Potential ecotoxicological effects of silver nanoparticles and silver sulphide on the endogeic earthworm Aporrectodea caliginosa (Savigny 1826).

Silver nanoparticles (AgNPs) are increasingly used in consumer products and subsequently arrive in wastewater systems, accumulating as silver sulphide (Ag2S) in the resulting biosolids, which are commonly spread onto agricultural fields as a fertiliser. Experiments were performed to investigate the effect of AgNPs, using the endogeic earthworm Aporrectodea caliginosa as a test organism. In an acute toxicity experiment, A. caliginosa were exposed to soil containing different concentrations of AgNPs (0, 1, 5, 10, 50, 100, 250, 500, 750, and 1000 mg kg-1 dry soil) and Ag2S (0, 10, 50, 100, 500, and 1000 mg kg-1 dry soil). Earthworm biomass and mortality were monitored. Earthworms exposed to 500, 750 and 1000 mg kg-1 fresh AgNPs had mortality rates of 20%, 60% and 70%, respectively. Changes in biomass were directly related to AgNP concentration. Exposure to Ag2S did not affect biomass or mortality. Further experiments used 0, 10, 50, 100 and 250 mg kg-1 AgNPs and 0, 50, 100, 500, and 1000 mg kg-1 Ag2S to evaluate sublethal effects on A. caliginosa. Avoidance behaviour in a linear gradient was evaluated after 14 days. Earthworms significantly preferred soil that was free of either AgNPs or Ag2S. The same concentrations were used to assess effects on cocoon production of A. caliginosa exposed to AgNPs and Ag2S. In the first 3 months of AgNP exposure, higher concentrations had a negative effect on cocoon production, but this effect diminished thereafter. Ag2S had no discernible effect on reproduction. Overall, introduction of AgNPs into the soil through the application of biosolids appears to be of low concern to the tested endogeic earthworm.

Mitonuclear discordance and patterns of reproductive isolation in a complex of simultaneously hermaphroditic species, the Allolobophora chlorotica case study.

Historical events of population fragmentation, expansion and admixture over geological time may result in complex patterns of reproductive isolation and may explain why, for some taxa, the study of mitochondrial (mt) and nuclear (nu) genetic data results in discordant evolutionary patterns. Complex patterns of taxonomic diversity were recently revealed in earthworms for which distribution is largely the result of paleogeographical events. Here, we investigated reproductive isolation patterns in a complex of cryptic species of earthworms in which discordant patterns between mt and nu genetic lineages were previously revealed, the Allolobophora chlorotica aggregate. Using four nu microsatellite markers and a fragment of the cytochrome c oxidase subunit I mt gene, we carried out a parentage analysis to investigate the mating patterns (i) between individuals belonging to two divergent mt lineages that cannot be distinguished with nu markers and (ii) between individuals belonging to lineages that are differentiated both at the mt and nu levels. Amongst the 157 field-collected individuals, 66 adults were used in cross-breeding experiments to form 22 trios based on their assignment to a mt lineage, and 453 obtained juveniles were genotyped. We showed that adults that mated with both their potential mates in the trio produced significantly more juveniles. In crosses between lineages that diverged exclusively at the mt level, a sex-specific pattern of reproduction characteristic to each lineage was observed, suggesting a possible conflict of interest concerning the use of male/female function between mating partners. In crosses between lineages that diverged both at the mt and nu levels, a high production of cocoons was counterbalanced by a low hatching rate, suggesting a post-zygotic reproductive isolation. Different degrees of reproductive isolation, from differential sex allocation to post-zygotic isolation, were thus revealed. Lineages appear to be at different stages in the speciation process, which likely explain the observed opposite patterns of mitonuclear congruence.

Assessment of avoidance behaviour by earthworms (Lumbricus rubellus and Octolasion cyaneum) in linear pollution gradients.

Avoidance behaviour by earthworms is recognised as a valuable endpoint in soil quality assessment and has resulted in the development of a standardised test (ISO 17512-1, 2008) providing epigeic earthworms with a choice between test and control soils. This study sought to develop and evaluate an avoidance test utilising soil-dwelling earthworms in linear pollution gradients with Visible Implant Elastomer (VIE) tags used to identify individual organisms. Sequential experiments were established in laboratory-based mesocosms (0.6m×0.13m×0.1m) that determined the relative sensitivities (in terms of associated avoidance behaviour) of Octolasion cyaneum and Lumbricus rubellus at varying levels of polluted soil and also assessed the influence of introduction point on recorded movement within gradients. In an initial gradient (0%, 25%, 50%, 75%, 100% polluted soil), both species exhibited a clear avoidance response with all surviving earthworms retrieved (after 7 days) from the unpolluted soil. In a less polluted gradient (0%, 6.25%, 12.5%, 18.75%, 25%) L. rubellus were retrieved throughout the gradient while O. cyaneum were located within the 0% and 6.25% divisions, suggesting a species-specific response to polluted soil. Results also showed that the use of a linear pollution gradient system has the potential to assess earthworm avoidance behaviour and could provide a more ecologically relevant alternative to the ISO 17512: 2008 avoidance test. However, further work is required to establish the effectiveness of this procedure, specifically in initial chemical screening and assessment of single contaminant bioavailability, where uptake of pollutants by earthworms could be measured and directly related to the point of introduction and retrieval.

Chemical Composition of Earthworm (Dendrobaena veneta Rosa) Biomass Is Suitable as an Alternative Protein Source.

The selected chemical composition (dry matter, ash, total protein, and crude fat) of the integumentary muscles of Dendrobaena veneta were determined, plus the dry matter (DM) percentage content of 17 amino acids and the profile (%) of fatty acids. Results were compared with a more fully studied earthworm, Eisenia fetida. In addition, the composition of exogenous amino acids was compared to the WHO standard for pork, beef, and chicken eggs. Both earthworm species were grown on the same kitchen waste, and protein composition was analyzed using the same methods. Studies indicated that the muscle of D. veneta was characterized by a high level of protein (76.82% DM). A similar content of exogenous amino acids was observed in the protein of both earthworms, but for phenylalanine and isoleucine, slightly higher levels were recorded for E. fetida. More histidine, lysine, threonine, isoleucine, and arginine were found in earthworms compared with chicken egg white. Fatty acids play an essential role in balancing human or animal feed and their content determines the dietary and nutritional value of the food. Both earthworm species contained the appropriate content of saturated and unsaturated acids. In D. veneta, a higher content of arachidonic acid was found, and in E. fetida, lauric, tridecanoic, and palmitic acids were present. Future issues of food security may force us to seriously consider earthworm protein for indirect or even direct human consumption.

Mechanistic Effect Modeling of Earthworms in the Context of Pesticide Risk Assessment: Synthesis of the FORESEE Workshop.

Earthworms are important ecosystem engineers, and assessment of the risk of plant protection products toward them is part of the European environmental risk assessment (ERA). In the current ERA scheme, exposure and effects are represented simplistically and are not well integrated, resulting in uncertainty when the results are applied to ecosystems. Modeling offers a powerful tool to integrate the effects observed in lower tier laboratory studies with the environmental conditions under which exposure is expected in the field. This paper provides a summary of the (In)Field Organism Risk modEling by coupling Soil Exposure and Effect (FORESEE) Workshop held 28-30 January 2020 in Düsseldorf, Germany. This workshop focused on toxicokinetic-toxicodynamic (TKTD) and population modeling of earthworms in the context of ERA. The goal was to bring together scientists from different stakeholder groups to discuss the current state of soil invertebrate modeling and to explore how earthworm modeling could be applied to risk assessments, in particular how the different model outputs can be used in the tiered ERA approach. In support of these goals, the workshop aimed at addressing the requirements and concerns of the different stakeholder groups to support further model development. The modeling approach included 4 submodules to cover the most relevant processes for earthworm risk assessment: environment, behavior (feeding, vertical movement), TKTD, and population. Four workgroups examined different aspects of the model with relevance for risk assessment, earthworm ecology, uptake routes, and cross-species extrapolation and model testing. Here, we present the perspectives of each workgroup and highlight how the collaborative effort of participants from multidisciplinary backgrounds helped to establish common ground. In addition, we provide a list of recommendations for how earthworm TKTD modeling could address some of the uncertainties in current risk assessments for plant protection products. Integr Environ Assess Manag 2021;17:352-363. © 2020 SETAC.

Aspects of the ecology of the earthworm Eisenia lucens (Waga 1857) studied in the field and in laboratory culture.

This work relates data from field sampling of Eisenia lucens and from laboratory-based culture. Field sampling used soil sorting and vermifuge extraction and took place in beech-dominated forests of southwest Poland. Initial work derived population estimates from four sub-communities of the forest looking for seasonal dynamics and later work employed targeted sampling in summer within rotting wood to obtain live specimens for laboratory culture. A preliminary examination within and below rotten wood during winter was also undertaken. In the laboratory, clitellate earthworms were kept at 20 °C, the substrate changed every 6 months, and the population examined. Cocoons were incubated individually at 15 °C, with number of hatchlings per cocoon and the mass of each determined. Hatchlings were grown at 15 °C in field-collected wood and compared with growth in a 1:1 volume ratio of wood and horse manure. Further hatchlings were fed with horse manure only (at 10 °C) and after 19 weeks, half were transferred to 15 °C. In the field, mature individuals varied significantly (p < 0.01) in biomass between 2 sampling sites where found, with an overall mean density across sites of 4.14 ± 3.53 m-2 with a mean biomass of 2.21 ± 1.93 g m-2. Numbers in soil varied over the sampling period, with a suggestion that this species moves from mineral soil to organic-rich dead wood as conditions permit. In summer, all life stages were recovered from rotting wood above the mineral soil. Sampling in winter found cocoons in rotting wood below snow. These hatched rapidly (within 2 weeks) when taken to the laboratory. Laboratory culture allowed maintenance of a population for 2 years. Mean cocoon mass was 50.6 mg with a mean of 2.9 hatchlings per cocoon and hatchling mass was inversely proportional to number per cocoon. Growth with 50% horse manure was significantly greater (p < 0.001) than with wood. Increased temperature from 10 to 15 °C brought more significantly (p < 0.05) rapid growth. To culture this species through its life cycle, a natural substrate is needed, but then it is necessary to acclimate the animals to something more easily obtainable. More work is needed from field sampling to fully understand the seasonal dynamics of this species, which utilises different parts of the soil profile throughout the year.