kill insect pests, with acetamiprid being the only neonicotinoid allowed to be applied outdoors in the EU. To evaluate honeybees' exposure to acetamiprid under field conditions as well as to test the use of in-situ tools to monitor pesticide residues, two honeybee colonies were installed in five Eucalyptus sp. plantations having different area where Epik® (active substance: acetamiprid) was applied as in a common spraying event to control the eucalyptus weevil pest. Flowers, fresh nectar, honey bees and colony products samples were collected and analyzed for the presence of acetamiprid residues. Our main findings were that (1) acetamiprid residues were found in samples collected outside the spraying area, (2) the amount of residues transported into the colonies increased with the size of the sprayed area, (3) according to the calculated Exposure to Toxicity Ratio (ETR) values, spraying up to 22 % of honeybees foraging area does not harm the colonies, (4) colony products can be used as a valid tool to monitor colony accumulation of acetamiprid and (5) the use of Lateral Flow Devices (LFDs) can be a cheap, fast and easy tool to apply in the field, to evaluate the presence of acetamiprid residues in the landscape and colony products.

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.

Application of a standard risk assessment scheme to a North Africa contaminated site (Sfax, Tunisia) -Tier 1.

Phosphorus is a critical element to agriculture, consequently global phosphate rock demand will remain rising to feed a growing world population. The beneficiation of phosphorous ore gives rise to several tons of a waste by-product [phosphogypsum (PG)] which valorisation is limited, within other reasons, by the risks posed to environment and human health. Although threatening, the accumulation in stacks is the only procedure so far practiced by several countries as a means to get rid of this industrial externality. As part of a NATO Science for Peace Project (SfP 983311) this study describes the application of an environmental risk assessment (ERA) framework, to assess the risks posed by a PG stack to the surrounding soils, in Sfax, Republic of Tunisia. The ERA followed a weight of evidence approach, supported by two lines of evidence (LoE): the chemical (ChemLoE) and the ecotoxicological (EcotoxLoE). Integrated risks point for risk values greater than 0.5 in soils collected in PG stack surrounding area. Soil salinization, has likely contributed to the exacerbation of risks, as well as to the lack of consistency between both LoEs. This study highlights the need of rethinking the weight given to each LoE in ERA, in areas where soil salinization is a reality.

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.

Environmental risk assessment of pesticides in tropical terrestrial ecosystems: Test procedures, current status and future perspectives.

Despite the increasing use of pesticides in tropical countries, research and legislative efforts have focused on their temperate counterparts. This paper presents a review of the literature on environmental risk assessment of pesticides for tropical terrestrial agroecosystems. It aims at evaluating potential differences in pesticide risk between temperate and tropical regions as well as to highlight research needs in the latter. Peculiarities of pesticide risks in tropical terrestrial agroecosystems are discussed in subsections 1) agricultural practices; 2) research efforts; 3) fate and exposure; 4) toxicity testing methods; and 5) sensitivity. The intensive and often inadequate pesticide application practices in tropical areas are likely to result in a relatively greater pesticide exposure in edge-of-field water bodies. Since pesticide fate may be different under tropical conditions, tropical scenarios for models estimating predicted environmental pesticide concentrations should be developed. Sensitivity comparisons do not indicate a consistent similar, greater or lower relative sensitivity of tropical soil organisms as compared to temperate organisms. However, several methods and procedures for application in the tropics need to be developed, which include: 1) identifying and collecting natural soils to be used as reference test substrates in tests; 2) identifying and discerning the range of sensitivity of native test species to soil contaminants; 3) developing test guidelines applicable to tropical/subtropical conditions; and 4) developing methods and procedures for higher tier testing for full development and implementation of environmental risk assessment schemes.

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.

Placing arbuscular mycorrhizal fungi on the risk assessment test battery of plant protection products (PPPs).

Arbuscular mycorrhizal fungi (AMF) are mutualistic symbionts considered a key group in soil systems involved in the provision of several ecosystem services. Recently they have been listed by EFSA as organisms to be included in the test battery for the risk assessment of plant protection product (PPPs). This study aimed to contribute to improve the ISO Protocol (ISO 10832: 2009) by assessing the feasibility of using other AMF species under different test conditions. Overall, results showed that AMF species Gigaspora albida and Rhizophagus clarus (selected out of five AMF species) are suitable to be used in spore germination tests using the ISO protocol (14 days incubation with sand or artificial soil as substrate) to test PPPs. However, several modifications to the protocol were made in order to accommodate the use of the tested isolates, namely the incubation temperature (28 °C instead of 24 °C) and the change of reference substance (boric acid instead of cadmium nitrate). The need for these changes, plus the results obtained with the three fungicides tested (chlorothalonil, mancozeb and metalaxyl-M) and comparisons made with literature on the relevance of the origin of AMF isolates in dictating the adequate test conditions, emphasize the importance of adjusting test conditions (AMF species/isolates and test temperature) when assessing effects for prospective risk assessment targeting different climatic zones. So, further studies should be conducted with different AMF species and isolates from different climatic regions, in order to better define which species/isolate and test conditions should be used to assess effects of a particular PPP targeting a given climatic zone.

Organic wastes as soil amendments - Effects assessment towards soil invertebrates.

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.

Soil ecotoxicology in Brazil is taking its course.

Soil ecotoxicology has been motivated by the increasing global awareness on environmental issues. Northern Hemisphere has been the main driver of this science branch; however, the number and quality of contributions from the Southern Hemisphere are increasing quickly. In this case study, Brazil is taken as an example of how soil ecotoxicology has developed over the last 30 years. It starts with a brief historical overview depicting the main events on soil ecotoxicology in the country. Following, an overview on the Brazilian legislation related to soil ecotoxicology is given, covering regulations with prospective focus, mainly on the registration of pesticides. Regulations with retrospective focus in contaminated areas are also given. Then, an outline of the actors in soil ecotoxicology and examples of prospective ecotoxicological studies performed with soil organisms and plants are given by stressor groups: pesticides, pharmaceuticals, metals, and residues. Experiences from retrospective studies, mainly looking at the assessment of industrial sites, are also covered. Emphasis is given on methodological aspects, pointing to needed actions, mainly regarding the different biotic and abiotic conditions of a tropical country. Finally, the last session discusses how soil ecotoxicology could be improved in methodological adaptations as well as legal requirements.

Ecological Risk Assessment of a Metal-Contaminated Area in the Tropics. Tier II: Detailed Assessment.

This study presents data on the detailed evaluation (tier 2) of a site-specific ecological risk assessment (ssERA) in a former smelter area contaminated with metals (Santo Amaro, Bahia, Brazil). Combining information from three lines of evidence (LoE), chemical (ChemLoE), ecotoxicological (EcotoxLoE) and ecological (EcoLoE), in the Triad approach, integrated risk values were calculated to rank sites and confirm the potential risk disclosed with tier 1. Risk values were calculated for the habitat and for the retention functions in each sampling point. Habitat function included the ChemLoE calculated from total metal concentrations. The EcotoxLoE was based on reproduction tests with terrestrial invertebrates (Folsomia candida, Enchytraeus crypticus, Eisenia andrei), shoot length and plant biomass (Avena sativa, Brassica rapa). For the EcoLoE, ecological parameters (microbial parameters, soil invertebrate community, litter breakdown) were used to derive risk values. Retention function included the ChemLoE, calculated from extractable metal concentrations, and the EcotoxLoE based on eluate tests with aquatic organisms (Daphnia magna reproduction and Pseudokirchneriella subcapitata growth). Results related to the habitat function indicated that the metal residues are sufficient to cause risk to biota, while the low metal levels in extracts and the general lack of toxicity in aquatic tests indicated a high soil retention capacity in most sampling points. Integrated risk of tier 2 showed the same trend of tier 1, suggesting the need to proceed with remediation actions. The high risk levels were related to direct toxicity to organisms and indirect effects, such as failure in the establishment of vegetation and the consequent loss of habitat quality for microorganisms and soil fauna. This study shed some light on the selection of tools for the tier 2 of an ssERA in tropical metal-contaminated sites, focusing on ecological receptors at risk and using available chemical methods, ecological surveys and ecotoxicity tests.

Ecotoxicological assessment of a dredged sediment using bioassays with three species of soil invertebrates.

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.

Improving ecological risk assessment in the Mediterranean area: selection of reference soils and evaluating the influence of soil properties on avoidance and reproduction of two oligochaete species.

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.

Avoidance tests in site-specific risk assessment--influence of soil properties on the avoidance response of Collembola and earthworms.

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.

Avoidance tests with Collembola and earthworms as early screening tools for site-specific assessment of polluted soils.

Avoidance tests with earthworms and collembolans were conducted to demonstrate their feasibility as early screening tools for assessing the toxic potential of metal-polluted soils. Four different soils, with different degrees of metal contamination, were obtained from an abandoned mining area. All possible paired combinations were assessed for an avoidance response by the organisms. Data revealed that both species were able to avoid the most contaminated soils at the center of the ore extraction and treatment areas compared to those collected further away from the mine. However, earthworms and springtails differed in sensitivity to metals, especially when testing the two most polluted soils that had different contamination profiles. Earthworms exhibited a more consistent, less variable response than springtails. Overall results showed that avoidance tests with collembolans and earthworms have the potential to be used as screening tools in ecological risk assessment schemes for contaminated land, to trigger other tests in case of concern. However, further method development is needed to reduce variability in the data, particularly in the Collembola assays, and to gain knowledge about the possible effects of soil properties on the outcome of the tests.