Session 3: Earthworms facing environmental pollutants: effects, responses and retroactions
(Tuesday, July
12 – 2:30 p.m. – 6:30 p.m.)



The effects of different gold mine tailings on growth, reproduction and avoidance-behaviour of earthworms

Mark Maboeta, Bryan McGuirk, Pieter Theron
Unit for Environmental Sciences and Management, North West University, Potchefstroom, Private Bag X6001, Potchefstroom 2520, South Africa

The mining industry plays a key role in the economic development of South Africa as one of the largest exporters of valuable resources such as, gold, platinum group metals and other metals and minerals. Unfortunately, mining produces large volumes of solid waste in the form of tailing disposal facilities (TDFs), which contain a variety ofmetals which are hazardous to the soil environment. The aim of this study was to use earthworm bioassays and avoidance-behaviour tests to assess the effects of gold minetailings on soil organisms. Six random soil samples were taken from four different sites on a gold mine viz. two different TDFs and two grassy pasture sites. Earthworms (Eisenia andrei) were exposed for a period of 28 days to the soils from these different sites. Soil from the TDFs were also diluted to25%, 50%, 75% and 100% respectively. Soil chemical analysis showed that the TDFs had the lowest pH levels and that only Cr concentrations were higher than proposed benchmarks in two of the sites. Earthworms exposed to the TDF material showed significantly (p < 0.05) lower earthworm biomass with a very low cocoon production. The avoidance-behaviour test showed similar results, where earthworms generally preferred naturals and control soils over the TDF material. It can be concluded, that even though the gold mine was inactive for a long period of time, it still remains a highly contaminated area.

keywords: Bioassays, earthworms, gold mines, soil, metals, tailing disposal facilities.


Effects of silver nanoparticles on the endogeic earthworm species Aporrectodea caliginosa

J. Kister1, C. N. Lowe1, C. Mellor1, E. D. Elphinstone2 and K. R. Butt1
University of Central Lancashire, School of Natural Science, Preston, United Kingdom
Myerscough College, Bilsborrow, Preston, PR3 0RY, UK

Silver nanoparticles (AgNPs) are increasingly used in consumer products due to their antimicrobial properties. Subsequently, they arrive in wastewater systems and accumulate in the resulting biosolids, which are commonly spread onto agricultural fields as a fertiliser. While the effects of AgNPs on earthworms has been well studied, the lack of consistency in both species of test organism and type of nanoparticle used makes direct comparison of results difficult. A series of 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 over two weeks to soil containing concentrations of newly purchased, in addition to aged AgNPs (0, 1, 5, 10, 50, 100, 250, 500, 750, and 1000 mg kg-1 dry soil). Earthworm biomass and mortality were monitored. All earthworms survived exposure to oxidised (aged) AgNPs, while those 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. Further experiments used 0, 10, 50, 100 and 250 mg kg-1 AgNPs 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 AgNPs. The same concentrations were used to assess effects on cocoon production of A. caliginosa exposed to AgNPs. Over 5 months, it was found that higher concentrations had a negative effect on cocoon production, but this effect diminished after 2 months.While high concentrations of AgNPs can prove toxic to A. caliginosa, it appears that they can acclimatise to lower concentrations.

keywords: Aporrectodea caliginosa; Avoidance behaviour; Ecotoxicology; Silver nanoparticles; Reproduction


The effect of coal ash and slag with enhanced content of natural ly occurring radionuclides on Eisenia fetida

Josip Nikolić1, Davorka Hackenberger Kutuzović1, Božena Skoko2, Branimir Hackenberger Kutuzović1,Goran Klobučar3, Matej Vucić3, Marko Štrok4, Leja Rovan4, Eva Kerčmar4, Ivica Prlić2
1  J. J. Strossmayer University, Department of Biology, Cara Hadrijana 8A, HR-31000 Osijek, Croatia12  Institute for Medical Research and Occupational Health, Ksaverska cesta 2, HR-10000, Zagreb, Croatia23  Faculty of Science, Department of Biology, Rooseveltov trg 6, HR-10000 Zagreb, Croatia34  Jožef Stefan Institute, Laboratory for radiochemistry, Jamova 39, 1000 Ljubljana, Slovenia4

Industrial residues and wastes with higher content of naturally occurring radionuclides represent a potential ecological risk to the biota residing in the vicinity of the disposal sites. Coal ash and slag landfills are widespread due to usage of coal as fuel in thermal power plants worldwide. Combustion of coal results in preconcentration of radionuclides naturally present in coal in its residual ash as well as the spread of the ash particles at greater distances. Earthworms represent one of the most important group of terrestrial organisms and are therefore often used in ecotoxicological studies. The aim of this study was to determine the influence of weathered coal ash and slag with enhanced content of radionuclides from uranium decay chain that were sampled in the area of the former chemical factory in Kaštela Bay (Croatia), on biochemical markers (protein, lipid and carbohydrate content, lipid peroxidation, GST and AChE activity), genotoxicity  markers  (Comet assay and micronucleus test), cytotoxicity (neutral red uptake assay) and reproduction of the earthworm Eisenia fetida. The results have shown that 28 days exposure to those substrates cause  oxidative stress, genotoxic and cytotoxic effects and inhibit reproduction. Compared to the control samples, earthworms exposed to the coal ash had significantly higher activity concentrations of Po-210, a member of uranium decay chain and one of the most radiotoxic naturally occurring radionuclide. Coelomic fluid expression had no significant impact on the activity concentrations of Po-210 in earthworms, indicating that polonium is mostly deposited in the soft tissues. 

keywords: Radionuclide; earthworm; biomarker;  reproduction; genotoxicity


Exploring the potential use of earthworms to mitigate microplastic pollution in the soil

K. Meng1, E. Huerta Lwanga1,3, M. van der Zee2, V. Geissen1
1  Soil Physics and Land Management Group, Wageningen University & Research, Droevendaalsesteeg 3, 6708PB Wageningen, The Netherlands
2  Wageningen Food&Biobased Research, BornseWeilanden 9, 6708WG Wageningen, The Netherlands
3  Agroecología, El Colegio de la Frontera Sur, Unidad Campeche, Av Polígono s/n, Cd. Industrial, Lerma, Campeche, Mexico

The accumulation of microplastics in the soil possesses risk to soil health. Efforts need to be made to alleviate microplastic pollution in the soil. Here, we explored the potential use of Lumbricus terrestris(Oligochaeta) to mitigate microplastic (Low-density polyethylene (LDPE), Polylactic acid (PLA) and Polybutylene adipate terephthalate (PBAT)) pollution in the soil, focusing on the ingestion behavior of this earthworm on different microplastics and the respective changes of those microplastics during the passage through the earthworm digestive tract. A 4-day Petri Dish test and a 35-day mesocosm test were conducted. No mortality was recorded in both tests, and the presence of microplastics in the soil did not show any negative impacts on the growth rate, ingestion rate, and activity of earthworms. In the Petri Dish test, we found that in a slightly microplastic-polluted soil (1%, w/w), Lumbricus terrestris could achieve an ingestion efficiency of 0.38-1.47, 1.18-2.73 and 0.89-2.15 mg MP g-1 worm d-1, for LDPE, PBAT and PLA microplastics respectively with a size range of 53-1000 μm (diameter). The ingestion capacity on microplastics showed a size-dependent manner. In the mesocosm test, microplastic size distributions in the bulk soil and in the worm gut were significantly different for LDPE and PBAT. After ingestion, the ratio of small LDPE microplastics (area 325-10,000 μm2) increased by 115.5% in the gut, while the ratio of small PBAT microplastics (area 325-10,000 μm2) decreased by 46.4% in the gut. In addition, the depolymerization of PBAT and PLA microplastics was observed in the gut. Overall, our results suggest that once ingested by the earthworm, microplastics would undergo fragmentation and potentially depolymerization in the gut, depending on the polymer type. Bioremediation of microplastic-polluted soil using earthworms is a promising approach.

keywords: Microplastics; Earthworm; Bioremediation; Depolymerization; Fragmentation


Earthworms facilitate the biotransformation of roxarsone and reduce its adverse effects on soil properties.

Yinsheng Li
Shanghai Jiao Tong University


Exploring the molecular mechanisms of pesticide tolerance of the soil engineer biodiversity by combining transcriptomics and proteomics analyses

Audrey Barranger1, Christophe Klopp2, Stéphanie Llopis1, Barbara Le Bot3, Gaëlle Saramito3, Emmanuelle Com4, Claudia Wiegand1 and Françoise Binet1
UMR CNRS ECOBIO 6553, University of Rennes 1, Campus de Beaulieu, 263 Avenue du Général Leclerc, 35042 Rennes Cedex, France
UR INRA 875 MIAT, GENOTOUL, 24 Chemin de Borde Rouge, 31326 Castanet-Tolosan Cedex, France
UMR Inserm 1085, IRSET, LERES, Avenue du Professeur Léon Bernard, 35043 Rennes Cedex, France
UMR Inserm 1085, IRSET, Protim, Proteomics Core Facility, University of Rennes 1, Campus de Beaulieu, 263 Avenue du Général Leclerc, 35042 Rennes Cedex, France

Environmental diffuse pollution by agrochemicals has become a major soil threat. In cropped fields, non-target organisms, such as earthworms are frequently exposed to different pesticide applications. Although soil engineer biodiversity declines in agro-ecosystems, certain endogeic species are still abundant and persist in intensively managed fields, suggesting they become tolerant to long-term anthropogenic pressure. We thus raised a first work hypothesis that populations of A. caliginosa earthworms derived from conventional managed field developed a tolerance to pesticides compared to those from organic managed field. If A. caliginosa species is tolerant, another correlative question is to decipher whether worm tolerance is based on physiological acclimation or whether it involves genetic adaptation, which can be inherited across generations. We set-up a multigenerational evolutionary toxicology experiment (F0, F1 and F2) to obtain 3 generations of earthworms and used a non-targeted strategy combining transcriptomics and proteomics to resolve the issue. A. caliginosa populations used in this study originated from two contrasting-managed fields, a conventionally versus an organic field. Adult worms have been cultured in chemical-free soil in the laboratory and experimentally exposed to epoxiconazole for 28 days, as relevant fungicide widely used. We were successful in performing the first de novo transcriptome assembly of the endogeic earthworm species A. caliginosa. We also showed that several molecular pathways involved in stress exposure were constitutively activated in earthworms of highly contaminated fields. These responses were linked to up-regulated genes involved in detoxification, oxidative stress and mitochondrial respiratory chain. Next step of the work will be to create de novo a protein database involved in pesticide response of soil engineer and identify the genes and proteins network responsible for tolerance in A. caliginosa species. This study will improve our understanding of the long-term impact of chronic exposure of soil engineers to low-dose multi-pollutants and assess the costs associated with this tolerance for populationsin natura.

keywords: Stress ecology ;Omics ; Earthworms ;  Pesticide residues ; Soil management


terrestrial fauna might be underestimated when based solely on active ingredient toxicity and standard earthworm tests

Jorge-Escudero Gabriella1,2, Pérez Polanco Mariana1, Lagerlöf Jan3, Pérez Carlos A.4, Míguez Diana.5
Departamento de Sistemas Ambientales, Facultad de Agronomía, Universidad de la República, Garzón 780, 12900 Montevideo, Uruguay.
Departamento de Suelos y Aguas, Facultad de Agronomía, Universidad de la República, Garzón 780, 12900 Montevideo, Uruguay.
Department of Ecology, Swedish University of Agricultural Sciences, P.O Box 7044, SE-75007, Uppsala, Sweden
Departamento de Protección Vegetal, EEMAC, Facultad de Agronomía, Universidad de la República, Ruta 3 km 363, 60.000 Paysandú, Uruguay.
Latitud – LATU Foundation, Technological Laboratory of Uruguay (LATU), Montevideo, Uruguay.

Ecosystem services provided by earthworms are lost when land management reduce their populations. Hence, the importance of a thorough assessment of management effect on this group. Particularly, commercial fungicide toxicological effects, among those of other pesticides, may be underestimated if assessed solely by the toxicity of their active ingredients. Combined effects of various active ingredients and/or other ingredients may be far more deleterious than expected. Therefore, this present study aimed to: (1) review the possible influence of other ingredients within the formulations of two commercial fungicides, by pondering the hazards of each one, at a screening level, referring to official information presented in ECHA (European Chemicals Agency), NCBI (National Center for Biotechnology Information) and in relevant earthworm toxicity test data found in the literature; (2) assess the sub-lethal effect of these commercial fungicides on Eisenia fetida;  and (3) assess the acute lethal effect of one commercial fungicide on both Glossoscolex rione and E. fetida. Pondering hazards of all components of the studied commercial formulations revealed that alongside with the toxic active ingredients come other ingredients, some of which are as toxic, or more toxic than the former, and may even be in higher concentrations. The results of the tests with E. fetida met the validation criteria of the ISO 11268 standards. The inhibition concentration of 10% of the progeny of E. fetida (IC10) was estimated at 133 mg kg-1 for PROSARO®, and 1544 mg kg-1 for SWING PLUS®. Both fungicides showed an effect of hormesis on the progeny, i.e. stimulation at low concentrations. Being the first work of toxicity carried out with the earthworm G. rione, it was found that this is probably much more sensitive to the fungicide PROSARO than E. fetida, with preliminary values ​​of lethal concentration at 14 d of 285 mg kg-1 for the former, and > 1000 mg kg-1 for the latter. However, studies with G. rione should be continued in order to adjust breeding and survival conditions in laboratory conditions.

keywords: Eisenia fetida; Glossoscolex rione;cocktail effect


Assessing the bioaccumulation of engineered nanomate rials (TiO2) in oligochaetes – can Eisenia fetida and Enchytraeus crypticus be used interchangeably

E.J. Eagles1, C.L. Schultz1, A.G. Etxabe1, C. Svendsen1, N. J. Clark2, J. Vassallo2, R. D. Handy2 and E. Lahive1.
1  UK Centre for Ecology and Hydrology, Wallingford, OX10 8BB, UK2  School of Biological and Marine Sciences, University of Plymouth, PL4 8AA, UK
2  School of Biological and Marine Sciences, University of Plymouth, PL4 8AA, UK

Engineered nanomaterials (ENMs) are chemical substances or materials that have increasingly been used in consumer products and for commercial applications. They can reach soil through direct addition or as trace constituents of industrial and domestic waste materials and sewage sludge applied as a soil conditioner. One of the key components of environmental risk assessment for ENMs is the assessment of their potential for bioaccumulation, subsequent transfer through and biomagnification within food chains. For terrestrial environments, the only regulatory test guideline (OECD test guideline 317) for invertebrates evaluates bioaccumulation in terrestrial oligochaetes using either earthworms (usually Eisenia fetida) or the potworm, Enchytraeus crypticus. However, there can be differences between how these two species handle chemicals and so can result in differing bioaccumulation profiles. Enchytraeus crypticus offer the advantage of requiring less materials for testing, making them more attractive from a regulatory perspective but data for Eisenia fetida is more commonly available. The aim of this study was to compare the uptake of TiO2 ENMs - a non-soluble, low toxicity ENM - from soil by these two species to assess how comparable the bioaccumulation profiles. The uptake and elimination of four different TiO2 ENMs is investigated both in Eisenia fetida and Enchytraeus crypticus over21 and 14 days, respectively. Animals are sampled in time for measurement of TiO2 tissue concentrations, biochemical indicators of stress and histological alterations throughout the uptake and elimination periods. The objective is to assess how comparable the bioaccumulation potential of the TiO2 ENMs with differing properties is in the two species. The biochemical measurements and histology further will allow the assessment of any damage in the organisms as a result of exposure.

keywords: Earthworm;nanoparticle; uptake; risk assessment


Evaluation of Existing Models for Estimating the Bioaccumulation of Pesticides in Earthworms Using a Large Experimental Dataset

Jun Li1,Mark E Hodson1,Colin D. Brown1,Roman Ashauer2 and Tania Alvarez2
Department of Environment and Geography, University of York, Heslington,York, YO10 5NG, UK
Syngenta Crop Protection AG, Basel 4058, Switzerland

Pesticides can accumulate in earthworms and exert adverse effects on earthworms and top predators (Van Hoeselet al., 2017; Reed et al., 2016; Reinecke and Reinecke, 2007). Standardized laboratory procedures for determining the bioaccumulation of contaminants into earthworms such as the OECD guideline 317 (OECD 2010) are very labour intensive and time consuming. There is an ongoing community effort (see ‘Mind the gap – on the way to a spatial and temporal explicit TKTD model for earthworms’;Roeben 2019) to produce a spatially and temporally explicit toxicokinetic-toxicodynamic (TKTD) model for the uptake of pesticides by earthworms. Several predictive models, including empirical and dynamic mechanistic models (Torralba-Sanchez, 2016; Jager, 1998; Belfroidet al., 1993; Connell and Markwell, 1990; Armitage and Gobas, 2007; Jager, 2004; Jager et al., 2003) have been developed during the last three decades to predict the bioconcentration or bioaccumulation of organic chemicals in earthworms. However, the applicability of these models in predicting body residues for a variety of pesticides and earthworm species in various soil environments is uncertain, in part due to the lack of independent data sets to test the models. We are currently generating kinetic uptake and elimination data, for five pesticides (lenacil, flutriafol, dieldrin, hexachlorobenzene and p,p’-DDT) with varying Kd values (0.2-3.4 L/kg, 0.7-10.7 L/kg, 8.9-145.4 L/kg, 178.8-2929 L/kg and 3535-57900 L/kg, respectively) by the earthworms Eisenia fetida, Aporrectodea caliginosa and Lumbricus terrestris in five contrasting soils. Here we present the L. terrestris data. The data are used to evaluate and refine existing models and identify chemical and soil properties influencing earthworm uptake. Refined models could be highly beneficial for environmental risk assessment of pesticides and provide a rapid solution for regulators to support environmental decision making.

keywords: Pesticide, Earthworm, Uptake, Mechanistic model



Metaphire californica with different exposure history through toxicokinetic modelling

Caide Huang1,2, Yan Ge1, Zhiqiang Shen1, Kun Wang1, Shizhong Yue1, Yuhui Qiao1
1  College of Resources and Environmental Science, China Agricultural University, Beijing 100193, China
2  UK Centre for Ecology and Hydrology, Wallingford, OX10 8BB, UK

Toxicokinetic (TK) model provides a new approach to mechanistically elucidate the natural variation of metal handling strategy by adaptive and sensitive earthworm populations. TK model was applied to explore the metal handling and resistance strategy of wild Metaphire californica with different historical exposure history through a 12-day re-exposure and another 12-day elimination incubation. M. californica populations showed different kinetic strategies for non-essential metals (Cd and Pb) and essential metals (Zn and Cu), which were closely related to their exposure history. M. californica from the most serious Cd-contaminated soil showed the fastest kinetic rates of both Cd uptake (K1 = 0.78gsoil/gworm/day) and elimination (K2 = 0.23 day−1), and also had the lowest Cd half-life (t1/2 = 3.01 day), which demonstrated the potential Cd-resistance of wild M. californica from Cd-contaminated soils. Besides, the comparative experiment showed totally different metal kinetics of laboratory Eisenia fetida from field M. californica, suggesting the impacts of distinct exposure history and species-specifical sensitivities. These findings provide a novel approach to identify and quantify resistance using TK model and also imply the risk of overlooking existing exposure background and interspecies extrapolation in eco-toxicological studies and risk assessments.

keywords: Earthworms, Heavy metals, Toxicokinetic, Resistance, Environmental risk assessment


Using genome resources to understand invertebrate pesticides sensitivity and mixture responses

David Spurgeon1, Stephen Short1,2, Alex Robinson1, Elma Lahive1, Claire Badder1,2, Peter Kille2
UK Centre for Ecology & Hydrology, MacLean Building, Benson Lane, Wallingford, Oxon, OX10 8BB, UK
Cardiff University, School of Bioscience, Museum Avenue, Cardiff, Wales, UK

Understanding why some species are more sensitive that others to a given chemical is perhaps the most fundamental challenge in ecotoxicology. In this talk, we will highlight how the growing volume of genome and transcriptome data can be mined to identify the nature of the toxicokinetic and toxicodynamic traits that govern sensitivity. Using examples from comparative studies with earthworms and between earthworms and other taxa, we will show how sensitivity can be attributed to the genome complement of species, the presence of key residues in receptor binding domains, and, for the first time, on tissues specific expression patterns.

We generated whole genomes and tissue specific transcritomes for multiple terrestrial annelid species. These genomic resources provided a basis for investigating the potential drivers of species sensitivity in three case studies of differential sensitivity for the organophosphate chlorpyrifos among earthworm compared to arthropods, among earthworm species for the neonicotinoid imidacloprid and of differential mixture effects between earthworms and other species for azole fungicides and pyrethroid insecticides.

Bioassays identified cases of differential sensitivity between different earthworms and compared also to other taxa (e.g. arthropods). Genomic approaches then allowed us to investigate the bases of these differences.

Chlorpyrifos. Earthworms (Lumbricus rubellus) showed an approximate order of magnitude lower sensitivity to chlorpyrifos than a soil arthropod (Folsomia candida). This difference could be linked to tissue specific cholinesterase orthologue expression.

Imidacloprid. A large-magnitude (>30-fold) difference in sensitivity for imidacloprid between earthworms. This difference can be explained by difference the expression of different isoforms of sub-unit of the nicotinic acetylcholine receptor that contain amino acid residue associated with strong or weak imidacloprid binding.

Mixtures. Difference in the degree of synergy found between different mixtures in earthworms compared to other invertebrate taxa. This difference has been linked to the potential for one chemical to affect the toxicokinetic of another by inhibiting phase one and two metabolism to different extents.

keywords: genomics; orthologue, toxicokinetics, toxicodynamics


Anti-oxidant, Anti-bacterial and Anti-fungal properties of gelatin derived from earthworm, Eudriluseugeniae

A.Sujithra Devi1, Nor Azwady Abd. Aziz1, Muskhazli Mustafa1, Shuhaimi Mustafa2, Teng Suk Kuan3
1 Department of Biology, Faculty of Science, Universiti Putra Malaysia, Selangor, Malaysia
2 InstitutPenyelidikanProduk Halal, Universiti Putra Malaysia, Selangor, Malaysia
3 School of Foundation, Xiamen University, Malaysia

Earthworm body contains high amount of proteins with high content of fibrinolytic, anti-coagulative, anti-oxidant and anti-microbial peptides. The latest breakthrough in cosmeceutical industry might come from earthworms due to the beneficial earthworm bioactive peptides with no previous study on earthworm collagen and gelatin. With the great demand of Halal and kosher market, an alternative high value collagen and gelatin can be derived from earthworms and the study on its proximate characters and bioactivity poses as a significant precursor for earthworm collagen and gelatin to be used in cosmeceuticals. In this study, Eudriluseugeniaewas cultured in five different treatmentsand all treatments of 10 replicates each were monitored in laboratory condition for 8 weeks. Extraction of collagen and gelatin was done and the study on the characters of earthworm collagen and gelatin for probable use in cosmeceuticals enhances the possibility of earthworm as an alternative collagen and gelatin source.

The character of collagen and gelatin obtained from earthworm were compared with secondary data obtained from common commercial sources. A proximate analysis on collagen extracted from earthworms showed that Eudriluseugeniaecontains a significant amount of earthworm collagen and gelatin. The gelatin extracted was further characterized for the presence of bioactive peptide and its stability.

Bioactivity test for anti-oxidant, anti-fungal and anti-bacterial was further carried out on earthworm gelatin obtained to determine bioactivity properties of the gel. Results showed a positive activity of more than 50% suggesting the presence of anti-oxidant (DPPH inhibition), anti-bacterial and anti-fungal properties in earthworm gelatin. This study is the first of its kind in the field of earthworm collagen and gelatin and it has shown profound characters in earthworm collagen and gelatin. This study has shown promising results in promoting the use of earthworm, Eudriluseugeniaeas an alternative source in obtaining beneficial and profitable value added collagen and gelatin that can be potentially used in cosmeceutical industry.

keywords: Collagen, gelatin, anti-bacterial, anti-fungal, anti-oxidant


What are the Toxicokinetic and Toxicodynamic Mechanisms Underlying the Synergistic Interactions between Chemicals in Mixture Exposures?

K. Noort1,2, S. Short1, H. Selck2, D. Spurgeon1
UK Centre for Ecology & Hydrology, MacLean Building, Benson Lane Wallingford, OX10 8BB, UK
Roskilde University, Universitetsvej 1, 4000 Roskilde, Denmark

In realistic environmental conditions, a large amount of toxic chemical substances occur together, generating interactive effects that can be synergistic, additive, or antagonistic. Due to the large number of combinations of chemicals that can occur together, assessment of chemical mixtures relies on the use of predictive models. These models, however, do not sufficiently consider the effects of mixtures, and cannot predict their interactive effects. Among these effects, synergistic interactions are seen as of greater importance for risk assessment practices, as these can increase toxic effects of a mixture to far beyond what is predicted.

Synergistic interactions are the outcome of some sort of interaction between chemicals, whereby either one affects how the other is taken up to the target site (toxicokinetic) or what it does at the target site (toxicodynamic). Processes that can be affected by chemicals to lead to synergism are bioavailability, uptake, internal transport, metabolization, binding at target site and excretion.

My binomial mixture studies shows that synergism occurs in the annelid Enchytraeuscrypticus while exposed to Prochloraz (azole fungicide) and either Cypermethrin (pyrethroid) or Azoxystrobin (strobilurin fungicide) in water medium. In my poster presentation I will show my most recent results on the mechanistic attribution of this synergistic interaction, focussing on annelid metabolic activity and its enzymatic immune systems against pesticides using a novel methodology for ex vivo testing of enzymatic activity in terrestrial invertebrates. I will explain this methodology and share my findings on its application in earthworms.


Earthworms facilitate the biotransformation of roxarsone and alter its adverse effects on soil properties

Songge DENG, Yizhao WU, Jiangping QIU, Yinsheng LI
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China

Roxarsone (ROX) poses potential eco-risk as inorganic arsenic released in soils, and environmental microbes are the major drivers for its biotransformation.Earthworms may contribute to the arsenic (As) biotransformation using their gut microbiota, but the microbial interaction of arsenic biotransformation genes (ABGs) between soil and earthworm gut had not been clarified to date.

We established the microcosm study with environmental related concentration of ROX, earthworm Eisenia fetida and farmland soil to: (i) characterize the distribution of ABGs and bacterial communities in soils and guts; (ii) identify the contribution of gut microbial ABGs to the biodegradation of ROX; (iii) analyze the multiple impacts of earthworms to soil properties under ROX contamination.

Significant shifts of bacteria, ABGs and As compounds were shown between soils and guts. ROX increased the bacterial abundance such asProteobacteriaand the As biotransformation functions of oxidation and demethylation. Earthworms elevated bacterial α-diversity, increased the capacity of As reduction and methylation, and reduced the ratio of arsenate (As-V) to arsenite (As-III) in soil. Further, earthworms decreased the half-life of ROX from 73 d to 26 d, and maintained the risk assessment code (RAC) as ‘moderate risk’ through enhancing As accumulation and volatilization. Earthworms modified the water stability of soil aggregates by increase of its mean weight diameter (MWD), which was significantly correlated to the soil properties of pH, EC, organic matters (OM), total and nitrogen (TN, and ), available potassium and phosphorus ( and Olsen-P), enzymic activities of phosphatase (NP), (UE), (SC) and (CAT). ROX could comparatively reduce the Olsen-P, UE and CAT, and also affect to other soil properties indirectly through inhibiting earthworm burrows. However, its negative impacts could be offset by earthworms.

This study suggest that earthworms contributed to microbial ABG functions, efficiently facilitated the ROX biodegradation and altered its adverse effects, providing insights into the biomonitoring and bioremediation for organic arsenic pollutants in soil.

Acknowledgments: This research was supportedby Livelihood Issues Science and Technology Projects Shanghai Minhang (2021 MH-MS17), and Key Projects of Shanghai Municipal Agricultural Commission (2021 No.2-2).

keywords: Eisenia fetida, gut microbiota, arsenic biotransformation genes, arsenic speciation, soil aggregates


Elucidation of Immune Responses of  Earthworm , Eudrilus eugeniae

S. Umamaheswari
Department of Biotechnolog, ManonmaniamSundarnr Universit, Tirunelveli - 627 012, TamilNadu, India

Eudrilus eugeniae is a common type of earthworm used for vermicomposting. The earthworms are protostomian animals possessing the true coelom of mesenchymal origin. The coelom is the main body cavity in most animals and is positioned inside the body to surround and contain the digestive tract and other organs. The coelomic fluid contains different enzymes (proteases, amylases,phosphatases) and hormones (auxins, cytokinins) and essential nutrients (K, Ca, Mg, Cl, Cu, P and Na) for the plant growth and increases disease resistance. The component of the coelomic fluid of earthworms defends the mini dentifying foreign substances and develops potency to fight against them. The coelomic fluid has many biological properties (cytotoxic, proteolytic, antimicrobial, hemolytic, haemo-agglutinating, mitogenic). The purpose of this study was to elucidate immune protein from E. eugeniaecoelomic fluid. The coelomic fluid extracted was stimulated by broth, Bacillus subtilis and Pseudomona saeruginosa and assessed for the antibacterial, haemolytic and proteolyticactivities. P. Aeruginosa stimulated CFrendereda maximum antibacterial, haemolytic and proteolytic activity. An additional new extraprotein observed in the P. aeruginosa stimulated CFwaspartially purified and identified to be similar to a putative testis-expressed protein13C from Rhinopithecus roxellanaon subjecting to BLAST analysis. The protein obtained from P. fluorescens isolated after 48 hours on stimulation of E. eugeniae CF was partially purified based on their haemolytic and proteolytic activities. The presence of phenazine-1-carboxylic acid in the P.fluorescens isolated protein was identified by LC-MS possessing therapeutic efficacy.In this context, the research was focused on the efficacy of phenazine-1-carboxylic acid isolated from the recurrent bacteria, P. fluorescens isolated from the  stimulated coelomic fluid to be used with the potential of wound healing.

keywords: Earthworm, Immune Response, Protein, BLAST Analysis


Spent coffee grounds as suitable replacement for standard soil in ecotoxicological test guidelines

Mendes Luís André and Dominguéz Jorge
Universidade de Vigo, GEA (Grupo de Ecoloxía Animal), 36310 Vigo, Spain

Eisenia andrei has recently been considered a suitable replacement for Eisenia fetida in ecotoxicological assays in OECD and ISO guidelines. This indicates that alternative materials and methods could potentially be used in standard procedures intended for the evaluation of toxicity of compounds. It also promotes the use of less time-consuming procedures, as well as reducing the costs and other limitations on performing ecotoxicological assessment, such as non-existence of appropriate species and availability of materials. In recent years, spent coffee grounds have been used to produce vermicompost and presented as a suitable additive to organic fertilizers, such as biochar. In addition, the physico-chemical characteristics of spent coffee grounds indicate the material as a suitable substrate for earthworms, with the organisms performing as well as in natural soil.

In order to maximize the use of spent coffee grounds as a potential alternative to soil and to optimize the current standard guidelines, standardized tests were performed with E. andrei in fresh and aged spent coffee grounds spiked with carbendazim; the results were compared with those obtained using currently recommended artificial soil. Avoidance, survival and reproduction were assessed.

While avoidance and survival did not differ significantly, earthworm reproduction was higher in the substrate composed of clean aged coffee grounds than in artificial soil. The substrates spiked with similar concentrations of carbendazim did not produce significant differences in these parameters.

The findings clearly indicate that aged spent coffee grounds have potential to be used as a substrate in ecotoxicological tests, and their eventual addition to the current guidelines.

keywords: Standard guidelines, organic matter, earthworms, circular economy


Effect of earthworms, submitted to urban amendments in agricultural context,
on nanoplastic distribution

Caudron Tiana(1), Pérès Guénola(1), Gigault Julien(2, 3), Davranche Mélanie(2), Benard Yannick(1), Fautrel Alain(4), Perraud Manuel(1), Pattier Maxime(2), Wahl Aurélie(2), Wang Jingjing(5), Baalousha Mohammed(5)
1  UMR SAS INRAe Institut Agro Rennes-Angers, 65 rue de Saint Brieuc, 35042 Rennes cedex, France
2  University of Rennes, CNRS, UMR GEOSCIENCES, Campus Beaulieu, 35042 Rennes cedex, France
3  International Research Laboratory, Laval University, Quebec, G1V 0A6, Canada
4 Plateforme H2P2, UAR Biosit, INSERM University Rennes 1, France
5 Center for Environmental Nanosciences and Risk, University of South Carolina, Columbia, South Carolina, United States

With the highlighting of the “great vortex” of plastic waste in two of the main oceans, the North Atlantic and the Pacific, plastic debris is gaining more and more attention from the scientific community. While the vast majority of scientific efforts have focused on these large oceanic gyres, until now continental systems (soil, water) have been studied too little regarding micro and nanoplastics pollution, and the consequences of this pollution on the functioning of the terrestrial ecosystem are still too little known.In the agricultural context, several sources of plastics are identified such as plastic mulch, protective tarpaulins, tunnels, wrappings, as well as amendments, leading to significant pollution. As part of the amendments, particular attention is given to amendments of urban origin. It is becoming urgent to improve knowledge on the effect of these plastic-rich amendments on ecosystem functioning, and a special interest is given on the effect of plastic on soil biodiversity especially earthworms community as earthworms are considering as key actors in soil functioning and soil heath.

In this context one aim of CINAPE project (ADEME funding) was to assess the impact of urban amendments rich in plastics (micro and nanoplastics) on the earthworm community. Two approaches were developed: i) a laboratory experiment where the biological responses of three earthworm species (L. terrestris, A c. chlorotica, L. centralis) were assessed; ii) in situ, two field campaigns were carried on in 2020 and 2022, comparing at each time two fallows: one which received urban amendments 15 years ago, and a non polluted fallow ; earthworms were sampled using hand sorting method completed with mustard. The laboratory experiment showed the sensitivity of L. centralis to urban amendment. Results from the first field campaign showed in tendency, but not significantly, a negative response of plastic amendment on earthworm community in terms of abundance and biomass, epigeic species being more sensitive than other ecological categories (anecic or endogeic). These results will be compared to the new field campaign.

keywords: Micro nanoplastic pollution; ecotoxicology; sub-lethal response; controlled conditions; in situ experiment


Effect of earthworms, submitted to urban amendments in agricultural context, on nanoplastic distribution

Pérès Guénola1, Julien Gigault2,3, Davranche Mélanie2, Benard Yannick1, Perraud Manuel1, Wahl Aurélie2, Wang Jingjing4, Baalousha Mohammed4
1  UMR SAS INRAe Institut Agro Rennes-Angers, 65 rue de Saint Brieuc, 35042 Rennes cedex, France
2  University of Rennes, CNRS, UMR GEOSCIENCES, Campus Beaulieu, 35042 Rennes cedex, France
3  International Research Laboratory, Laval University, Quebec, G1V 0A6, Canada
4 Center for Environmental Nanoscience and Risk, Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina, United States

Plastic pollution is gaining more and more attention from the scientific community. While the vast majority of scientific efforts have focused on oceanic gyres, less attention has been given to continental systems (e.g., soil, water). Additionally, most studies focus on microplastics; that is plastic debris greater than several micrometers or even several hundred micrometers. However, few studies focus on the size continuum of nanoplastics to microplastics. In the agricultural context, several sources of plastics are identified leading to significant pollution. As part of the agricultural amendments, particular attention is given to amendments of urban origin. It is becoming urgent to improve knowledge on the effect of these plastic-rich amendments on soil functioning, and in retrospect to improve our knowledge of the effect of soil organisms on plastic evolution and distribution. Regarding soil organisms, earthworms are recognised as key actors in soil functioning due to their feeding activity and foraging activity, but their effect on nanoplastic distribution is still unknown.

In this context, the aims of CINAPE project “Characterisation and Identification of the Impact of NAnoplastic on Environment” (funding ADEME – French Agency for Ecological Transition) were to assess i) the impact of urban amendments rich in plastics (micro and nanoplastics) on the earthworm foraging activity (cast production) and ii) the effect of earthworms on nanoplastic distribution in the casts and in the earthworm’s body.

A microcosm approach was carried during two months under controlled conditions to compare the effect of plastic-rich amendments on the foraging activity of three earthworms species (L. terrestris, A c. chlorotica, L. centralis). Afterwards, nanoplastic evaluation in the casts and earthworm’s body was done; heterogeneity along earthworm body was assessed by considering three parts of the body: i) before the male pore, ii) the clitellum part, and iii) after the clitellum part. 

The evaluation of nanoplastic in the different compartments (cast, earthworm’s body) was possible due to the development of innovative analytical methods.

keywords: Nanoplastic distribution; nanoplastic accumulation; casts; earthworm body


Factors affecting bioaccumulation of heavy metals in the earthworm genus Aporrectodea Örley, 1881 in croplands

Robabeh Latif1, Atabak Roohi Aminjan2, Maria J. I. Briones3
Farzanegan Campus, SemnanUniversity, Semnan, Iran
Department of Biology, Faculty of Science, Bu-Ali Sina University, Hamedan, Iran
Departamento de Ecología y Biología Animal, Universidad de Vigo, Spain

Heavy metal bioaccumulation in earthworms can be used as an ecological indicator forheavy metal contamination in soils. In this study, the levels of Pb and Cd were analyzed in the body tissues of two endogeicearthworm species:Aporrectodeacaliginosaand A. roseacollected from three agricultural soils (turnip cabbage, beet, and fallow).At each system, soil samples for earthworm hand-sorting (quadrats of 50´50×25 cm) and analyses of organic matter and metal contents together with  those of edible parts of agricultural plants were randomly collected at each field. The amounts of heavy metals were measured using flame atomic absorption spectrophotometry, organic matter content by loss on ignition (at 550 ºC for 4h) and soil Ca concentrationsby Systronics Flame Photometer.The results showed that A. caliginosa was the dominant species in the turnip cabbage cropland, whereas A. rosea was dominant in the beet farmland; however, in the fallow system, both species had similar densities. The concentrations of Cd and Pb in earthworms were higher in the croplands than in the fallow. The concentrations of both Cd and Pb were significantly higher in the A. caliginosaspecimens collected fromtheturnip cabbage and fallow lands, than in the soil samples from the same sites. By contrast, no significant differences were observed in the metal accumulations of the earthworm speciescollected from the fallow, although higher levels of Pb and Cd were measured in A. rosea and A. caliginosa, respectively. In addition, we found that the Pb concentration in earthworms was negatively correlated to calcium concentration in the soils, whereas the significant increase inorganic matter contents observed inthe soils withturnip cabbage (compared to the fallow) led to a higher accumulation of Cd in A. rosea; the opposite trend was observed for Pb accumulation in A. caliginosa. These results suggest that crop identity, calcium concentrations and organic matter contentsplay a key role in the uptake of heavy metals by earthworms.

keywords: Aporrectodea caliginosa; A. rosea; Pb; Cd; Calcium; Organic matter


E. crypticus as a model organism for high-throughput agrochemical toxicity testing

Alex Robinson1, Sylvan Bart2, Stephen Short1, Elma Lahive1, Emily Eagles1, Pete Kille3, Dave Spurgeon1
1  UK Centre for Ecology and Hydrology, Wallingford OX10 8BB
2  MO-ECO2 (Modelling and data analyses for ecology and ecotoxicology), Paris, France
3  University of Cardiff, Cardiff, CF10 3AT

With the large number of novel chemicals being synthesised, reliable methods are required for assessing invertebrate sensitivity to toxins, in order to identify chemicals or combinations of interest. The ideal toxicity testing protocol would have the following characteristics: Precise, Repeatable, High throughput, Test species of ecological importance, Environmentally relevant.

Enchytraeids make ideal model organisms for soil invertebrate toxicity testing. Cultures are reliable to maintain, individuals are convenient to handle, and genetic testing has found them to be near clonal, minimising variability.Enchytraeids and annelids in general are important ecologically playing a key role in soil functioning.  Soil testing following the OECD protocol, is highly consistent, however the test takes 14 days, and only allows for toxicity at a single time point to be determined.

It is impossible to optimise all the aspects of environmental toxicity testing within a single protocol. Instead, by compromising on environmental relevance, highly precise repeatable data can be rapidly generated to compare relative toxicities of chemicals, or identify compounds of interest worthy of further investigations.

We used the aquatic enchytyaeid test proposed by Roembke & Knacker (1989) to test the susceptibility of E. crypticus to 16 commonly used agrochemicals, covering insecticides, fungicides and herbicides, comparing effect concentrations at different time points.

The tests were highly repeatable and sensitive enough to discern slopes on most dose – response relationships with fungicides and cypermethrin showing the greatest toxicity. Chlorpyrifos and neonicotinoids were found to have very low toxicity, as did all herbicides tested apart from glyphosate.

Having identified compounds of interest, responses were compared with those for 2 species of arthropod, with the order of toxicity for the compounds being found to be very different. Transcriptomics and biochemical assays were then used to understand potential reasons for differential sensitivity, allowing future predictions to be made.

keywords: Enchytraeids; Ecotoxicology; Agrochemicals; Bioassays; Sensitivity


Endogeic earthworms facing pesticide residues in agricutural landscapes: bioaccumulation and population risk assessment

Audrey Barranger1, Romain Georges1, Olivier Delhomme2, Maurice Millet2, Florent Lafay3, Emmanuelle Vulliet3, Gaëlle Daniele3and Françoise Binet1
UMR CNRS ECOBIO 6553, University of Rennes 1, Campus de Beaulieu, 263 Avenue du Général Leclerc, 35042 Rennes Cedex, France
UMR CNRS ICPEES 7515, University of Strasbourg, 25 rue Becquerel, 67087 Strasbourg Cedex 2, France
UMR CNRS ISA 5280, University of Lyon 1 Campus Claude Bernard, 5 rue de la Doua, 69100 Villeurbanne, France

With the advent of modern agriculture, cultivated fields have become deeply man-modified ecosystems in which high yields are achieved through conventional management based on intensive use of chemicals (pesticides, fertilisers) and frequent soil tillage.  Recent studies have alerted on the impact of modern agriculture on soil biodiversity and its functions, particularly through the use of pesticides. Although agricultural soils are the main entry of pesticides, large-scale monitoring studies (i.e., number of sites and compounds under study above 10) on soil contamination are still very rare, especially on currently used pesticides. There is consequently a lack of knowledge on the dynamics and fate of the contamination in agricultural areas. Fauna in terrestrial ecosystems is directly exposed topesticides when spreading products, or indirectly via aerial drift or secondary poisoning by feeding on contaminated plants or prey. Assessing the transfer of pesticides from soil to fauna inhabiting it is one direct way to provide accurate information onbioaccumulation and to determine the potential effect of chemicals on ecosystem health.In this study, adopting a synchronic approach, 27french agricultural fields were sampled (9 permanent meadows, 9 conventional fields and 9 organic fields), out of which 6 cropped fields were three timemonitoredover two years through a diachronic approach. Soil and earthworms were concomitantly sampled and analysed for pesticide residues. Multi-class pesticides (73 selected pesticides) and glyphosate and its metabolite AMPA have been determined by LC-MS/MS.Results in endogeic earthwormsshowed that45 out of the 75 targeted pesticides were detected at least once and up to 16 molecules have been detected in one composite sample (3 pooled earthworms). Among them, the most often detected pesticides are the insecticide imidacloprid and the herbicide metolachlor ESA, detected respectively in 85 and 60% of the biological samples. We showed that earthworms faced several chemicals at high dose in their natural environment.Next step will be to calculate pesticide transfer from soil to earthworm tissues. Ultimately, management practices will be linked to life history traits of endogeic community such as individual weight or life stage to highlight potential toxicity of soil pesticide contamination.

keywords: Chemical contamination ; Agrosystems ;  Soil engineer ; Field management ; Ecosystem health


Optimisation of quantum dot biosynth esis in the earthworm D. Veneta

Norah Alkhayyal1, Mark Green2, Klaus Suhling2, Carl Hobbs3 and Stephen Sturzenbaum1
1  Department of Analytical, Environmental and Forensic Science, King’s College London
2  PhysicsDepartment, King’s College London
3  Histology and Imaging Department, King’s College London

Semi-conductor quantum dots (QDs) are utilised in various fields including biological applications such as bioimaging since they are more photostable and larger than organic dyes. QDs can be chemically synthesized in the lab using heavy metal precursors, however, this requires specialist glassware, solvents, inert gases, and heating. The earthworm, however, is capable of biosynthesising quantum dots via their intrinsic metal detoxification pathway which involves the transportation of metals by metallothioneins (MTs) and glutathione reductase to the chloragogenous tissue surrounding the gut1. The research presented now focuses on the method development to increase the yield of highly luminescent QDs. This was achieved by exposing the earthworms (Dendrobaenaveneta) to a mixture of cadmium and tellurium precursors at concentrations 50, 100 and 200 mg/kg and also to mercury and tellurium precursors at 200 mg/kg of soil for four weeks. The concentration of metals in the soil and worms was determined via an inductively coupled plasma mass spectrometer (ICP-MS) and the MTs protein and metals were localized on cross sections of the worm via imaging mass cytometry (IMC). The generated nanoparticles were collected using two different methods, extraction from chloragogenous tissue by dissecting the worm and brushing off the cells and a less invasive method by collecting coelomic fluid excreted through dorsal pores via electro-stimulation. The metallothionein gene expression was analyzed in the chloragogenous and coelomic cells using quantitative polymerase chain reaction (qPCR). The absorbance and photoluminescence were measured by absorbance and fluorescence spectrometry to estimate the size of the collected QDs. Their colour was confirmed after exciting the samples to ultraviolet lamp under short wavelength 365 nm. Changes in fluorescence lifetime of the QDs were determined by fluorescence lifetime imaging microscopy (FLIM). By optimizing the fluorescence lifetime and quantum yield we aim to enhance the quality and biocompatibility of QDs in imaging science.

1. Sturzenbaum,S.R.,Hockner, M.,  Panneerselvam, A., Levitt, J., Bouillard, J-S.,  Taniguchi, S.,   Dailey, L-A .,Khanbeig, A.R.,Rosca, E. V.,  Thanou, M.,  Suhling, K., Zayats, A. V. and  Green, M. (2013).Biosynthesis of luminescent quantum dots in an earthworm. Nature Nanotechnology (8) 58-60.

keywords: D. veneta, Cadmium, tellurium, quantum dots and metallothionein


ioaccessibility in predators

Shizhong Yue1, Li Jia 2, Ruiping Wang1
Shandong Key Laboratory of Biophysics, Institute of Biophysics, Dezhou University, Dezhou, 253023, PR China
2  ISTO UMR7327, CNRS-Université d'Orléans-Brgm, Orléans, 45071, France

Introduction : The increase of soil selenium (Se) level caused by natural and anthropogenic reasons is a potential ecological threat. Earthworms canuptake and conversion inorganic Se into organic components [1], which are easily synthesized into macromolecular organic substances. Resulting in poor excretion and Seaccumulation in worms. This earthworm-source Se might generate a potential ecological risk to its predators. This research aims to evaluate thebioaccumulation and excretion capacity of Se(IV) inEisenia fetida and investigate the bioaccessibility of earthworm-derived Se in poultry/birds.

Methods : Adult E. fetida(650-800 mg)were exposed to selenite spiked soil (0/2.5/5/10/20 mg Se/kg, respectively) for 14 d(uptake phase) followed by another 14 d (elimination phase) to the original clean soil. The bioaccessibility of earthworm-source Se at 14 and 28 days were evaluated by an in vitro gastrointestinal digestion model [2].Samples weredigested by HNO3-H2O2 and total Se concentrations were determined. The bioaccessibility of earthworm Se was calculated by the bioaccessibleSe (Se amount in digestive juice) divide by the total Se.

Results mg/kg and11.42, 15.56, 24.83, 40.50, and 57.68 mg Se/kg DW at14 and 28 d, respectively. Results indicated that Seresidualsin earthworms reached 66.41 -79.66%.Additionally, earthworm-derived Se was highly bioaccessible to poultry/birds (66.42-84.74%)atthe uptake  and elimination phases. Interestingly, Se bioaccessibility in 14 d in 5, 10, and 20 mg/kg groups were higher than in 28 d, demonstrating that the bioaccessible Se increased after the excretion stage.

reservoir. Earthworm-derived Se has a high bioaccessibility to poultry/birds and existing great potential ecological risk to their predators, especially those at higher trophic levels.


[1] Yue S.Z., et al., 2019. Selenium accumulation, speciation and bioaccessibility in selenium-enriched earthworm (Eisenia fetida). Microchem J.145:1-8

[2] Greiner, R., 2021. Limitations of an in vitro model of the poultry digestive tract on the evaluation of the catalytic performance of phytases. J Sci Food Agric.101(6), 2519-2524.

keywords: earthworm; selenium;excrete; bioaccessibility; ecological risk


Understanding the oxidative stress response of Eisenia fetida after exposure to copper in soil amended with industrial byproducts

N. Alsarawi; J. Whalen; G. Sunahara
Department of Natural Resource Sciences, Macdonald Campus of McGill University

Copper is well known for its toxicity to earthworms based on dose-response at the organismal level (i.e., survival, growth, reproduction) and metabolic activities such as respiration. Since copper toxicity is associated with an excess of reactive oxygen species in the affected organisms, the oxidative stress response should be a sensitive indicator of toxicity. The purpose of this work is to understand the oxidative stress of Eisenia fetida after exposure to copperin soil amended with industrial byproducts. It is hypothesized that with increasing copper concentrations, the earthworms will undergo oxidative stress resulting from the imbalance between reactive oxygen species (ROS) and antioxidants enzymes, superoxide dismutase (SOD) and catalyst (CAT), because of the accumulation of copper in the metal-binding protein metallothionein. The experimental procedure involves performing survival, growth, and reproduction experiments conducted according to OECD guidelines, and by quantifying the degree of DNA damage, measuring ROS levels, SOD and CAT activities, and by conducting LC/MS analysis on metabolite biomarkers in earthworms’ coelomic fluid. The bioassays will be conducted on earthworms exposed to copper in soil amended with papermill biosolids, sludge incinerated ash, and municipal dried sludge. We expect that the oxidative stress response will be a robust indicator of contamination, and that this approach will account for the presence of multiple contaminants, which is a limitation in traditional dose-exposure-response studies.

keywords: OECD bioassays; oxidative stress; DNA damage; metabolite biomarkers


Effects of  pesticide mixtures toward Lumbricus Rubellus and Enchytraeus crypticus.

Sylvain Bart1,2, Kevin Noort1, Alex Robinson1, Emily Eagles1, Stephen Short1, Elma Lahive1, David J Spurgeon1
1  UK Centre for Ecology and Hydrology, Wallingford OX10 8BB
2  MO-ECO2 (Modelling and data analyses for ecology and ecotoxicology), Paris, France

Due to agricultural practices, soil organisms are exposed to pesticide mixtures in soil. The complexity of the ecotoxicological assessment of mixtures toward non-target soil organisms is overlaid by the fact that interactions may occur, leading to more of less effects than expected. We here aimed at testing the effects of different pesticide mixtureswhichwere found to be synergistic (cypermethrin and prochloraz, azoxystrobin and prochloraz), on various aquatic species, to assess if similar interactions could occurinsoil species: earthworms and enchytraeids. We exposed Lumbricusrubellus in natural soils for 28 days to pesticides separately; cypermethrin (50-1500 mg/kg), azoxystrobin (31.25-250 mg/kg), and prochloraz (5-625 mg/kg), and to binary mixtures. For all treatments (singles and mixtures), we monitored body mass and survival over time (at day 7, 14, 21 and 28). We also exposed Enchytraeus crypticusin LUFA 2.2 soils for 14 days to the same pesticides(single and mixture exposures), and monitored the survival over time (at day 3, 7 and 14). For both species, the survival data were analyzed with the General Unified Threshold model for Survival (GUTS); toxicokinetic-toxicodynamic models (TKTD models) which simulate the time-course of processes leading to toxic effects on organisms. We used the single exposures data to calibrate the models, then, we predicted the mixture effects and compared them to the observed mixture effects.We found lethal and sublethal effects, but no clear synergism could be identified in the tested mixtures for these two soil organisms.

keywords: Mixture hazard; azole fungicide; pyrethroid; effect modelling


Earthworm risk assessment for plant protection products registration: Identification of suitable options to address the future tiered approach and need for further research

Blondel C., Brulle F., Perrat E., Ravat J., Farama E.,Chaton P.- F., Mazerolles V.
ANSES French Agency for Food, Environmental and Occupational Health & Safety, 14 Rue Pierre et Marie Curie 94700 Maisons-Alfort, France

In the frame of the European regulation (EC) No 1107/2009, an ecotoxicological regulatory risk assessment for Plant Protection Products (PPP) is mandatory. It typically follows a tiered approach, based on a large range of organism tests, following the latest guidelines. Regarding the soil organisms when exposure cannot be excluded, risk assessment covers from worst-case situations (i.e.based on conservative estimates and toxicity laboratory studies) to more realistic assessment (i.e. based on field studies). Currently, for earthworms the first-tier risk assessment is based upon chronic toxicity data from laboratory studies (i.e. Eisenia fetida, using artificial OECD substrate). These laboratory studies are performed in controlled conditions following recommendation of dedicated guidelines (e.g.: OECD, ISO, academic standards). Toxicity values are then compared to the predicted environmental concentration in soil. In case of unacceptable risk, according to the Tiered Approach ((EC) No 1107/2009), field studies should be performed to assess the potential effects on earthworm communities in more realistic conditions. However, to date no other refinement options are formally available. The guidance on the risk assessment of PPP for soil organisms is going to be revised in the upcoming years. A recent EFSA scientific opinion (2017) proposed a new framework for risk assessment of soil organisms and arevised protection goals defined for soil organism groups. This opinion also identified intermediate tier approaches as a focus for future research that need development, standardized test designs and further guidance.  More precisely, two intermediate tiered approaches (option A or B) were identified.The intermediate tier A corresponds to laboratory experiments performed to address the sensitivity of species belonging to one group of organisms whereas intermediate tier B deals with natural communities assembled in microcosm-like experiments.

The aim of the poster will be manifold to (i) identify existing study designs and risk assessment tools that could be used for the intermediate tiers; (ii) identify technical limitations and gaps and (iii) propose recommendations for further research and development to better address the earthworms risk assessment in the upcoming years.

keywords: Earthworm risk assessment; plant protection product; intermediate-tier options; regulatory risk assessment


Microplastics in Lumbricus terrestris middens/casts and surrounding urban soil

Bruno Ćaleta, Davorka Hackenberger Kutuzović, Branimir Hackenberger Kutuzović
Department of Biology, Josip Juraj Strossmayer University of Osijek, Cara Hadrijana 8a, 31000 Osijek

Continuous accumulation and low degradation rates of plastic waste in the environment indicate the cumulative effect that microplastics particles could have on the environment. The impact of microplastics pollution on terrestrial ecosystems, relative to aquatic ecosystems, has been poorly investigated. Also, the impact of soil microplastics on soil-dwelling organisms is not well understood. Therefore, the main objective of this study was to investigate whether Lumbricus terrestris concentrates the microplastics with its behaviour. To investigate the interaction of microplastics and L. terrestris, topsoil samples and earthworm casts/middens samples were collected at different locations that included agricultural land and green urban areas. Microplastics from soil and cast samples were extracted by density separation using aqueous zinc (II) chloride solution (5M). Isolated particles were counted, and their surface areas and diameters were analyzed and quantified using ImageJ 1.5. Mean microplastics count in soil was 977.76 ± 726.39 particles kgsoil-1 and in earthworm casts 655.89 ± 265.11 particles kgcast.-1. Mean surface area of microplastics isolated from soil samples was 0.482 ± 0.765 mm2 and from earthworm casts 0.529 ± 0.660 mm2. On average, diameter of microplastic particles was 1.102 ± 0.703 mm in particles isolated from soil and 1.205 ± 0.705 mm in particles from earthworm casts. Previous studies have shown that earthworms can accelerate biodegradation of microplastics in the soil by ingestion. Therefore, it was expected that the count of microplastics in earthworm casts would exceed the count of microplastics in soil samples. Also, it was expected that dimensions of microplastics would be smaller in size in earthworm casts compared to microplastics in soil. However, this research has shown the opposite, which could be explained by the preferential retention of smaller microplastics within the earthworm organism.

keywords: soil microplastics; earthworm cast; biodegradation; Lumbricus terrestris


Irrigation with treated wastewater can impact soil quality:
a sentinel invertebrate approach

Chubin Zhang1, Leon Barron2, Stephen Sturzenbaum1
1  Analytical, Environmental& Forensic Sciences Department, School of Cancer and Pharmaceutical Sciences, Faculty of Life Sciences & Medicine, King's College London, UK
2  Environmental Research Group, School of Public Health, Faculty of Medicine, Imperial College London, UK

The impact of pharmaceutically active compounds (PhACs), especially psychiatric drugs, entering the terrestrial environment via wastewater irrigation and biosolid application are yet to be understood.This is due to the lack of understanding of transport kinetics in the soil and the availability of reliable quantitative trace analytical methods. Earthworms, one of the essential indicator species of soil health, accumulate biosolid fertilisers and wastewater contaminants that exert negative impacts on soil quality. One key aim of this project was to focus on antidepressant drugs (carbamazepine, citalopram and other tricyclic antidepressants) found regularly in soil and explore their impact on earthworm ecotypesin terms of uptake dynamics and molecular and life cycle endpoints. This was achieved by constructing a simplified soil column system irrigated with 1µg/L for 3 months to quantify the time-dependent transportation of the drugs and determine their fate in Dendrobaenaveneta, Lumbricusrubellus and Lumbricusterrestris. Trace CECs in fortified soil and worms were extracted by pressurised liquid extraction (PLE) and analysedby liquid chromatography-tandem mass spectrometry (LC-MS/MS). Theglobal transcriptional responses were assessed by RNAsequencing(RNASeq) and confirmed by qPCR. A notable accumulation of carbamazepine was observed in the posterior tissues, which was also transported from the gut to other anterior organs (e.g., cerebral ganglion). RNASeq uncovered specific transcripts, most notably the sterol regulatory-element binding proteins (SREBPs) were markedly altered by drug exposure. The absence of a CYP3A4 response (a homolog of the human drug metabolism pathway) indicated the presence of a potential unique metabolic process in earthworms. Overall, this project was able to dissect differences in the transport kinetics of wastewater-derived pharmaceuticals within the soil matrix and uncover resultant molecular-genetic and lifecycle changes within exposed earthworms.

keywords: Earthworms; Antidepressants; RNASeq; Soil column