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Soil contamination by antibiotics is a global issue of great concern that contributes to the rise of bacterial antibiotic resistance and can have toxic effects on non-target organisms. This study evaluated the variations of molecular, cellular, and histological parameters in Eisenia fetida earthworms exposed to sulfamethazine (SMZ) and tetracycline (TC), two antibiotics commonly found in agricultural soils. The earthworms were exposed for 14 days to a series of concentrations (0, 10, 100, and 1000 mg/kg) of both antibiotics. SMZ and TC did not affect the survival of E. fetida, however, other effects at different levels of biological complexity were detected. The two highest concentrations of SMZ reduced the viability of coelomocytes. At the highest TC concentration, there was a noticeable decline in cell viability, acetylcholinesterase activity (neurotoxicity), and the relative presence of mucopolysaccharides in the epidermis (mucous production). Glutathione S-transferase activity decreased in all TC treatments and at the highest SMZ concentration. However, levels of malondialdehyde and protein carbonyls did not change, suggesting an absence of oxidative stress. Tetracycline was neurotoxic to E. fetida and changed the integrity of the epidermis. Both antibiotics altered the intestinal microbiota of E. fetida, leading to a reduction in the relative abundance of bacteria from the phyla Proteobacteria and Bacteroidetes, while causing an increase in the phylum Actinobacteroidota. All observed changes indicate that both SMZ and TC can disrupt the earthworms' immune system and gut microbiome, while fostering the growth of bacteria that harbour antibiotic resistance genes. Finally, both antibiotics exerted additional metabolic and physiological effects that increased the vulnerability of E. fetida to pathogens., 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In this work, an accurate analytical method was developed for the simultaneous analysis of twenty-seven antimicrobials (AMs) in earthworms using liquid chromatography coupled to a triple quadrupole mass spectrometry detector (UHPLC-MS/MS). Adequate apparent recoveries (80-120 %) and limits of quantification (LOQ) (1 μg·kg -1 - 10 μg·kg -1 ) were obtained, with the exception of norfloxacin (34 μg·kg -1 ). The method was applied to evaluate the accumulation of sulfamethazine (SMZ) and tetracycline (TC) in earthworms after performing OECD-207 toxicity test, in which Eisenia fetida (E. fetida) organisms were exposed to soils spiked with 10 mg·kg -1 , 100 mg·kg -1 or 1000 mg·kg -1 of SMZ and TC, individually. The results confirmed the bioaccumulation of both AMs in the organisms, showing a greater tendency to accumulate SMZ since higher bioconcentration factor values were obtained for this compound at the exposure concentrations tested. In addition, the degradation of both AMs in both matrices, soils and earthworms was studied using liquid chromatography coupled to a q-Orbitrap high resolution mass spectrometry detector. Thirteen transformation products (TPs) were successfully identified, eight of them being identified for the first time in soil/earthworm (such as 4-Amino-3-chloro-n-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide or 4-(dimethylamino)-1,11,12a-trihydroxy-6,6-dimethyl-3,7,10,12-tetraoxo-3,4,4a,5,5a,6,7,10,12,12a-decahydrotetracene-2-carboxamide, among others) and their formation/degradation trend over time was also studied. Regarding the biological effects, only SMZ caused changes in earthworm growth, evidenced by weight loss in earthworms exposed to concentrations of 100 mg·kg -1 and 1000 mg·kg -1 . Riboflavin decreased at all concentrations of SMZ, as well as at the highest concentration of TC. This indicates that these antibiotics can potentially alter the immune system of E. fetida. This research represents a significant advance in improving our knowledge about the contamination of soil by AM over time. It investigates the various ways in which earthworms are exposed to AMs, either by skin contact or ingestion. Furthermore, it explores how these substances accumulate in earthworms, the processes by which earthworms break them down or metabolise them, as well as the resulting TPs. Finally, it examines the potential effects of these substances on the environment., 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Plant Protection Products (PPP) raise concerns as their application may cause effects on some soil organisms considered non-target species which could be highly sensitive to some pesticides. The European Food and Safety Authority (EFSA), in collaboration with the Joint Research Centre (JRC) of the European Commission, has developed guidance and a software tool, Persistence in Soil Analytical Model (PERSAM), for conducting soil exposure assessments. EFSA PPR Panel has published recommendations for the risk assessment of non-target soil organisms. We have used PERSAM for calculating PPPs predicted environmental concentrations (PECs); and used the estimated PEC for assessing potential risks using Toxicity Exposure Ratios (TER) for selected soil organisms and good agricultural practices. Soil characteristics and environmental variables change along a latitudinal axis through the European continent, influencing the availability of PPP, their toxicity upon soil biota, and hence, impacting on the risk characterization. Although PERSAM includes as input geographical information, the information is aggregated and not further detailed in the model outputs. Therefore, there is a need to develop landscape based environmental risk assessment methods addressing regional variability. The objective was to integrate spatially explicit exposure (PECs) and effect data (biological endpoints i.e. LC 50 , NOEC, etc.) to estimate the risk quotient (TER) of four PPP active substances (esfenvalerate, cyclaniliprole, picoxystrobin, fenamidone) on non-target species accounting European landscape and agricultural variability. The study was focused on the effects produced by the above-mentioned pesticides on two soil organisms: E. fetida earthworms and Folsomia sp. collembolans. After running PERSAM assuming a worst case application of PPPs, PECs in total soil and pore water were obtained for different depths in northern, central and southern European soils. With this data, soil variability and climatic differences among soils divided in three large Euroregions along a latitudinal transect (Northern, Central, Southern Europe) were analysed. Summarising, a trend to accumulate higher PECs and TERs in total soil was observed in the north decreasing towards the south. Higher PECs and TERs could be expected in pore water in southern soils, decreasing towards the north. The risk disparity between pollutant concentrations at different soils compartments should be taken into account for regulatory purposes, as well as the potential landscape variabilities among different Euroregions., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.)

Increasing soil loss and the scarcity of useful land requires new reusing strategies. Thus, recovery of polluted soils recovery offers a chance for economic and social regeneration. With this objective, different soil cleaning technologies have been developed during the last few decades. On one hand, classical physical and/or chemical technologies can be found which are efficient, but have high costs and impacts upon ecosystems. On the other hand, biological methods (such as phytoremediation, bioremediation and vermiremediation) are relatively cost effective and eco-friendly, but also more time-consuming. These biological methods and their yields have been widely studied but little is known about the interaction between different soil cleaning methods. The combination of different biological strategies could lead to an improvement in remediation performance. Hence, in the present work, different micro-, vermi- and phyto-remediation combinations are applied in a sewage sludge polluted landfill in Gernika-Lumo (Basque Country) which was used as a disposal point for decades, in search of the treatment (single) or combination (dual or triple) of treatments with best remediation yields. Eight experimental groups were applied (n=3) placing earthworms (E), bacteria (B), plants (P), bacteria+earthworms (B+E), bacteria+plants (B+P), plants+earthworms (P+E) plants+bacteria+earthworms (P+B+E) and a non-treated (N.T.) group in the experimental plot (Landfill 17), for 12 months. In order to assess the efficiency of each treatment, a complete characterization (chemical and ecotoxicological) was carried out before and after remediation. Results showed high removal rates for dieldrin (between 50% and 78%) in all the experimental groups. In contrast, removal rates around 20-25% were achieved for heavy metals (Cd 15%-35%; Ni 24%-37%; Pb 15%-33%; Cr 7%-39%) and benzo(a)pyrene (19.5%-28%). The highest reductions were observed in dual (P+E, B+E) and triple (P+B+E) treatments. The best elimination yields were obtained after P+B+E treatment, as highlighted by the battery of ecotoxicological tests and bioassays performed with earthworms, plants and bacteria., Competing Interests: Declaration of competing interest We declare that we do not have any conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Soils contaminated by organic and inorganic pollutants like Cr(VI) and lindane, is currently a main environmental challenge. Biological strategies, such as biostimulation, bioaugmentation, phytoremediation and vermiremediation, and nanoremediation with nanoscale zero-valent iron (nZVI) are promising approaches for polluted soil health recovery. The combination of different remediation strategies might be key to address this problem. For this reason, a greenhouse experiment was performed using soil without or with an organic amendment. Both soils were contaminated with lindane (15 mg kg -1 ) and Cr(VI) (100 or 300 mg kg -1 ). After one month of aging, the following treatments were applied: (i) combination of bioaugmentation (actinobacteria), phytoremediation (Brassica napus), and vermiremediation (Eisenia fetida), or (ii) nanoremediation with nZVI, or (iii) combination of biological treatments and nanoremediation. After 60 days, the wellness of plants and earthworms was assessed, also, soil health was evaluated through physico-chemical parameters and biological indicators. Cr(VI) was more toxic and decreased soil health, however, it was reduced to Cr(III) by the amendment and nZVI and, to a lesser extent, by the biological treatment. Lindane was more effectively degraded through bioremediation. In non-polluted soils, nZVI had strong deleterious effects on soil biota when combined with the organic matter, but this effect was reverted in soils with a high concentration of Cr(VI). Therefore, under our experimental conditions bioremediation might be the best for soils with a moderate concentration of Cr(VI) and organic matter. The application of nZVI in soils with a high content of organic matter should be avoided except for soils with very high concentrations of Cr(VI). According to our study, among the treatments tested, the combination of an organic amendment, biological treatment, and nZVI was shown to be the strategy of choice in soils with high concentrations of Cr(VI) and lindane, while for moderate levels of chromium, the organic amendment plus biological treatment is the most profitable treatment., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Several ecotoxicological studies assessed metal toxicity upon soil biota and other communities but were mainly focused on the study of a single chemical and usually under optimal conditions of temperature. Meanwhile an increasing global warming is leading to new scenarios by combining different stress factors; chemical stress and thermal stress. Presently, this study aims to assess the joint effects produced by cadmium and elevated temperature on earthworms different levels of biological complexity. Eisenia fetida earthworms were maintained at 19 °C and 26 °C and simultaneously exposed to four Cd concentrations (1.25, 2.5, 25 and 125 mg Cd/Kg soil) for 14 (Short term exposure) and 56 days (reproduction test). Endpoints were addressed at different levels of biological complexity: reproductive impairment (cocoons and juvenile productions), Cd tissue accumulation, mortality of adults, weight loss and cytotoxic effects (coelomocyte viability). In the Short term exposure, increase in temperature produced a larger accumulation of Cd. Hence, earthworms exposed to 125 mg Cd/kg soil under heat stress (26 °C) showed a two fold higher Cd accumulation comparing to those at 19 °C. Earthworms exposed to moderate-high concentrations of Cd (2.5-125 mg Cd/kg) and maintained at 26 °C showed severe weight loss and high mortality rates. The neutral red uptake capacity of coelomocytes extruded from earthworms exposed to the highest Cd concentration decreased after 14 d at 19 °C, and more markedly at 26 °C. The reproduction impairment (decreased number of cocoons) was enhanced after exposure to concentrations higher than 2.5 mg Cd/kg at 26 °C, and after exposure to 125 mg Cd/kg at 19 °C. Earthworm reproduction capability is highly vulnerable to the effect of toxicants at elevated temperatures and sublethal concentrations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The current use and development of applications with silver nanoparticles (Ag NPs) could lead to potential inputs of these NPs to soils. Consequently, it is crucial to understand the ecotoxicological risks posed by Ag NPs in the terrestrial compartment. In the present investigation, the effects produced by PVP-PEI coated Ag NPs were assessed in Eisenia fetida earthworms in comparison with the soluble form (AgNO 3 ). Earthworms were exposed for 1, 3 and 14 days to a range of sublethal concentrations of Ag (0, 0.05 and 50mg/kg) and at each exposure time, apart from mortality and weight loss of individuals, metallothionein (MT) protein concentration and catalase (CAT) activity were quantified in earthworm tissues. In addition, cellular and molecular level endpoints (cell viability, absolute and relative trophic indices and transcription levels of catalase-cat- and metallothionein-mt-) were measured in coelomocytes extruded from exposed earthworms. Despite the lack of effects in traditional endpoints (mortality and weight loss), Ag NPs and AgNO 3 posed changes at lower levels of biological complexity (biochemical, cellular and molecular levels). Both Ag forms induced similar changes in the metal detoxification mechanism (MT, mt) and in the antioxidant response system (CAT, cat) of E. fetida. In contrast, Ag form dependant cytotoxicity and subpopulation ratio alterations (eleocytes/amoebocytes) were recorded in extruded coelomocytes. Complementarily, the use of coelomocytes to assess molecular level endpoints represented a relevant alternative for development of non-invasive biomarkers., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Despite the increasing interest in silver nanoparticles toxicity still few works dealt with the hazards of nanosized Ag in soils (either dissolved in pore water or coupled to colloids) although disposal of biosolids in landfills has been reported as the major source of silver nanoparticles in terrestrial environments. Presently, Eisenia fetida was used to assess the toxicity of 5 nm sized PVP-PEI coated silver nanoparticles in soil through the implementation of different exposure media Standard Toxicity Tests (Paper Contact and Artificial Soil -OECD-207- and Reproduction -OECD-222- Tests) together with cellular biomarkers measured in extruded coelomocytes. In order to decipher the mode of action of silver nanoparticles in soil and the uptake routes in earthworms, special attention was given to the Ag accumulation and distribution in tissues. High Ag accumulation rates, weight loss, and mortality due to the disruption of the tegument could be the result of a dermal absorption of Ag ions released from silver nanoparticles (Paper Contact Test). However, autometallography showed metals mainly localized in the digestive tract after Artificial Soil Test, suggesting that Ag uptake occurred mostly through soil ingestion. That is, silver nanoparticles attached to soil colloids seemed to be internalized in earthworms after ingestion of soil and transferred to the digestive gut epithelium where at high doses they have triggered severe effects at different levels of biological complexity.

A suite of organisms from different taxonomical and ecological positions is needed to assess environmentally relevant soil toxicity. A new bioassay based on Dictyostelium is presented that is aimed at integrating slime molds into such a testing framework. Toxicity tests on elutriates and the solid phase developmental cycle assay were successfully applied to a soil spiked with a mixture of Zn, Cd, and diesel fuel freshly prepared (recently contaminated) and after 2 yr of aging. The elutriates of both soils provoked toxic effects, but toxicity was markedly lower in the aged soil. In the D. discoideum developmental cycle assay, both soils affected amoeba viability and aggregation, with fewer multicellular units, smaller fruiting bodies and, overall, inhibition of fruiting body formation. This assay is quick and requires small amounts of test soil, which might facilitate its incorporation into a multispecies multiple-endpoint toxicity bioassay battery suitable for environmental risk assessment in soils. Environ Toxicol Chem 2016;35:1413-1421. © 2015 SETAC., (© 2015 SETAC.)

Lanestosa is a chronically polluted site (derelict mine) where the soil (Lanestosa (LA) soil) exceeds screening values (SVs) of regulatory policies in force (Basque Country; Europe) for Zn, Pb and Cd. A scenario-targeted toxicity assessment was carried out on the basis of a multi-endpoint bioassay approach. Acute and chronic toxicity bioassays were conducted with selected test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates and with bioaccumulation studies in earthworms. Besides, the toxicity profile was compared with that of the mine runoff (RO) soil and of a fresh artificially polluted soil (LAAPS) resembling LA soil pollutant profile. Extractability studies in LA soil revealed that Pb, Zn and Cd were highly available for exchange and/or release into the environment. Indeed, Pb and Zn were accumulated in earthworms and LA soil resulted to be toxic. Soil respiration, V. fischeri, vegetative and developmental cycles of D. discoideum and survival and juvenile production of E. fetida were severely affected. These results confirmed that LA soil had unacceptable environmental risk and demanded intervention. In contrast, although Pb and Zn concentrations in RO soil revealed also unacceptable risk, both metal extractability and toxicity were much lower than in LA soil. Thus, within the polluted site, the need for intervention varied between areas that posed dissimilar risk. Besides, since LAAPS, with a high exchangeable metal fraction, was the most toxic, ageing under in situ natural conditions seemingly contributed to attenuate LA soil risk. As a whole, combining multi-endpoint bioassays with scenario-targeted analysis (including leaching and ageing) provides reliable risk assessment in soils posing unacceptable environmental risk according to SVs, which is useful to optimise the required intervention measures.

Coelomocytes comprise the immune system of earthworms and due to their sensitivity responding to a wide range of pollutants have been widely used as target cells in soil ecotoxicology. Recently, in vitro assays with primary cultures of coelomocytes based in the neutral red uptake (NRU) assay have been developed as promising tools for toxicity assessment chemical in a reproducible and cost-effective manner. However, NRU showed a bimodal dose-response curve previously described after in vivo and in vitro exposure of earthworm coelomocytes to pollutants. This response could be related with alterations in the relative proportion of coelomocyte subpopulations, amoebocytes and eleocytes. Thus, the aims of the present work were, first, to establish the toxicity thresholds that could be governed by different cell-specific sensitivities of coelomocytes subpopulations against a series of metals (Cu, Cd, Pb, Ni), and second to understand the implication that coelomocyte population dynamics (eleocytes vs. amoebocytes) after exposure to pollutants can have on the viability of coelomocytes (measured by NRU assay) as biomarker of general stress in soil health assessment. Complementarily flow cytometric analyses were applied to obtain correlative information about single cells (amoebocytes and eleocytes) in terms of size and complexity, changes in their relative proportion and mortality rates. The results indicated a clear difference in sensitivity of eleocytes and amoebocytes against metal exposure, being eleocytes more sensitive. The bimodal dose-response curve of NRU after in vitro exposure of primary cultures of coelomocytes to metals revealed an initial mortality of eleocytes (decreased NRU), followed by an increased complexity of amoebocytes (enhanced phagocytosis) and massive mortality of eleocytes (increased NRU), to give raise to a massive mortality of amoebocytes (decrease NRU). A synergistic effect on NRU was exerted by the exposure to high Cu concentrations and acidic pH (elicited by the metal itself), whereas the effects on NRU produced after exposure to Cd, Ni and Pb were due solely to the presence of metals, being the acidification of culture medium meaningless.

Coelomocytes, immunocompetent cells of lumbricids, have received special attention for ecotoxicological studies due to their sensibility to pollutants. Their in vitro responses are commonly quantified after in vivo exposure to real or spiked soils. Alternatively, quantifications of in vitro responses after in vitro exposure are being studied. Within this framework, the present study aimed at optimizing the neutral red uptake (NRU) assay in primary culture of Eisenia fetida coelomocytes for its application in soil toxicity testing. Optimized assay conditions were: earthworm depuration for 24 h before retrieving coelomocytes by electric extrusion; 2 × 10(5) seeded cells/well (200 µl) for the NRU assay and incubation for 1 h with neutral red dye. Supplementation of the culture medium with serum was not compatible with the NRU assay, but coelomocytes could be maintained with high viability for 3 days in a serum-free medium without replenishment. Thus, primary cultures were used for 24 h in vitro toxicity testing after exposure to different concentrations of Cd, Cu, Ni and Pb (ranging from 0.1 to 100 μg/ml). Primary cultures were sensitive to metals, the viability declining in a dose-dependent manner. The toxicity rank was, from high to low, Pb > Ni > Cd > Cu. Therefore, it can be concluded that the NRU assay in coelomocytes in primary cultures provides a sensitive and prompt response after in vitro exposure to metals.

Eisenia fetida is a model species for soil health assessment and different biomarkers that detect either the presence of bioavailable contaminants or their biological effect have been developed. These parameters are performed in a target tissue or whole earthworm, without considering the marked zonation in histological organisation, enzyme activities and gene expression pattern existing along the body. Thus, the present work was aimed at (a) characterising the morphofunctional heterogeneity along the digestive tract of E. fetida in tissue morphology and turnover, lysosomal enzyme markers (β-glucuronidase, β-GUS; hexosaminidase, HEX), lipofuscin contents (LPF) and metallothionein (MT) and catalase (CAT) gene expression; and (b) determining whether the responsiveness to Cd exposure varies among tissues and along the digestive tract. HEX and β-GUS exhibited a heterogeneous distribution pattern along and across the digestive tract and Cd exposure caused a marked decrease of HEX and an increase of β-GUS activity. Likewise, the significant decrease of cell turnover and the induction of MT transcription were was zone-dependent. Therefore, in was concluded that the consideration of the zonation when applying biomarker for toxicity assessment would reduce the intrinsic variability that results from overlooking the marked morphofunctional heterogeneity that exists in annelids along their body axis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Toxicity profiles of two soils (a brownfield in Legazpi and an abandoned iron mine in Zugaztieta; Basque Country) contaminated with several metals (As, Zn, Pb and Cu in Legazpi; Zn, Pb, Cd and Cu in Zugaztieta) and petroleum hydrocarbons (in Legazpi) were determined using a multi-endpoint bioassay approach. Investigated soils exceeded screening values (SVs) of regulatory policies in force (Basque Country; Europe). Acute and chronic toxicity bioassays were conducted with a selected set of test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates, as well as with bioaccumulation studies in earthworms. The sensitivity of the test species and the toxicity endpoints varied depending on the soil. It was concluded that whilst Zugaztieta soil showed very little or no toxicity, Legazpi soil was toxic according to almost all the toxicity tests (solid phase Microtox, D. discoideum inhibition of fruiting body formation and developmental cycle solid phase assays, lettuce seed germination and root elongation test, earthworm acute toxicity and reproduction tests, D. discoideum cell viability and replication elutriate assays). Thus, albeit both soils had similar SVs, their ecotoxicological risk, and therefore the need for intervening, was different for each soil as unveiled after toxicity profiling based on multiple endpoint bioassays. Such a toxicity profiling approach is suitable to be applied for scenario-targeted soil risk assessment in those cases where applicable national/regional soil legislation based on SVs demands further toxicity assessment.

The social amoeba Dictyostelium discoideum has been proposed for assessing stress responses to pollutants in soil and it has already been successfully employed in the aquatic environment. Presently, we developed the DDDC assay (D. discoideum developmental cycle assay) for both soil toxicity assessment and soil health screening. The DDDC assay is primarily aimed at determining the capacity of D. discoideum to undergo its developmental programme forming a fruiting body, measured in terms of fruiting body formation inhibition and fruiting body size factor, which may be considered an indication of its ecological fitness (potential for spore dispersal). A second objective of the solid phase DDDC assay is to identify potential mechanisms of toxic action on the developmental cycle, for which three checkpoints are examined: (a) aggregation arrest, (b) migration arrest, and (c) culmination arrest. Presently, conditions for the DDDC assay such as soil texture, soil water content, soil pH, food availability and incubation time were investigated and optimized. In addition, both solid and liquid phase variants of the DDDC assay were applied to assess the toxicity of Hg, at regulatory concentrations. The developmental cycle and ecological fitness were affected from the exposure to 0.3 mg Hg/kg dry-wt soil onwards. The DDDC assay has been shown to be a high sensitivity test., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This is a pilot study for assessing soil ecosystem health in chronically polluted sites on the basis of a 3-tier approach (screening+scoring+understanding) designed to be cost-effective and scientifically based, and to provide straightforward advice and support to managers and stakeholders involved in environmental protection. For the initial screening (Tier 1), the use of a highly sensitive, low-cost biomarker such as neutral red uptake (NRU) in earthworm coelomocytes is proposed. In sites where an alteration in NRU has been established, the stress level may be further assessed by utilising a suite of low-cost and rapid biomarkers of effect integrated in an integrative biological response (IBR) index to obtain an objective (scored) assessment of the induced stress syndrome (Tier 2). The IBR/n index is based on the integration of biomarkers at different levels of biological organisation. Acyl-CoA oxidase activity (AOX), catalase activity (CAT), lipofuscin optical density (LOD%), NRU and the mean epithelial thickness (MET) have been used to calculate the IBR/n index. Biomarkers are determined in earthworms, Eisenia fetida, exposed ex situ to real soils (three mining sites and a reference) for 3, 10 and 17d. The 3d NRU (Tier 1) provided signal of stress. After 3d, PCA, based on the suite of biomarkers (Tier 2), discriminated reference and polluted sites according to toxicity profiles and at 17d, the most polluted site is segregated from less polluted and reference sites. Soils were classified as harmful, unhealthy (not apparently toxic) or healthy. Soils were investigated by microarray transcriptomics (Tier 3), to understand the causes (aetiology) and consequences (prognosis) of health impairment. Tier 3 discriminates, according to stress syndrome traits, soils that did not fall into the category of highly stressed and revealed the main agent causing toxicity at each site by identifying the toxicity mechanisms and biological responses., (Copyright © 2012 Elsevier B.V. All rights reserved.)