Embryotoxicity of silver nanomaterials (Ag NM300k) in the soil invertebrate Enchytraeus crypticus - Functional assay detects Ca channels shutdown.

Despite that silver (Ag) is among the most studied nanomaterials (NM) in environmental species and Ag's embryotoxicity is well known, there are no studies on Ag NMs embryotoxicity in soil invertebrates. Previous Full Life Cycle (FLC) studies in Enchytraeus crypticus, a standard soil invertebrate, showed that Ag materials decreased hatching success, which was confirmed to be a hatching delay effect for silver nitrate (AgNO3) and mortality for Ag NM300K. In the present study, we aimed to investigate if the impact of Ag takes place during the embryonic development, using histology and immunohistochemistry. E. crypticus cocoons were exposed to a range of concentrations of Ag NM300K (0-10-20-60-115 mg Ag/kg) and AgNO3 (0-20-45-60-96 mg Ag/kg) in LUFA 2.2 soil, in an embryotoxicity test, being sampled at days 1, 2, 3 and 6 (3, 4, 5 and 7 days after cocoon laying). Measured endpoints included the number of embryonic structures, expression of transferrin receptor (TfR) and L type calcium channels (LTCC) through histological and immunohistochemistry analysis, respectively. Results confirmed that Ag materials affected the embryonic development, specifically at the blastula stage (day 3). The expression and localization of TfR in E. crypticus was shown in the teloblasts cells, although this transcytosis mechanism was not activated. Ag affected calcium (Ca) metabolism during embryonic development: for AgNO3, LTCC was initially activated, compensating the impact, for Ag NM300K, LTCC was not activated, hence no Ca balance, with irreversible consequences, i.e. terminated embryonic development. An Adverse Outcome Pathway was drafted, integrating the mechanisms here discovered with previous knowledge.

Confirmatory assays for transient changes of omics in soil invertebrates - Copper materials in a multigenerational exposure.

Environmental risk assessment (ERA) based on effects caused by chronic and longer term exposure is highly relevant. Further, if mechanistic based approaches (e.g. omics) can be included, beyond apical endpoints (e.g. reproduction), the prediction of effects increases. For Cu NMs (and CuCl2) this has been studied in detail, covering multi-omics and apical effects using the soil standard species Enchytraeus crypticus. The intermediate level effects like cell/tissue and organ alterations represent a missing link. In the present study we aimed to: 1) perform long term exposure to Cu materials (full life cycle and multigeneration, 46 and 224 days) to collect samples; 2) perform histology and immunohistochemistry on collected samples at 12 time points and 17 treatments; 3) integrate all levels of biological organization onto an adverse outcome pathway (AOP) framework. CuO NMs and CuCl2 caused both similar and different stress response, either at molecular initiating events (MIE) or key events (KEs) of higher level of biological organization. Cell/Tissue and organ level, post-transcriptional and transcriptional mechanisms, through histone modifications and microRNA related protein, were similarly affected. While both Cu forms affected the Notch signalling pathway, CuCl2 also caused oxidative stress. Different mechanisms of DNA methylation (epigenetics) were activated by CuO NMs and CuCl2 at the MIE.

Developing an epigenetics model species - From blastula to mature adult, life cycle methylation profile of Enchytraeus crypticus (Oligochaete).

DNA methylation is an epigenetic mechanism of particular importance in developmental biology, but methylation also varies along organisms' life cycle. Recent studies have deliberated copper (Cu) exposure induced epigenetic changes in Enchytraeus crypticus, a standard species belonging to one of the most common and important genera of soil invertebrates in many ecosystems. There is however no information on how DNA methylation levels change within the life cycle of this species. We here investigate the global DNA methylation profile along the life cycle of E. crypticus and compare this to the expression of target genes involved in methylation. Results showed that after the lowest DNA methylation level at day 3 (early embryonic stage, blastula) there was an increase by day 7 (organogenesis) after which levels were maintained at days 11, 18 and 25. DNA methyltransferase associated protein 1 (DMPA1) and Methyl Binding Domain 2 (MBD2) gene expression was highest during embryo stages (3 to 7 days), then decreasing (11, 18 days) and finally unregulated in adults (25 days). Hence, we here show that DNA methylation in E. crypticus changes among the different life stages, from cocoons to adults. Such information is a key knowledge to use this endpoint and tool in an ecotoxicology context. This means that it is almost implicit that gene expression levels are age specific for a given stressor. It seems logic to recommend to always compare individuals with the same age between treatments, and to be careful when extrapolating results among life stages. Once, we understand more of these effects we may even be able to predict which life stage is more sensitive to specific stressors. An experimental design that aims to cover epigenetics of stressors in a multigenerational exposure, including transgenerational effects, should ensure the synchronous age of organisms for sampling analysis purposes.

Multigenerational Exposure to WCCo Nanomaterials-Epigenetics in the Soil Invertebrate Enchytraeus crypticus.

It has become clear how important it is to assess longer term effects of (nano) materials in the environment given the current evidence showing how epigenetics drives response mechanisms. Here we studied global DNA methylation in standard soil invertebrate Enchytraeus crypticus over 224 days when exposed to nanostructured tungsten carbide cobalt (WCCo nanomaterials (NMs)) and to cobalt (CoCl2) in a multigenerational experiment. In order to assess the transgenerational effect, we used a multigenerational (MG) test design consisting of four generations in spiked soil followed by two generations in clean soil. Results showed that MG exposure to WCCo NMs caused global DNA methylation to increase, which continued in unexposed generations and was associated with an increase in reproduction (phenotypic effect). In general, WCCo NMs caused more (and more consistent) methylation than CoCl2.

Epigenetic effects of (nano)materials in environmental species - Cu case study in Enchytraeus crypticus.

Chemical stressors can induce epigenomic changes, i.e., changes that are transferred to the next generation, even when the stressor is removed. Literature on chemical induced epigenetic effects in environmental species is scarce. We here provide the first results on epigenetic effects caused by nanomaterials with an environmental OECD standard soil model species Enchytraeus crypticus species. We assessed the epigenetic potential in terms of global DNA methylation, gene-specific methylation via bisulfite sequencing and MS-HRM (Methylation Sensitive - High Resolution Melting), and gene expression qPCR for genes involved in DNA methylation, histone modifications, non-coding RNA and stress response mechanisms). We have exposed E. crypticus in a multigenerational (MG) test design to Cu (copper oxide nanomaterials (CuO NMs) and copper salt (CuCl2)). To link possible epigenetic effects to population changes, we used exposure concentrations (ECx) that caused a 10% and 50% reduction in the reproductive output (10% and 50% are the standards for regulatory Risk Assessment), the organisms were exposed for five consecutive generations (F1-F5) plus two generations after transferring to clean media (F5-F7), 7 generations in a total of 224 days. Results showed that MG exposure to Cu increased global DNA methylation and corresponded with phenotypic effects (reproduction). Gene expression analyses showed changes in the epigenetic, stress and detoxification gene targets, depending on the generation and Cu form, also occurring in post-exposure generations, hence indicative of transgenerational effects. There were in general clear differences between organisms exposed to different Cu-forms, hence indicate nanoparticulate-specific effects.

High-throughput gene expression in soil invertebrate embryos - Mechanisms of Cd toxicity in Enchytraeus crypticus.

Gene expression can vary with the organisms' life stage. It is known that embryos can be more sensitive to toxicant exposure, as previously demonstrated for Enchytraeus crypticus (Oligochaeta) exposed to cadmium (Cd), known to cause embryotoxicity and hatching delay. It was shown that Ca enters embryos via the L-type Ca channels in the cocoon membrane, this being affected in Cd exposed embryos (Cd-Ca competition is well-known). In the present study, the embryotoxic mechanisms of Cd were studied via high-throughput gene expression for E. crypticus. Cocoons (1-2 days old), instead of the adult organism, were exposed in Cd spiked LUFA 2.2 soil during 1 day. Results showed that Cd affected Ca homeostasis which is implicated in several other molecular processes. Several of the major modulators of Cd toxicity (e.g., impaired gene expression, cell cycle arrest, DNA and mitochondrial damage) were identified in the embryos showing its relevancy as a model in ecotoxicogenomics. The draft Adverse Outcome Pathway was improved. Previously was hypothesized that gene regulation mechanisms were activated to synthesize more Ca channel proteins - this was confirmed here. Further, novel evidences were that, besides the extracellular competition, Cd competes intracellularly which causes a reduction in Ca efflux, and potentiates Cd embryotoxicity.

Multigenerational effects of copper nanomaterials (CuONMs) are different of those of CuCl2: exposure in the soil invertebrate Enchytraeus crypticus.

Nanomaterials (NMs) are recommended to be tested in longer term exposures. Multigenerational (MG) studies are scarce and particularly important because effects can be transferred to the next generation. The current risk assessment framework does not include MG effects and this is a caveat for persistent materials. Here, the effects of copper NMs (CuONMs) and copper salt (CuCl2) were assessed in a MG exposure (4 generations in spiked soil + 2 generations in clean soil, F1 to F7 generations in total), with the standard soil model Enchytraeus crypticus, using relevant reproduction test effect concentrations (EC10, EC50), monitoring survival and reproduction. This represented ca. 1 year continuous exposure tests. MG effects varied with effect concentration and test materials: CuONMs caused increased toxicity for EC10 exposed organisms (EC50 did not change), and transfer to clean media reset effects, whereas CuCl2 reduced toxicity for EC10 and EC50, but the transfer to clean media "revived" the initial effects, i.e. close to EC50 levels in F7. Clearly CuONMs and CuCl2 cause different mechanisms of toxicity or response in the long term, not predictable based on short term or one generation studies. The present contributes for the improvement of risk assessment, adding important information for the long term exposure and effects.

Effects of copper oxide nanomaterials (CuONMs) are life stage dependent - full life cycle in Enchytraeus crypticus.

Copper oxide nanomaterials (CuONMs) have various applications in industry and enter the terrestrial environment, e.g. via sewage sludge. The effects of CuONMs and copper chloride (CuCl2) were studied comparing the standard enchytraeid reproduction test (ERT) and the full life cycle test (FLCt) with Enchytraeus crypticus. CuONMs mainly affected growth or juveniles' development, whereas CuCl2 mainly affected embryo development and/or hatching success and adults survival. Compared to the ERT, the FLCt allowed discrimination of effects between life stages and provided indication of the underlying mechanisms; further, the FLCt showed increased sensitivity, e.g. reproductive effects for CuONMs: EC10 = 8 mg Cu/kg and EC10 = 421 mg Cu/kg for the FLCt and the ERT respectively. The performance of the FLCt is preferred to the ERT and we recommend it as a good alternative to assess hazard of NMs. Effects of CuONMs and CuCl2 are life stage dependent and are different between Cu forms.

Effects of Ag nanomaterials (NM300K) and Ag salt (AgNO3) can be discriminated in a full life cycle long term test with Enchytraeus crypticus.

Information on effects of silver nanoparticles on soil invertebrates, especially using long-term exposures, is scarce. In this study we investigated the effects of the reference Ag (NM300K) (compared to AgNO3) using the full life cycle test (FLCt) of the soil invertebrate Enchytraeus crypticus. Results showed that effects were higher compared to the standard reproduction test, which is shorter and does not cover the FLC. Both Ag forms caused a reduction on hatching success, juvenile and adult survival and reproduction with similar ECx. Differences between AgNO3 and Ag NM300K could be discriminated using the FLCt: AgNO3 decreased hatching success was shown to be a delay in the process, whereas Ag NM300K caused irreversible effects during the same time frame. These effects may have occurred during the embryo development, hatching (inhibition) or survival of hatched juveniles. Ag NM300K caused non-monotonic concentration-response effect as observed by the high effect of the lowest concentration (20mgkg-1). It is known that dispersion is higher at lower concentrations - this could explain the increased effect at low concentration. Non monotonic responses are well described in the literature, where effects of high cannot predict for low concentrations, hence special attention should be given for NMs low concentration effects.

Mechanisms of phenanthrene toxicity in the soil invertebrate, Enchytraeus crypticus.

Polycyclic aromatic hydrocarbons (PAHs) continue to cause environmental challenges as a result of their release into the environment by a great variety of anthropogenic activities and their accumulation in soils. Studies were conducted on the toxicological effect of the model PAH phenanthrene using the soil invertebrate model Enchytraeus crypticus at the individual, tissue, and molecular levels. Animals were exposed for 2 d and 21 d to phenanthrene concentrations corresponding to the (previously estimated) 3-wk effective concentration, 10% (EC10) and EC50 for effects on reproduction. Gene expression profiling did not reveal a typical phenanthrene-induced biotransformation signature, as it usually does in arthropods and vertebrates. Instead, after 2 d of exposure, only general metabolic processes were affected, such as translation and adenosine triphosphate synthesis-coupled electron transport. Histological sections of tissues of 2-d exposed animals showed no deviations from control. In contrast, after prolonged exposure of up to 21 d, histopathological effects were found: chloragogenous cells were highly vacuolated and hypertrophic. This was corroborated by differential expression of genes related to immune response and oxidative stress at the transcriptomic level. The data exemplify the complexity and species-specific features of PAH toxicity among soil invertebrate communities, which restricts read-across and extrapolation in the context of soil ecological risk assessment. Environ Toxicol Chem 2016;35:2713-2720. © 2016 SETAC.

Development of an embryotoxicity test for Enchytraeus crypticus--the effect of Cd.

The existing standard enchytraeid reproduction test (ERT) concerns the assessment of effects on survival and reproduction. In the present study we optimized and propose an embryotoxicity test using Enchytraeus crypticus. Cadmium (Cd) was used as a test substance. Endpoints evaluated were embryo development, number of embryonic structures, Calcium (Ca) channels quantification and hatching success with macroscopic monitoring, histological and immunohistochemistry analysis. Results showed that Cd is embryotoxic for this species, causing a decrease in the hatching success (EC50=3.1mg/kg), a delay or disruption in formation of embryonic structures depending on concentrations (<5mg Cd/kg or ⩾16mg Cd/kg). Results from immunohistochemistry suggest a competitive binding between Cd and Ca for Ca channels, resulting in changes in Ca homeostasis. The use of the E. crypticus embryotoxicity test with the combination of histological and immunohistological tools provided a good option towards mechanistic information enhancing the importance of these tests to evaluate the hazard of chemicals and possible use in risk assessment.

Enchytraeid Reproduction Test(PLUS): hatching, growth and full life cycle test--an optional multi-endpoint test with Enchytraeus crypticus.

Soil ecotoxicity standard tests for invertebrates are usually limited to the assessment of endpoints like survival and reproduction. Adverse effects may occur at other developmental stages, e.g. embryo development, hatching or growth. The species Enchytraeus crypticus is a model organism in the standard soil ecotoxicology test, where survival and reproduction are assessed. In the present study we optimized the test method to include additional endpoints. The proposed test started with synchronized age organisms', and included additionally hatching success, growth, maturation status and full life cycle. This allowed for the calculation of cocoon production and population growth rate. Results indicated that Cd is embryotoxic, main effect occurs on the embryo developmental stage and maturity. Further, the full life cycle test can discriminate between pre- and post-embryo formation. The increased sensitivity and full life cycle detail level makes it potentially useful for novel materials e.g. nanomaterials where the mode of action and hence effect target is unknown.

Non-avoidance behaviour in enchytraeids to boric acid is related to the GABAergic mechanism.

Soil invertebrates, e.g. enchytraeids, are known to be able to avoid unfavourable conditions, which gives them an important ecological advantage. These organisms possess chemoreceptors that can detect stressors, which in turn activate responses such as avoidance behaviour. We studied the avoidance behaviour in response to boric acid (BA) using enchytraeids. Results showed not only no avoidance, but that increasing concentrations seemed to have an "attraction" effect. To study the underlying mechanism, a selection of genes targeting for neurotransmission pathways (acetylcholinesterase (AChE) and gamma-aminobutyric acid receptor (GABAr)) were quantified via quantitative real-time polymerase chain reaction (qPCR). Evidences were that BA is neurotoxic via the GABAergic system mechanism where it acts as a GABA-associated protein receptor (GABAAR) antagonist possibly causing anaesthetic effects. This is the first time that (non)avoidance behaviour in invertebrates was studied in relation with the GABAergic system. We strongly recommend the combination of such gene and/or functional assay studies with the avoidance behaviour test as it can bring many advantages and important interpretation lines for ecotoxicity with minor effort.

Thyroid disruption in the lizard Podarcis bocagei exposed to a mixture of herbicides: a field study.

Pesticide exposure has been related with thyroid disrupting effects in different vertebrate species. However, very little is known about the effects of these compounds in reptiles. In the Mediterranean area, lacertid lizards are the most abundant vertebrate group in agroecosystems, and have been identified as potential model species for reptile ecotoxicology. The aim of this study was to understand if the herbicides applied in corn fields have thyroid disruptive effects in the lizard Podarcis bocagei. Adult male lizards were captured in north-western Portugal in corn fields treated with herbicides (exposed sites), and in organic agricultural fields (reference sites). Thyroid and male gonad morphology and functionality, and testosterone levels were investigated through histological, immunohistochemical and biochemical techniques. Lizards from exposed locations displayed thyroid follicular lumens with more reabsorption vacuoles and significantly larger follicular area than those from reference fields. Furthermore, testes of lizards from exposed locations had significantly larger seminiferous tubule diameters, significantly higher number of spermatogenic layers and displayed an up-regulation of thyroid hormone receptors when compared with lizards from reference areas. These findings strongly suggest that the complex mixture of herbicides that lizards are exposed to in agricultural areas have thyroid disrupting effects which ultimately affect the male reproductive system. Alachlor, which has demonstrated thyroid effects in mammals, may be largely responsible for the observed effects.

Biomarkers of exposure and effect in a lacertid lizard (Podarcis bocagei Seoane) exposed to chlorpyrifos.

In Europe, reptiles have been recently included in environmental risk-assessment processes for registration of plant-protection products. However, data on toxicity effects of most compounds are lacking. Chlorpyrifos is the most commonly used organophosphorus insecticide worldwide. In the present study, the authors exposed a lacertid lizard, Podarcis bocagei, to sublethal concentrations of chlorpyrifos. Individuals were exposed through spiked food for a period of 20 d (low dose 0.12 mg/kg/d, high dose 1.57 mg/kg/d). After exposure, various biomarkers of exposure and effect were evaluated, including the activities of glutathione S-transferase and enzymes involved in the glutathione redox cycle, glutathione concentrations, activities of esterases, liver and testes histopathologies, as well as locomotory and predatory behavior. The results indicate that sublethal, subchronic exposure to chlorpyrifos can affect P. bocagei in a dose-dependent manner. Adverse effects occurred at both the subindividual and individual levels, including inhibition of carboxylesterases and cholinesterases (ChEs), liver histopathological changes, and altered predatory behaviors. Animals exposed to chlorpyrifos took more time to capture and subdue prey items. The results suggest a link between effects at subindividual levels of organization with those observed at the whole individual level after exposure to environmentally realistic dosages of chlorpyrifos.

The use of a lacertid lizard as a model for reptile ecotoxicology studies: part 2--biomarkers of exposure and toxicity among pesticide exposed lizards.

As part of a wider study examining the impacts of corn pesticides on lacertid lizards in north-western Portugal, we examined various physiological, biochemical, and histological biomarkers of exposure and effect among field populations of Podarcis bocagei. Biomarkers included body condition index, standard metabolic rate, locomotor performance, parasitization, glutathione oxidative pathways and related enzyme activity, lipid peroxidation and liver and testis histology. Few of the various biomarkers investigated provided statistically significant evidence of toxic effect. However, using a weight of evidence approach, we conclude that pesticides are affecting lizards living in the vicinity of pesticide exposed corn agriculture sites. Lizards from these locations present a profile of animals under metabolic stress with reduced condition indices, increased standard metabolic rate, lower incidence of hepatocyte vacuolation, altered iron metabolism, increased activation of GSH oxidation pathways, and even increased prevalence of hemoparasites.

The use of a lacertid lizard as a model for reptile ecotoxicology studies--part 1 field demographics and morphology.

At the European level, lacertid lizards have been proposed as potential model species for reptile ecotoxicology. We studied demographic and morphological aspects of natural field subpopulations of Podarcis bocagei inhabiting similar agricultural habitats which were either regularly exposed to pesticides, or not. Parameters examined in this study included population size and density, sex ratio, adult body size, fluctuating asymmetry in femoral pores and parasite prevalence. In general, we detected few statistically significant differences between the exposed and reference subpopulations. Although field situations are ecologically complex and factors other than pesticides may be acting, the absence of observable effects on field subpopulations is probably indicative that lizards are coping or compensating for this level of exposure.