Soil properties influence the toxicity and availability of Zn from ZnO nanoparticles to earthworms.

To develop models that support site-specific risk assessment for nanoparticles (NPs), a better understanding of how NP transformation processes, bioavailability and toxicity are influenced by soil properties is needed. In this study, the influence of differing soil properties on the bioavailability and toxicity of zinc oxide (ZnO) NPs and ionic Zn to the earthworm Eisenia fetida was investigated. Earthworms were exposed to ZnO_NPs and ionic Zn, between 100 and 4400 mg Zn/kg, in four different natural soils (organic matter content: 1.8-16.7%, soil pH: 5.4-8.3, representing sandy loam to calcareous soils). Survival and reproduction were assessed after 28 and 56 days, respectively. Zn concentrations in soil pore waters were measured while labile concentrations of Zn were measured using an in-situ dynamic speciation technique (diffusive gradient in thin films, DGT). Earthworm Zn tissue concentrations were also measured. Soil properties influenced earthworm reproduction between soil controls, with highest reproductive output in soils with pH values of 6-7. Toxicity was also influenced by soil properties, with EC50s based on total Zn in soil ranging from 694 to >2200 mg Zn/kg for ZnO_NP and 277-734 mg Zn/kg for ionic Zn. Soil pore water and DGT measurements showed good agreement in the relative amount of Zn extracted across the four soils. Earthworms exposed to ZnO_NPs survived higher Zn concentrations in the soils and had higher tissue concentrations compared with ionic Zn exposures, particularly in the high organic content calcareous soil. These higher tissue concentrations in ZnO_NP exposed earthworm could have consequences for the persistence and trophic mobility of Zn in terrestrial systems and need to be further investigated to elucidate if there any longer-term risks associated with sustained input of ZnO_NP to soil.

A Kinetic Approach for Assessing the Uptake of Ag from Pristine and Sulfidized Ag Nanomaterials to Plants.

Nanomaterials (NMs) are thermodynamically unstable by nature, and exposure of soil organisms to NMs in the terrestrial environment cannot be assumed constant. Thus, steady-state conditions may not apply to NMs, and bioaccumulation modeling for uptake should follow a dynamic approach. The one-compartment model allows the uptake and elimination of a chemical to be determined, while also permitting changes in exposure and growth to be taken into account. The aim of the present study was to investigate the accumulation of Ag from different Ag NM types (20 nm Ag0 NMs, 50 nm Ag0 NMs, and 25 nm Ag2 S NMs) in the crop plant wheat (Triticum aestivum). Seeds were emerged in contaminated soils (3 or 10 mg Ag/kg dry soil, nominal) and plants grown for up to 42 d postemergence. Plant roots and shoots were collected after 1, 7, 14, 21, and 42 d postemergence; and total Ag was measured. Soil porewater Ag concentrations were also measured at each sampling time. Using the plant growth rates in the different treatments and the changing porewater concentrations as parameters, the one-compartment model was used to estimate the uptake and elimination of Ag from the plant tissues. The best fit of the model to the data included growth rate and porewater concentration decline, while showing elimination of Ag to be close to zero. Uptake was highest for Ag0 NMs, and size did not influence their uptake rates. Accumulation of Ag from Ag2 S NMs was lower, as reflected by the lower porewater concentrations. Environ Toxicol Chem 2021;40:1861-1872. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Evidence-based logic chains demonstrate multiple impacts of trace metals on ecosystem services.

Trace metals can have far-reaching ecosystem impacts. In this study, we develop consistent and evidence-based logic chains to demonstrate the wider effects of trace metal contamination on a suite of ecosystem services. They demonstrate knock-on effects from an initial receptor that is sensitive to metal toxicity, along a cascade of impact, to final ecosystem services via alterations to multiple ecosystem processes. We developed logic chains to highlight two aspects of metal toxicity: for impacts of copper pollution in soil ecosystems, and for impacts of mercury in freshwaters. Each link of the chains is supported by published evidence, with an indication of the strength of the supporting science. Copper pollution to soils (134 unique chains) showed a complex network of pathways originating from direct effects on a range of invertebrate and microbial taxa and plants. In contrast, mercury pollution on freshwaters (63 unique chains) shows pathways that broadly follow the food web of this habitat, reflecting the potential for mercury bioaccumulation. Despite different pathways, there is considerable overlap in the final ecosystem services impacted by both of these metals and in both ecosystems. These included reduced human-use impacts (food, fishing), reduced human non-use impacts (amenity value) and positive or negative alterations to climate regulation (impacts on carbon sequestration). Other final ecosystem goods impacted include reduced crop production, animal production, flood regulation, drinking water quality and soil purification. Taking an ecosystem services approach demonstrates that consideration of only the direct effects of metal contamination of soils and water will considerably underestimate the total impacts of these pollutants. Construction of logic chains, evidenced by published literature, allows a robust assessment of potential impacts indicating primary, secondary and tertiary effects.

Extending standard testing period in honeybees to predict lifespan impacts of pesticides and heavy metals using dynamic energy budget modelling.

Concern over reported honeybee (Apis mellifera spp.) losses has highlighted chemical exposure as a risk. Current laboratory oral toxicity tests in A. mellifera spp. use short-term, maximum 96 hour, exposures which may not necessarily account for chronic and cumulative toxicity. Here, we use extended 240 hour (10 day) exposures to examine seven agrochemicals and trace environmental pollutant toxicities for adult honeybees. Data were used to parameterise a dynamic energy budget model (DEBtox) to further examine potential survival effects up to 30 day and 90 day summer and winter worker lifespans. Honeybees were most sensitive to insecticides (clothianidin > dimethoate ≫ tau-fluvalinate), then trace metals/metalloids (cadmium, arsenic), followed by the fungicide propiconazole and herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). LC50s calculated from DEBtox parameters indicated a 27 fold change comparing exposure from 48 to 720 hours (summer worker lifespan) for cadmium, as the most time-dependent chemical as driven by slow toxicokinetics. Clothianidin and dimethoate exhibited more rapid toxicokinetics with 48 to 720 hour LC50s changes of <4 fold. As effects from long-term exposure may exceed those measured in short-term tests, future regulatory tests should extend to 96 hours as standard, with extension to 240 hour exposures further improving realism.

Life-history effects of arsenic toxicity in clades of the earthworm Lumbricus rubellus.

Exposures of Lumbricus rubellus to a series of arsenic concentrations in soil were used to assess life-stage (juvenile, adult) and genotype specific sensitivities, to calculate population growth rate (λ) and to assess patterns of As accumulation. Significant mortality was seen in juveniles at 125 mg/kg As, while growth and maturation was affected from 36 mg/kg and above. In adults, cocoon production at the highest concentration (125 mg/kg) was significantly reduced. Phylogenetic analysis was performed by comparison of mitochondrial sequences to establish genotypic variation among juveniles. Three clades with more than 7.5% divergent were described, with 70% of earthworms belonging to a single clade. Date of and mass at maturation was significantly different between clades, but clades were not differentially As sensitive. Parameter λ was reduced at 36 mg/kg As and was negative at 125 mg/kg As, suggesting impacts and population stability and potential extinction at environmentally relevant concentrations.

A metabolomics based test of independent action and concentration addition using the earthworm Lumbricus rubellus.

A major challenge in ecotoxicology is to understand the effects of multiple toxicants on organisms. Here we assess the effects on survival, weight change, cocoon production and metabolism caused by exposure to two similarly acting (imidacloprid/thiacloprid) and two dissimilarly acting (chlorpyrifos/Nickel) chemicals on the earthworm Lumbricus rubellus. We assessed the standard models of concentration addition (CA) and independent action (IA), in conjunction with a metabolomics based approach to elucidate mechanisms of effect. For imidacloprid and thiacloprid the reproductive effects indicated probable additivity. Although this suggests joint effects through a similar mechanism, metabolite changes for each pesticide actually indicated distinct effects. Further, earthworms exposed to a 0.5 toxic unit equitoxic mixture demonstrated metabolic effects intermediate between those for each pesticide, indicating a non-interactive, independent joint effect. For higher effect level mixtures (1 and 1.5 toxic units), metabolite changes associated with thiacloprid exposure began to dominate. The metabolomic effects of the two dissimilarly acting chemicals were distinct, confirming separate modes of action and both proved more toxic than anticipated from previous studies. In the mixtures, phenotypic effects were in accordance with IA estimates, while metabolite changes were dominated by Ni effects, even though chlorpyrifos contributed most to reproductive toxicity. This could be attributed to the greater systematic effect of Ni when compared to the more specifically acting chlorpyrifos.

Modelling the joint effects of a metal and a pesticide on reproduction and toxicokinetics in Lumbricid earthworms.

It is important to understand the aetiology of interactive mixtures effects (i.e. synergism and antagonism) if results from known cases are to be extrapolated to untested combinations. The key role of toxicokinetics in determining internal concentrations at target sites means that understanding chemical uptake in mixtures is an essential requirement for mechanistic understanding of interactions. In this paper, a combined approach using mixture toxicity testing, toxicokinetic studies and modelling has been used to address the link between joint toxicity and internal concentration. The study is conducted in Lumbricid earthworms with a binary mixture of a metal (nickel) and an organophosphate insecticide (chlorpyrifos) not a priori expected to show interactive toxicity. As expected from their dissimilar modes of action and detoxification, exposure to combinations of nickel and chlorpyrifos resulted in additive toxicity. Measurement of internal concentrations indicated that both chemicals were rapidly accumulated (within 3 days) to equilibrium. When exposed as a mixture, Ni uptake followed the same pattern as found for the single chemical. This was not the case for chlorpyrifos which showed a faster rate of uptake and elimination and a slightly higher equilibrium concentration in a mixture. That the difference in chlorpyrifos kinetics in the mixture did not result in interactive toxicity highlights the need to assess chemical toxicodynamics as well as toxicokinetics. Measurement of chlorpyrifos-oxon identified the presence of this toxic form but implementation of more complex approaches encompassing toxicogenomics and epigenetics are ultimately needed to resolve the toxicokinetic to toxicodynamic link for these chemicals.

Comparative transcriptomic responses to chronic cadmium, fluoranthene, and atrazine exposure in Lumbricus rubellus.

Transcriptional responses of a soil-dwelling organism (the earthworm Lumbricus rubellus) to three chemicals, cadmium (Cd), fluoranthene (FA), and atrazine (AZ), were measured following chronic exposure, with the aim of identifying the nature of any shared transcriptional response. Principal component analysis indicated full or partial separation of control and exposed samples for each compound but not for the composite set of all control and exposed samples. Partial least-squares discriminant analysis allowed separation of the control and exposed samples for each chemical and also for the composite data set, suggesting a common transcriptional response to exposure. Genes identified as changing in expression level (by the least stringent test for significance) following exposure to two chemicals indicated a substantial number of common genes (> 127). The three compound overlapping gene set, however, comprised only 25 genes. We suggest that the low commonality in transcriptional response may be linked to the chronic concentrations (approximately 10% EC50) and chronic duration (28 days) used. Annotations of the three compound overlapping gene set indicated that genes from pathways most often associated with responses to environmental stress, such as heat shock, phase I and II metabolism, antioxidant defense, and cation balance, were not represented. The strongest annotation signature was for genes important in mitochondrial function and energy metabolism.

Human and environmental risk assessment of pharmaceuticals: differences, similarities, lessons from toxicology.

The presence of human and veterinary pharmaceuticals in the environment has caused increasing concern due their effects on ecological receptors. Improving the risk assessment of these compounds necessitates a quantitative understanding of their metabolism and elimination in the target organism (toxicokinetics), particularly via the ubiquitous cytochrome P-450 (CYP) system and their mechanisms of toxicity (toxicodynamics). This review focuses on a number of pharmaceuticals and veterinary medicines of environmental concern, and the differences and similarities between ecological and human risk assessment. CYP metabolism is discussed with particular reference to its ubiquity in species of ecological relevance. The important issue of pharmaceutical mixtures is discussed to assess how emerging technologies such as ecotoxicogenomics may assist in moving towards a more mechanism-based environmental risk assessment of pharmaceuticals.

Toxicological and biochemical responses of the earthworm Lumbricus rubellus to pyrene, a non-carcinogenic polycyclic aromatic hydrocarbon.

The effects of the polycyclic aromatic hydrocarbon (PAH) pyrene on earthworms were investigated in contact and soil tests. In addition to measuring toxic effects on survival and reproduction, Ethoxyresorufin-o-deethylase (EROD) activity and catalase activity were also studied as possible biomarkers of toxic stress. The survival data indicated that LC50 values were 0.0068 mg/ml for the contact test, and 283 mg/kg in the soil test. Cocoon production rate was significantly reduced compared to controls at 160, 640 and 2560 mg/kg in the soil test. No EROD activity could be detected in preliminary studies using control and exposed animals from the contact test, so this assay was not used to the soil test. Catalase activity was shown to be significantly lower at 640 mg/kg in the soil test compared to all other treatments and the control. When compared to toxicological data for other soil invertebrates, Lumbricus rubellus has an intermediate sensitivity in respects of survival and a lower sensitivity for reproductive effects, although the soil used in this study had a higher organic content than previous studies, meaning that the sensitivity of this species may be underestimated in comparison to previous published data for other soil invertebrates.

Measurement of annetocin gene expression: a new reproductive biomarker in earthworm ecotoxicology.

The emergence of new technologies from the genomics revolution will transform the potential application of biomarkers to assess how pollutants impact people, animals, and ecosystems. Genetic databases provide a huge resource from which candidate molecular biomarkers can be identified and, subsequently, exploited to address these issues. However, a major challenge is to link these novel molecular indices to ecologically relevant whole-organism life-cycle traits (such as reproduction and growth). Such a functional link is provided by annetocin, previously characterized as a member of the vasopressin/oxytocin superfamily of neuropeptides. It is expressed in annelid worms within the neurons of the central nervous system and has been shown to be involved in the induction of egg-laying behavior. This paper outlines the validation of annetocin as a novel biomarker of reproductive fitness in the earthworm Eisenia fetida. The design of primer pairs targeted toward oligochaete annetocin has facilitated the isolation of a full-length annetocin cDNA from this species. Optimization of a real-time quantitative PCR procedure exploiting the fluorescent DNA-binding molecule, Sybr Green, has allowed the measurement of annetocin transcript levels over a range covering six orders of magnitude. Using this approach, gene expression was measured in earthworms exposed to soils polluted with high concentrations of zinc and lead. Traditional growth and reproductive indices, including cocoon production, were also recorded and related to the molecular parameter. The future use of annetocin as a molecular genetic biomarker in terrestrial ecotoxicology is discussed.

A review of lysosomal membrane stability measured by neutral red retention: is it a workable earthworm biomarker?

The ability of biomarkers to integrate effects of chemicals on biota has lead to increased calls for their application in assessing the status of polluted ecosystems. In tandem there has been an increase in our knowledge of the ecophysiological responses of keystone species to pollutants, which has allowed the development of a number of promising methods. In contrast to the number of biomarker development studies, the number of biomarker validation studies has remained limited. This paper redresses this imbalance by drawing together data from studies that have used the earthworm lysosomal membrane stability response (measured using the neutral red retention assay). This review first gives a short history of the biomarker's development. Second, it sets published applications of the technique against established criteria for a "good" biomarker (i.e., dose-response relationship, sensitivity, ecological relevance, confounding factors, chemical specificity, species differences, time-response relationship, methodological concerns, and overall public/regulator confidence, and acceptance). Discussion of the biomarker's suitability to each criterion is followed by an overall evaluation of its workability for routine soil quality assessment and caveats for its use.

Responses of earthworms (Lumbricus rubellus) to copper and cadmium as determined by measurement of juvenile traits in a specifically designed test system.

In this study, the effects of two metals, copper and cadmium, on the growth and development of juvenile Lumbricus rubellus were measured in a toxicity test in which individuals were grown in isolation. This design had a number of advantages over traditional test systems for earthworms. Importantly, the test is specifically designed to measure two juvenile traits (survival over and length of the juvenile period) that have been shown to have a high sensitivity for determining population growth rate. The test system also maximizes replication, while allowing time-series-based monitoring of individual growth. For both metals, significant exposure-dependent effects on survival, growth, development time, and (less certainly) maturation weight were observed. Comparisons of the relative toxicity of the two metals indicated different concentration-response relationships. For copper, hormesis was found at low levels, while only at the highest soil concentration tested (10.07 micromol g(-1)) were (severe) toxic effects present. For cadmium, hormesis was also evident at the lowest concentration tested; however, at soil levels above this, a graded concentration-dependent toxic effect was apparent. These differences in the exposure response patterns can be (tentatively) explained in terms of the mechanisms for handling copper (an essential metal for earthworms) and cadmium (a putative nonessential element). The applicability of the test for routine measurement of chemical effects on ecologically relevant juvenile traits is also outlined and future developments are discussed.

Developing a new method for soil pollution monitoring using molecular genetic biomarkers.

Physiological responses to environmental stressors may induce changes in gene expression as part of an organism's homeostatic mechanisms. Thus molecular genetic biomarkers have the potential to be used for monitoring sublethal chemical exposure in ecosystems. This paper describes a methodological assessment of the suitability of a protocol to monitor selected biomarkers. The TaqMan real-time quantitative polymerase chain reaction was used to measure gene transcription in earthworms (Lumbricus rubellus) maintained on control or cadmium- or copper-spiked soil. Changes in the expression of two target genes, that encoding metallothionein isoform 2 (MT-2) and that encoding the mitochondrial large ribosomal subunit (MLRS), were quantified against the internal control gene beta-actin. The protocol used produced reliable and reproducible results. Transcript levels displayed qualitative and quantitative differences in the responses to the two metal ions. MLRS gene levels were unaffected by exposure to cadmium but displayed a response to high levels of copper. Conversely, cadmium greatly induced MT-2 gene expression, but copper only altered transcription of this gene at high exposure concentrations. This study demonstrates that it is now technically feasible to use gene expression as an index of pollution exposure in environmentally relevant organisms.

The development of genetically inherited resistance to zinc in laboratory-selected generations of the earthworm Eisenia fetida.

The capacity of species to adapt both physiologically and genetically to contaminants may allow populations to persist in polluted environments. Such 'adaptation' can have important implications for risk assessment, since it may mean that prediction based on extrapolation of toxicity studies with naïve populations may prove invalid for long-term contaminated sites. To investigate the evolution of zinc resistance in Eisenia fetida, worms from a previously unexposed population (parent) were selected and reared over two generations (F1, F2) while exposed to zinc in the laboratory. Relative sensitivities of unexposed and selected generations were then compared by exposing parent, F1 and F2 individuals to zinc in contact filter papers tests. Calculation of effect concentrations from this work indicated differences in sensitivity to zinc for successive generations, with higher toxicity values (LC(50), LC(90), LC(99)) found for the selected worms. The increases in resistance found for F1 and F2 worms were confirmed in a discriminating dose study. In addition to comparing the sensitivities of the parent, F1 and F2 generations for zinc, toxicity tests were also conducted with copper to assess if there was evidence of cross-resistance between the two metals. Results indicated similar increases in resistance to copper to those found for zinc. Mechanisms underlying the increased metal resistance were studied in toxicokinetic experiments. Results indicated no clear trends between the three generations indicating that physiological responses, other than differences in kinetic parameters, are responsible for the increased resistance found in the selected worms.

Effects of metal-contaminated soils on the growth, sexual development, and early cocoon production of the earthworm Eisenia fetida, with particular reference to zinc.

Juvenile Eisenia fetida (Savigny) were exposed for 20 weeks to an uncontaminated soil and to soils contaminated with cadmium, copper, lead, and zinc collected from seven sites at different distances from a smelting works at Avonmouth, southwest England. The survival, growth (= weight after 5 weeks exposure), time to sexual maturation (= percentages of adults present after 8 weeks), and reproduction (= number of cocoons produced by the worms) were compared with soil metal concentrations. Of the parameters measured, growth and sexual maturation time had the lowest EC50 values. The effects of metal-contaminated soils could be attributed both to the direct toxicity of the metals and to changes in the "scope for growth" of the exposed worms. A comparison of the results with those of an earlier toxicity test conducted with adult worms indicated that juveniles are more sensitive to metals than adults. Significant toxic effects on the growth and sexual maturation times of juveniles were detected in soils from sites for which no significant effects on the cocoon production of adults could be detected. The greater sensitivity of juvenile worms indicates the importance of considering effects on a variety of life history stages when conducting a risk assessment of the effects of pollutants in soils. Although E. fetida does not occur naturally in soils at Avonmouth, the present study provides evidence to support the suggestion that pollution from the smelter is responsible for the absence of worms within 2 km of the factory. Results presented in this paper, and from previous studies, suggest the observed absence is due to the effects of zinc on the growth and maturation of juveniles and the cocoon production rate of adult worms.

Extrapolation of the laboratory-based OECD earthworm toxicity test to metal-contaminated field sites.

: The effects of cadmium, copper, lead and zinc on survival, growth, cocoon production and cocoon viability of the earthworm Eisenia fetida (Savigny) were determined in three experiments. In experiment 1, worms were exposed to single metals in standard artificial soil. For experiment 2, worms were maintained in contaminated soils collected from sites at different distances from a smelting works situated at Avonmouth, south-west England. In experiment 3, worms were exposed to mixtures of metals in artificial soil at the same concentrations as those present in the field soils. A survey of earthworm populations was carried out also. Population densities and species diversities of earthworms declined with proximity to the smelting works. No earthworms were found within 1 km of the factory. Comparison of toxicity values for the metals determined in the experiments indicated that zinc is most likely to be limiting earthworm populations in the vicinity of the works. Zinc was at least ten times more toxic to E. fetida in artificial soil than in contaminated soils collected from the field. This difference was probably due to the greater bioavailability of zinc in the artificial soil. The results are discussed in the context of setting 'protection levels' for metals in soils based on laboratory toxicity data.

Effects of cadmium, copper, lead and zinc on growth, reproduction and survival of the earthworm Eisenia fetida (Savigny): Assessing the environmental impact of point-source metal contamination in terrestrial ecosystems.

The earthworm Eisenia fetida (Annelida: Oligochaeta) was exposed to a geometric series of concentrations of cadmium, copper, lead and zinc in artificial soil using the OECD recommended protocol. Mortality, growth and cocoon production were measured over 56 days to determine LC50 and EC50 values. No observed effect concentrations (NOECs) were also estimated. Furthermore, the percentage of viable cocoons and number of juveniles emerging per cocoon was recorded. Cocoon production was more sensitive than mortality for all the metals, particularly cadmium and copper for which NOEC reproduction values were an order of magnitude lower than those for NOEC mortality. However, there was no significant effect of metals on the viability of cocoons. The weights of earthworms declined in all treatments (including the controls) during the experiment. This was probably due to the lack of suitable food in the OECD standard soil medium used. It was concluded that future experiments should include animal manure in the test medium. The LC50, EC50 and NOEC values determined in this study were compared with concentrations of metals in soils in the vicinity of a smelting works at Avonmouth, southwest England. The 14-day LC50 for zinc in Eisenia fetida was exceeded in soils covering an area of 75 km2 around the works, compared to 4.2 km2 for copper and 4.7 km2 for lead. Soil values for cadmium did not exceed the LC50 value anywhere in the region. Similar estimates of relative effects on reproduction confirmed that zinc is most likely to be responsible for the absence of earthworms from sites close to the Avonmouth works. However, the OECD standard test overestimated the potential effects of metals on populations, since earthworms can be found as close as 1 km from the smelting works. The discrepancy between test and field observations was probably due to the greater availability of the metals in the artificial soil.