The effects of pollution by multiple metals derived from long-term smelting activities on soil mite communities in arable soils under different land use types in East China.

Soil pollution represents a threat to soil biodiversity and to soil and human health. However, many ecotoxicological issues, such as the impact of heavy metal pollution on the soil mite community and its spatial distribution in areas with complex environmental factors, are not fully understood. Here, an investigation was conducted in an arable area (about 11 km2) enclosed by surrounding mountains. The study area was contaminated with potentially toxic metals derived from copper smelting that was functioning for over 10 years. The area comprised four land use types: woodlands, dry fields, paddy fields, and wastelands, and was divided into 141 study sites each with an area of 6.25 ha. The soil metal (Cu, Zn, Pb, and Cd) contents, pH, and organic matter were determined and their distributions were established. Furthermore, soil mite (Acari) community properties (species richness, individual abundance, and Shannon-Wiener diversity index) were determined, and the distributions of total species number and abundance were ascertained. Soil metal pollution strongly reduced soil mite community, but the effects depended on mite groups or species and their sensitivity to different metals as well as land use types. CANOCO analysis revealed that the order Oribatida was more highly correlated with soil metal contents, whereas the other three orders responded to soil metal contents depending on land use types, mite properties, or metals. SADIE method indicated that the coordinate relationship between mite species number and metal concentration was more negative (4-25% of the study sites) than positive (4-12%). The metal pollution levels in the soil were evaluated by single and integrated pollution and ecological risk indices.

Analysis of heat and cold tolerance of a freeze-tolerant soil invertebrate distributed from temperate to Arctic regions: evidence of selection for extreme cold tolerance.

Tolerance to thermal extremes is critical for the geographic distributions of ectotherm species, many of which are probably going to be modified by future climatic changes. To predict species distributions it is important to understand the potential of species to adapt to changing thermal conditions. Here, we tested whether the thermal tolerance traits of a common freeze-tolerant potworm were correlated with climatic conditions and if adaptation to extreme cold constrains the evolutionary potential for high temperature tolerance. Further, we tested if evolution of thermal tolerance traits is associated with costs in other fitness traits (body size and reproduction). Lastly, we tested if slopes of temperature-survival curves (i.e., the sensitivity distribution) are related to tolerance itself. Using 24 populations of the potworm, Enchytraeus albidus Henle (Enchytraeidae), collected from a wide range of climatic conditions, we established a common garden experiment in which we determined high and low temperature tolerance (using survival as endpoint), average reproductive output and adult body size. Heat tolerance was not related to environmental temperatures whereas lower lethal temperature was about 10 °C lower in Arctic populations than in populations from temperate regions. Reproduction was not related to environmental temperature, but was negatively correlated with cold tolerance. One explanation for the trade-off between cold tolerance and reproduction could be that the more cold-hardy populations need to channel energy to large glycogen reserves at the expense of less energy expenditure for reproduction. Adult body size was negatively related to environmental temperature. Finally, the slopes of temperature-survival curves were significantly correlated with critical temperature limits for heat and cold tolerance; i.e., slopes increased with thermal tolerance. Our results suggest that relatively heat-sensitive populations possess genetic variation, leaving room for improved heat tolerance through evolutionary processes, which may alleviate the effects of a warmer future climate in the Arctic. On the other hand, we observed relatively narrow sensitivity distributions (i.e., less variation) in the most heat tolerant populations. Taken together, our results suggest that both cold and heat tolerance can only be selected for (and improved) until a certain limit has been reached.

Key to Ptychopteridae (Diptera) larvae of Northern Europe, with notes on distribution and biology.

A key to larvae of Ptychopteridae (phantom crane flies) is provided for species inhabiting Northern Europe. The key encompasses seven species, including the previously undescribed larvae of Ptychoptera longicauda (Tonnoir 1919). Larval specimens examined were primarily sampled from sites in Denmark. Larvae were associated with correctly identified adults based on DNA barcode (COI) sequence. In the development of the key, a wide suite of morphological characteristics were examined and evaluated for their utility to separate species. Current distribution maps are provided for all species occurring within Northern Europe. Based on records of larvae from Denmark and Finland, notes on larval habitats are given. We also present flight periods for all species examined during this study. Finally, the status of the enigmatic species Ptychoptera obscura (Peus 1958) is briefly discussed.

The effect of Bt crops on soil invertebrates: a systematic review and quantitative meta-analysis.

The ongoing debate about the ecological effects of Bt-crops calls for thorough reviews about the impact on soil biodiversity and their ecosystem services. Transgenic Bt-crops have been genetically modified by inserting a Bacillus thuriengensis gene so the plant expresses a Cry toxin aimed for insect crop pests. Non-target soil invertebrates are particularly recognized for their contribution to plant nutrient availability and turnover of organic matter and it is therefore relevant to protect these invertebrate taxa. A number of studies have compared the population abundance and biomass of soil invertebrates in agricultural fields planted with genetically modified Bt crops and their conventional counterparts. Here, were review and analyze a selection of studies on Protista, nematodes, Collembola, mites, enchytraeids, and earthworms systematically to empower the evidence for asking the question whether population abundances and biomasses of soil invertebrates are changed by Bt crops compared to conventional crops. 6110 titles were captured, of which 38 studies passed our inclusion criteria, and a final number of 22 publications were subject to data extraction. A database with 2046 records was compiled covering 36 locations and the Bt types Cry1Ab, Cry1Ac, Cry3Bb1 and Cry3Aa. Comparative effect sizes in terms of Hedges' g were calculated irrespectively of statistical significance of effects of the source studies. Cry effects on populations were compared across the studies in a meta-analysis employing a hierarchical Bayesian approach of weighted data according to the level of replication. The temporal development of effect sizes was modelled, thereby taking into account the variable duration of the field experiments. There was considerable variation among soil invertebrate orders, but the sample size was insufficient and the sample heterogeneity too large to draw any credible conclusions on the effect of Cry at the order level. However, across orders there was no significant effect of Cry on soil invertebrates.

Insights into the earthworm gut multi-kingdom microbial communities.

Earthworms are widely known to impact soil health, having a key role in nutrient cycling and are often referred to as soil engineers. They are vital for soil microbial assemblages particularly through their feeding and burrowing activity in soil. Earthworms feed on soil organic matter and litter, and the resulting casts alter the soil microbial community. However, the gut microbiome of earthworms remains less known. In this study, we used amplicon sequencing of the 16S rRNA gene for bacteria and 18S rRNA gene for eukaryotes to assess the gut community assemblages of earthworm species within three genera Aporrectodea, Allolobophora and Lumbricus that represent different life forms sharing the same habitat. The objective was to compare the gut microbiome profiles of eukaryotic and prokaryotic organisms to assess significance of earthworm life forms, and to explore the cross kingdom networks in an attempt to identify keystone species. We found a high eukaryotic diversity with a dominance of the SAR supergroup along with fungi and metazoan in the earthworm gut. The bacterial community were dominated by members of Proteobacteria, Acidobacteria, Actinobacteria, Firmicutes, and Verrucomicrobia. The eukaryotic and prokaryotic communities showed similar differences in alpha diversity, being lowest in Lumbricus herculeus. The beta diversity showed earthworm species as a key factor in shaping gut microbiomes with L. herculeus harboring distinct microbiomes compared to species of Aporrectodea caliginosa, A. longa, A. tuberculata and Allolobophora chlorotica. Cross kingdom networks showed high interactions between several protist and bacterial OTUs. In conclusion, this study suggested that the community assemblages of gut microbiomes were shaped by earthworm species and life form, and such assemblage consists of cross kingdom interactions among eukaryotes and prokaryotes.

Status and trends of terrestrial arthropod abundance and diversity in the North Atlantic region of the Arctic.

The Circumpolar Biodiversity Monitoring Programme (CBMP) provides an opportunity to improve our knowledge of Arctic arthropod diversity, but initial baseline studies are required to summarise the status and trends of planned target groups of species known as Focal Ecosystem Components (FECs). We begin this process by collating available data for a relatively well-studied region in the Arctic, the North Atlantic region, summarising the diversity of key terrestrial arthropod FECs, and compiling trends for some representative species. We found the FEC classification system to be challenging to implement, but identified some key groups to target in the initial phases of the programme. Long-term data are scarce and exhibit high levels of spatial and temporal variability. Nevertheless, we found that a number of species and groups are in decline, mirroring patterns in other regions of the world. We emphasise that terrestrial arthropods require higher priority within future Arctic monitoring programmes.

Assessment of toxicity of heavy metal-contaminated soils toward Collembola in the paddy fields supported by laboratory tests.

Effects on soil Collembola of Cu, Zn, Pb, and Cd pollution from Cu smelters over 40 years were investigated in paddy fields from an area of Eastern China. We compared the field effects to those observed in single-species laboratory tests employing the hemiedaphic collembolan Folsomia candida and the epedaphic Sinella curviseta obtained from laboratory cultures and exposed to field-collected polluted soil. The results indicated that different collembolan species responded differently to the pollution in the fields and could be divided into sensitive, indifferent, and tolerant types accordingly. The abundance of sensitive species decreased as the pollution increased, but this was not the same for indifferent and tolerant species. The dominant species changed from sensitive to tolerant species as the pollution increased. The reproduction of F. candida and S. curviseta was most sensitive to the contaminated soil compared to growth and survival; the sensitivity of the two species was similar. The growth was more sensitive than the survival for F. candida but not for S. curviseta. The growth and survival of F. candida were much more sensitive than those of S. curviseta. Sensitivity of field populations of F. candida (EC10 31 [15-46]) and hemiedaphic species Folsomia quadrioculata (EC10 52 [0.7-102]) were comparable with sensitivity of the reproduction of F. candida in the single-species tests (EC10 21 [14-27]), suggesting that single-species test based on laboratory cultures and field soil could be used to link laboratory and field data and then reflect the field situation. S. curviseta could be used as an epedaphic species in single-species tests and F. quadrioculata as an indicator species for assessment of field effect.

Wood ash application increases pH but does not harm the soil mesofauna.

Application of bioash from biofuel combustion to soil supports nutrient recycling, but may have unwanted and detrimental ecotoxicological side-effects, as the ash is a complex mixture of compounds that could affect soil invertebrates directly or through changes in their food or habitat conditions. To examine this, we performed laboratory toxicity studies of the effects of wood-ash added to an agricultural soil and the organic horizon of a coniferous plantation soil with the detrivore soil collembolans Folsomia candida and Onychiurus yodai, the gamasid predaceous mite Hypoaspis aculeifer, and the enchytraeid worm Enchytraeus crypticus. We used ash concentrations spanning 0-75 g kg-1 soil. As ash increases pH we compared bioash effects with effects of calcium hydroxide, Ca(OH)2, the main liming component of ash. Only high ash concentrations above 15 g kg-1 agricultural soil or 17 t ha-1 had significant effects on the collembolans. The wood ash neither affected H. aculeifer nor E. crypticus. The estimated osmolalities of Ca(OH)2 and the wood ash were similar at the LC50 concentration level. We conclude that short-term chronic effects of wood ash differ among different soil types, and osmotic stress is the likely cause of effects while high pH and heavy metals is of minor importance.

Urban and industrial land uses have a higher soil biological quality than expected from physicochemical quality.

Despite their importance both in soil functioning and as soil indicators, the response of microarthropods to various land uses is still unclear. The aim of this study is to assess the effect of land use on microarthropod diversity and determine whether a soil's biological quality follows the same physicochemical quality-based gradient from forest, agriculture-grassland, agriculture-arable land, vineyards, urban vegetable gardens to urban, industrial, traffic, mining and military areas. A database compiling the characteristics of 758 communities has been established. We calculated Collembola community indices including: species richness, Pielou's evenness index, collembolan life forms, the abundance of Collembola and of Acari, the Acari/Collembola abundance ratio, and the Collembolan ecomorphological index. Results show that agricultural land use was the most harmful for soil microarthropod biodiversity, whilst urban and industrial land uses give the same level of soil biological quality as forests do. Furthermore, differences between the proportions of Acari and ecomorphological groups were observed between land uses. This study, defining soil microarthropod diversity baselines for current land uses, should therefore help in managing and preserving soil microarthropod biodiversity, especially by supporting the preservation of soil quality.

Interaction webs in arctic ecosystems: Determinants of arctic change?

How species interact modulate their dynamics, their response to environmental change, and ultimately the functioning and stability of entire communities. Work conducted at Zackenberg, Northeast Greenland, has changed our view on how networks of arctic biotic interactions are structured, how they vary in time, and how they are changing with current environmental change: firstly, the high arctic interaction webs are much more complex than previously envisaged, and with a structure mainly dictated by its arthropod component. Secondly, the dynamics of species within these webs reflect changes in environmental conditions. Thirdly, biotic interactions within a trophic level may affect other trophic levels, in some cases ultimately affecting land-atmosphere feedbacks. Finally, differential responses to environmental change may decouple interacting species. These insights form Zackenberg emphasize that the combination of long-term, ecosystem-based monitoring, and targeted research projects offers the most fruitful basis for understanding and predicting the future of arctic ecosystems.

The effects of straw or straw-derived gasification biochar applications on soil quality and crop productivity: A farm case study.

Thermal gasification of straw is a highly efficient technology that produces bioenergy and gasification biochar that can be used as a soil amendment, thereby returning non-renewable nutrients and stable carbon, and securing soil quality and crop productivity. A Danish on-farm field study investigated the impact of traditional straw incorporation vs. straw removal for thermal gasification bioenergy production and the application of straw gasification biochar (GB) on soil quality and crop production. Two rates of GB were applied over three successive years in which the field was cropped with winter wheat (Triticum aestivum L.), winter oilseed rape (Brassica napus L.) and winter wheat, respectively, to assess the potential effects on the soil carbon pool, soil microorganisms, earthworms, soil chemical properties and crop yields. The application of GB did not increase the soil organic carbon content significantly and had no effect on crop yields. The application of straw and GB had a positive effect on the populations of bacteria and protists, but no effect on earthworms. The high rate of GB increased soil exchangeable potassium content and soil pH indicating its potassium bioavailability and liming properties. These results suggest, that recycling GB into agricultural soils has the potential to be developed into a system combining bioenergy generation from agricultural residues and crop production, while maintaining soil quality. However, future studies should be undertaken to assess its long-term effects and to identify the optimum balance between straw removal and biochar application rate.

Pesticide Side Effects in an Agricultural Soil Ecosystem as Measured by amoA Expression Quantification and Bacterial Diversity Changes.

BACKGROUND AND METHODS: Assessing the effects of pesticide hazards on microbiological processes in the soil is currently based on analyses that provide limited insight into the ongoing processes. This study proposes a more comprehensive approach. The side effects of pesticides may appear as changes in the expression of specific microbial genes or as changes in diversity. To assess the impact of pesticides on gene expression, we focused on the amoA gene, which is involved in ammonia oxidation. We prepared soil microcosms and exposed them to dazomet, mancozeb or no pesticide. We hypothesized that the amount of amoA transcript decreases upon pesticide application, and to test this hypothesis, we used reverse-transcription qPCR. We also hypothesized that bacterial diversity is affected by pesticides. This hypothesis was investigated via 454 sequencing and diversity analysis of the 16S ribosomal RNA and RNA genes, representing the active and total soil bacterial communities, respectively. RESULTS AND CONCLUSION: Treatment with dazomet reduced both the bacterial and archaeal amoA transcript numbers by more than two log units and produced long-term effects for more than 28 days. Mancozeb also inhibited the numbers of amoA transcripts, but only transiently. The bacterial and archaeal amoA transcripts were both sensitive bioindicators of pesticide side effects. Additionally, the numbers of bacterial amoA transcripts correlated with nitrate production in N-amended microcosms. Dazomet reduced the total bacterial numbers by one log unit, but the population size was restored after twelve days. The diversity of the active soil bacteria also seemed to be re-established after twelve days. However, the total bacterial diversity as reflected in the 16S ribosomal RNA gene sequences was largely dominated by Firmicutes and Proteobacteria at day twelve, likely reflecting a halt in the growth of early opportunists and the re-establishment of a more diverse population. We observed no effects of mancozeb on diversity.

Species composition of a soil invertebrate multi-species test system determines the level of ecotoxicity.

A soil multi-species, SMS, experimental test system consisting of the natural microbial community, five collembolan species and a predatory mite along with either Enchytraeus crypticus or the earthworm Eisenia fetida were exposed to α-cypermethrin. A comparison of the performance of these two types of SMSs is given to aid the development of a standard test system. E. fetida had a positive effect on the majority of the species, reducing the negative insecticide effect. E. fetida affected the species sensitivity and decreased the degradation of the insecticide due to the organic matter incorporation of earthworm food. After 8 weeks, the EC50 was 0.76 mg kg(-1) for enchytraeids and ranged between 2.7 and 18.9 mg kg(-1) for collembolans, more sensitive than previously observed with single species. Changes observed in the community structure and function illustrates the strength of a multi-species test system as an ecotoxicological tool compared to single species tests.

Microarray detection and qPCR screening of potential biomarkers of Folsomia candida (Collembola: Isotomidae) exposed to Bt proteins (Cry1Ab and Cry1Ac).

The impact of Bt proteins on non-target arthropods is less understood than their effects on target organisms where the mechanism of toxic action is known. Here, we report the effects of two Bt proteins, Cry1Ab and Cry1Ac, on gene expression in the non-target collembolan, Folsomia candida. A customized microarray was used to study gene expression in F. candida specimens that were exposed to Cry1Ab and Cry1Ac. All selected transcripts were subsequently confirmed by qPCR. Eleven transcripts were finally verified, and three of them were annotated. The responses of all eleven transcripts were tested in specimens for both Cry1Ab and Cry1Ac at a series of concentrations. These transcripts were separated into two and three groups for Cry1Ab and Cry1Ac, respectively, depend on their expression levels. However, those eleven transcripts did not respond to the Bt proteins in Bt-rice residues.

Laboratory assessment of the impacts of transgenic Bt rice on the ecological fitness of the soil non-target arthropod, Folsomia candida (Collembola: Isotomidae).

Transgenic rice expressing Bacillus thuringiensis (Bt) endotoxins (Bt rice) for pest control is considered an important solution to food security in China. However, tests for potential effects on non-target soil organisms are required for environmental risk assessment. The soil collembolan Folsomia candida L. (Collembola: Isotomidae) is a potential non-target arthropod that is often used as a biological indicator in bio-safety assessments of transgenic crops. In the present study, the roots, stems, and leaves of Bt rice were exposed to F. candida under laboratory conditions, with survival, reproduction and growth of the collembolan as ecological fitness parameters. Significant differences in ecological fitness were found among the different treatments, including differences in the plant parts and varieties of non-Bt rice, presumably as the result of three factors: gene modification, plant parts and rice varieties. The fitness of F. candida was less affected by the different diets than by the exposure to the same materials mixed with soil. Our results clearly showed that there was no negative effect of different Bt rice varieties on the fitness of F. candida through either diet or soil exposure.

Decrease in catalase activity of Folsomia candida fed a Bt rice diet.

Here we report the effects of three Bt-rice varieties and their non-Bt conventional isolines on biological traits including survival, reproduction, and the activities of three antioxidant enzymes superoxide dismutase, catalase and peroxidase, in the Collembolan, Folsomia candida. The reproduction was significantly lower when fed Kemingdao and Huahui1 than those feeding on their non-GM near-isogenic varieties Xiushui and Minghui63 respectively, this can be explained by the differences of plant compositions depended on variety of rice. The catalase activity of F. candida was significantly lower when fed the Bt-rice variety Kemingdao compared to the near-isogenic non-Bt-rice variety Xiushui. This suggests that some Bt-rice varieties may impose environmental stress to collembolans. We emphasize that changes in activity of antioxidant enzymes of non-target organisms are important in understanding the ecological consequences for organisms inhabiting transgenic Bt-rice plantations.

NORMA-Gene: a simple and robust method for qPCR normalization based on target gene data.

BACKGROUND: Normalization of target gene expression, measured by real-time quantitative PCR (qPCR), is a requirement for reducing experimental bias and thereby improving data quality. The currently used normalization approach is based on using one or more reference genes. Yet, this approach extends the experimental work load and suffers from assumptions that may be difficult to meet and to validate. RESULTS: We developed a data driven normalization algorithm (NORMA-Gene). An analysis of the performance of NORMA-Gene compared to reference gene normalization on artificially generated data-sets showed that the NORMA-Gene normalization yielded more precise results under a large range of parameters tested. Furthermore, when tested on three very different real qPCR data-sets NORMA-Gene was shown to be best at reducing variance due to experimental bias in all three data-sets compared to normalization based on the use of reference gene(s). CONCLUSIONS: Here we present the NORMA-Gene algorithm that is applicable to all biological and biomedical qPCR studies, especially those that are based on a limited number of assayed genes. The method is based on a data-driven normalization and is useful for as little as five target genes comprising the data-set. NORMA-Gene does not require the identification and validation of reference genes allowing researchers to focus their efforts on studying target genes of biological relevance.

Solid-phase microextraction (SPME) as a tool to predict the bioavailability and toxicity of pyrene to the springtail, Folsomia candida, under various soil conditions.

The porewater concentrations of pyrene were estimated by a negligible depletive solid-phase microextraction (SPME) method. The effects of organic matter (OM) and soil aging on the bioavailability of pyrene in soil were investigated by generation of reproductive effect concentrations (EC50) for the euedaphic springtail, Folsomia candida, under various test conditions. The soil used was a sandy loam soil with natural OM content of 2.6% (Askov soil). It was enriched with increasing organic matter concentrations of 5%, 10%, and 20% and was aged for 0, 56, and 112 days. The EC50 values of the springtails increased with increasing OM and aging of the soil. The increase of the OM content in the soil reduced the extractability of pyrene by SPME, as well as the toxicity of pyrene. An aging effect was demonstrated in Askov soil, EC50 values increased with increased contact time. The amounts of pyrene extracted by SPME were significantly affected by the soil concentration. Porewater concentrations determined by SPME decreased with increasing OM and aging. However, the pyrene EC50 porewater concentration remained largely constant at approximately 23 microg L(-1). The results demonstrated that the SPME method is a useful indicator for bioavailability to soil microarthropods.

Effects of acclimation temperature on thermal tolerance and membrane phospholipid composition in the fruit fly Drosophila melanogaster.

Adaptative responses of ectothermic organisms to thermal variation typically involve the reorganization of membrane glycerophospholipids (GPLs) to maintain membrane function. We investigated how acclimation at 15, 20 and 25 degrees C during preimaginal development influences the thermal tolerance and the composition of membrane GPLs in adult Drosophila melanogaster. Long-term cold survival was significantly improved by low acclimation temperature. After 60 h at 0 degrees C, more than 80% of the 15 degrees C-acclimated flies survived while none of the 25 degrees C-acclimated flies survived. Cold shock tolerance (1h at subzero temperatures) was also slightly better in the cold acclimated flies. LT50 shifted down by ca 1.5 degrees C in 15 degrees C-acclimated flies in comparison to those acclimated at 25 degrees C. In contrast, heat tolerance was not influenced by acclimation temperature. Low temperature acclimation was associated with the increase in proportion of ethanolamine (from 52.7% to 58.5% in 25 degrees C-acclimated versus 15 degrees C-acclimated flies, respectively) at the expense of choline in GPLs. Relatively small, but statistically significant changes in lipid molecular composition were observed with decreasing acclimation temperature. In particular, the proportions of glycerophosphoethanolamines with linoleic acid (18:2) at the sn-2 position increased. No overall change in the degree of fatty acid unsaturation was observed. Thus, cold tolerance but not heat tolerance was influenced by preimaginal acclimation temperature and correlated with the changes in GPL composition in membranes of adult D. melanogaster.

Probabilistic risk assessment for linear alkylbenzene sulfonate (LAS) in sewage sludge used on agricultural soil.

Deterministic and probabilistic risk assessments were developed for commercial LAS in agricultural soil amended with sewage sludge. The procedure done according to ILSI Europe's Conceptual Framework [Schowanek, D., Carr, R., David, H., Douben, P., Hall, J., Kirchmann, H., Patria, L., Sequi, P., Smith, S., Webb, S.F., 2004. A risk-based methodology for deriving quality standards for organic contaminants in sewage sludge for use in agriculture-conceptual Framework. Regul. Toxicol. Pharmacol. 40 (3), 227-251], consists of three main steps. First, the most sensitive endpoint was determined. This was found to be the chronic ecotoxicity of LAS to soil invertebrates and plants. Additional endpoints, such as potential for plant uptake and transfer in the food chain, leaching to groundwater, surface erosion run-off, human health risk via drinking water, plant consumption and soil ingestion were also systematically evaluated but were all assessed to be of little toxicological significance. In the second step, a back-calculation was conducted from the Predicted No-Effect Concentration in soil (PNECsoil) to a safe level of LAS in sludge (here called 'Sludge Quality Standard'; SQS). The deterministic approach followed the default agricultural soil exposure scenario in the EU-Technical Guidance Document (TGD). The SQS for LAS was calculated as 49 g/kg sludge Dry Matter (DM). In order to assess the potential variability as a result of varying agricultural practices and local environmental conditions, two probabilistic exposure assessment scenarios were also developed. The mean SQS was estimated at 55 and 27.5 g/kg DM for the homogeneous soil mixing and soil injection scenarios, respectively. In the final step, the resulting SQS values were evaluated for consistency and relevance versus available information from agricultural experience and field tests. No build-up, adverse impact on soil fertility, agronomic performance, or animal/human health have been reported for agricultural fields which have received sludge with high LAS levels for up to 30 years. Distribution statistics of LAS concentrations in anaerobically digested sewage sludge measured across Europe were created (mean value: 5.56 g LAS/kg sludge DM). When compared to the above mean SQS values, adequate risk characterisation ratios of 0.08-0.2 were found. The 'ecological risk' parameter calculated for anaerobic sludge from the probabilistic approaches was below 3%. A regulatory Limit Value for LAS of 2.60 g/kg sludge DM was originally proposed in the 3rd Draft of the Working Document on Sludge [CEC, 2000b. Working Document on Sludge. Third Draft, Brussels 27 April 2000, DG. Environment, 18 p.]. The current assessment, based on an updated dataset and a refined assessment procedure, suggests that the need for a limit value for LAS in sewage sludge cannot be substantiated on a risk basis.