Reproduction Effect Concentrations Research Articles

Molecular mechanisms of zinc toxicity in the potworm Enchytraeus crypticus, analysed by high-throughput gene expression profiling

Zinc (Zn) is known to be relatively toxic to some soil-living invertebrates including the ecologically important enchytraeid worms. To reveal the molecular mechanisms of zinc toxicity we assessed the gene expression profile of Enchytraeus crypticus (Enchytraeidae), exposed to the reproduction effect concentrations EC10 and EC50, over 4 consecutive days, using a high-throughput microarray (species customized). Three main mechanisms of toxicity to Zn were observed: 1) Zn trafficking (upregulation of zinc transporters, a defence response to regulate the cellular zinc level), 2) oxidative stress (variety of defence mechanisms, triggered by Reactive Oxygen Species (ROS)), and 3) effects on the nervous system (possibly the primary lesion explaining the avoidance behaviour and also why enchytraeids are relatively susceptible to Zn). The adverse outcome at the organism level (reproduction EC50) could be predicted based on gene expression (male gonad development, oocyte maturation), with Zn at the EC50 affecting processes related to higher stress levels. The gene expression response was time-dependent and reflected the cascade of events taking place over-time. The 1 to 4 days of exposure design was a good strategy as it captured the time for sequence of events towards zinc adverse outcomes in E. crypticus.

High-throughput transcriptomics: Insights into the pathways involved in (nano) nickel toxicity in a key invertebrate test species

Nickel nanoparticles (NiNPs) have an estimated production of ca. 20 tons per year in the US. Nickel has been risk-assessed for long in Europe, but not NiNPs, hence the concern for the environment. In the present study, we focused on investigating the mechanisms of toxicity of NiNPs and the comparison to NiNO3. The high-throughput microarray for the soil ecotox model Enchytraeus crypticus (Oligochaeta) was used. To anchor gene to phenotype effect level, organisms were exposed to reproduction effect concentrations EC20 and EC50, for 3 and 7 days. Results showed commonly affected pathways between NiNPs and NiNO3, including increase in proteolysis, apoptosis and inflammatory response, and interference with the nervous system. Mechanisms unique to NiNO3 were also observed (e.g. glutathione synthesis). No specific mechanisms for NiNPs were found, which could indicate that longer exposure period (>7 days) is required to capture the peak response to NiNPs. A mechanisms scheme is assembled, showing both common and unique mechanisms to NiNO3 and NiNPs, providing an important framework for further, more targeted, studies.

The Enchytraeus crypticus stress metabolome – CuO NM case study

The stress metabolome provides a thorough insight into the signals and hence mechanisms of response of organisms. This is an excellent tool to advance the understanding of interactions, especially for substances like nanomaterials (NMs), for which there is an urgent need for alternative methods for hazard assessment. The metabolome of Enchytraeus crypticus was studied for the first time. The case study, CuO NM (and CuCl2) covered exposure along a time frame [0–7–14 days (d)] and two reproduction effect concentrations (EC10 and EC50). High-performance liquid chromatography-mass spectrometry based method (HPLC-MS) was used, with reversed phase (RP) separation and mass spectrometric detection in positive and negative modes. Metabolite profiling of Cu materials yielded 155 and 382 metabolite features in positive and negative modes, respectively, showing an expression related with time, material, and ECx. The number of differentially expressed metabolites (DEMs) decreased with exposure time (14 d) for CuO NM, whereas for CuCl2 EC50 it increased. Overall, almost all DEMs are down-regulated for CuO NM and up-regulated for CuCl2 (both modes). Early effects were mainly related to amino acids and later to lysophospholipids (down-regulation). Furthermore, the underlying mechanisms of CuO NM toxicity (e.g. neurotransmission, nucleic acids generation, cellular energy, and immune defense) differ from CuCl2, where later metabolomic responses are mostly linked to the metabolism of lipids and fewer to amino acids. This study reports a large scale metabolome profiling for E. crypticus and identifies potential markers of Cu materials, which can help to align intelligent testing strategies and safer-by-design materials.

High-throughput tool to discriminate effects of NMs (Cu-NPs, Cu-nanowires, CuNO3, and Cu salt aged): transcriptomics in Enchytraeus crypticus

The current testing of nanomaterials (NMs) via standard toxicity tests does not cover many of the NMs specificities. One of the recommendations lays on understanding the mechanisms of action, as these can help predicting long-term effects and safe-by-design production. In the present study, we used the high-throughput gene expression tool, developed for Enchytraeus crypticus (4 × 44k Agilent microarray), to study the effects of exposure to several copper (Cu) forms. The Cu treatments included two NMs (spherical and wires) and two copper-salt treatments (CuNO3 spiked and Cu salt field historical contamination). To relate gene expression with higher effect level, testing was done with reproduction effect concentrations (EC20, EC50), using 3 and 7 days as exposure periods. Results showed that time plays a major role in the transcriptomic response, most of it occurring after 3 days. Analysis of gene expression profiles showed that Cu-salt-aged and Cu-nanowires (Nwires) differed from CuNO3 and Cu-nanoparticles (NPs). Functional analysis revealed specific mechanisms: Cu-NPs uniquely affected senescence and cuticle pattern formation, which can result from the contact of the NPs with the worms’ tegument. Cu-Nwires affected reproduction via male gamete generation and hermaphrodite genitalia development. CuNO3 affected neurotransmission and locomotory behavior, both of which can be related with avoidance response. Cu salt-aged uniquely affected phagocytosis and reproductive system development (via different mechanisms than Cu-Nwires). For the first time for Cu (nano)materials, the adverse outcome pathways (AOPs) drafted here provide an overview for common and unique effects per material and linkage with apical effects.

Does long term low impact stress cause population extinction?

This study assessed and monitored 40 consecutive reproduction tests - multigenerational (MG) - of continuous exposure to Cd (at 2 reproduction Effect Concentrations (EC): EC10 and EC50) using the standard soil invertebrate Folsomia candida, in total 3.5 years of data were collected. Endpoints included survival, reproduction, size and metallothionein (MTc) gene expression. Further, to investigate adaptation to the toxicant, additional standard toxicity experiments were performed with the MG organisms of F6, F10, F26, F34 and F40 generations of exposure. Exposure to Cd EC10 caused population extinction after one year, whereas populations survived exposure to Cd EC50. Cd induced the up-regulation of the MTc gene, this being higher for the higher Cd concentration, which may have promoted the increased tolerance at the EC50. Moreover, EC10 induced a shift towards organisms of smaller size (positive skew), whereas EC50 induced a shift towards larger size (negative skew). Size distribution shifts could be an effect predictor. Sensitivity increased up to F10, but this was reverted to values similar to F0 in the next generations. The maximum Cd tolerance limits of F. candida increased for Cd EC50 MG. The consequences for risk assessment are discussed.

Effect of Cu and Ni on cellular energy allocation in Enchytraeus albidus.

Effects of nickel and copper on Enchytraeus albidus (Oligochaeta) were investigated using the cellular energy allocation approach. This methodology is used to evaluate the energetic status of an organism and is indicative of its overall condition. Enchytraeids were exposed to the reproduction Effect Concentrations (EC50 and EC90), and the parameters measured were the total energy reserves available (protein, carbohydrate and lipid budgets) and the energy consumption [based on electron transport system activity] which were further integrated to obtain the cellular energy allocation over different periods of exposure (0-2, 2-4 and 4-8 days). Carbohydrates (in comparison to lipids and proteins) were the only energy source mobilized in the case of nickel within 8 days of exposure. For copper exposure, protein budgets were also strongly reduced. Energy consumption increased in a time and dose-dependent way for copper and in the longer exposure period (4-8 days) at the EC90 for Ni exposure, indicating that this is a good biomarker for effects of short-time metal exposure, while cellular energy allocation was only significantly reduced for the EC90 of copper (4-8 days) and EC50 of nickel (2-4 days). The effects of nickel at concentrations causing 50 and 90 % decrease in reproduction were likely not due to the changes in cellular energy allocation within 8 days of exposure.

Cellular Energy Allocation to Assess the Impact of Nanomaterials on Soil Invertebrates (Enchytraeids): The Effect of Cu and Ag.

The effects of several copper (Cu) and silver (Ag) nanomaterials were assessed using the cellular energy allocation (CEA), a methodology used to evaluate the energetic status and which relates with organisms’ overall condition and response to toxic stress. Enchytraeus crypticus (Oligochatea), was exposed to the reproduction effect concentrations EC20/50 of several Cu and Ag materials (CuNO3, Cu-Field, Cu-Nwires and Cu-NPs; AgNO3, Ag NM300K, Ag-NPs Non-coated and Ag-NPs PVP-coated) for 7 days (0-3-7d). The parameters measured were the total energy reserves available (protein, carbohydrate and lipid budgets) and the energy consumption (Ec) integrated to obtain the CEA. Results showed that these parameters allowed a clear discrimination between Cu and Ag, but less clearly within each of the various materials. For Cu there was an increase in Ec and protein budget, while for Ag a decrease was observed. The results corroborate known mechanisms, e.g., with Cu causing an increase in metabolic rate whereas Ag induces mitochondrial damage. The various Cu forms seem to activate different mechanisms with size and shape (e.g., Cu-NPs versus Cu-Nwires), causing clearly different effects. For Ag, results are in line with a slower oxidation rate of Ag-NMs in comparison with Ag-salt and hence delayed effects.

数種類の有機化学物質に対する淡水性甲殻類<i>Daphnia magna</i> および<i>Ceriodaphnia dubia </i>の慢性毒性による感受性比較

The current worldwide regulatory guideline requires evaluation of the chronic effects of chemicals on water flea Daphnia magna. Based on the life cycle of D.magna, chronic tests with survival and reproductive endpoints requires a 21 days exposure period. Ceriodaphnia dubia, a common freshwater invertebrate similar to the D.magna, has a shorter life cycle and therefore reproductive (and survival) endpoints have been established following about 8 days of exposure. No observed effect concentrations for reproduction were obtained for D.magna and C.dubia following exposure to general 10 organic compounds (ie. aromatic amines as pharmaceutical, chlorinated aromatic compound, polyaromatic compound and alkyl ester) according to the OECD TG 211 and the biological test method of Environmental Canada. These data were evaluated to determine whether one organism might be considered more sensitive. Reproduction data obtained from C.dubia chronic test provide similar sensitivity to D.magna in this study. Based on these data, C.dubia may be used as a cost-effective alternative and representative invertebrate species when assessing the potential risk on the organic compounds.

Profiling transcriptomic response of Enchytraeus albidus to Cu and Ni: Comparison with Cd and Zn

Metals are among the most common contaminants in soils in Europe. Although their effects are relatively well known regarding survival and reproduction to soil invertebrates, their mode of action is poorly understood. Enchytraeus albidus is a model organism in ecotoxicology and with the development of a gene library for this species, transcriptomic studies are now possible. The main aim of this study is to understand the Cu and Ni mechanisms of response in E. albidus, in comparison with Cd and Zn (already studied). E. albidus were exposed to Cu and Ni for 4 days to the reproduction effect concentrations EC50 and EC90. Results indicate that Cu and Ni have similar mechanisms of toxicity. When comparing four elements (hierarchical clustering) it was possible to observe a clear separation of Cd from all other metals. This separation correlates with the available information from other species regarding the toxicokinetics of the tested elements.

Gene Expression Responses Linked to Reproduction Effect Concentrations (EC10,20,50,90) of Dimethoate, Atrazine and Carbendazim, in Enchytraeus albidus

BackgroundMolecular mechanisms of response to pesticides are scarce and information on such responses from soil invertebrates is almost inexistent. Enchytraeus albidus (Oligochaeta) is a standard soil ecotoxicology model species for which effects of many pesticides are known on survival, reproduction and avoidance behaviour. With the recent microarray development additional information can be retrieved on the molecular effects.Methodology/Principal FindingsExperiments were performed to investigate the transcription responses of E. albidus when exposed to three pesticides – dimethoate (insecticide), atrazine (herbicide) and carbendazim (fungicide) – in a range of concentrations that inhibited reproduction by 10%, 20%, 50% and 90% (EC10, EC20, EC50 and EC90, respectively). The goal of this study was to further identify key biological processes affected by each compound and if dose-related. All three pesticides significantly affected biological processes like translation, regulation of the cell cycle or general response to stress. Intracellular signalling and microtubule-based movement were affected by dimethoate and carbendazim whereas atrazine affected lipid and steroid metabolism (also by dimethoate) or carbohydrate metabolism (also by carbendazim). Response to DNA damage/DNA repair was exclusively affected by carbendazim.ConclusionsChanges in gene expression were significantly altered after 2 days of exposure in a dose-related manner. The mechanisms of response were comparable with the ones for mammals, suggesting across species conserved modes of action. The present results indicate the potential of using gene expression in risk assessment and the advantage as early markers.

Chronic Exposure of the Oligochaete Lumbriculus variegatus to Polycyclic Aromatic Compounds (PACs): Bioavailability and Effects on Reproduction

This study aimed to monitor PAC availability to the oligochaete Lumbriculus variegatus during 28 days of exposure to spiked sediments, in order to obtain reliable chronic effect concentrations for reproduction. Sediment toxicity tests were performed using three pairs of PAC isomers: two homocyclic compounds (anthracene and phenanthrene), two azaarenes (acridine and phenanthridine), and the two main transformation products of the azaarenes (acridone and phenanthridone). During the experiment, available PAC concentrations in pore water (estimated using solid phase microextraction) decreased more than total PAC concentrations in the sediment. Relating effect concentrations to PAC concentrations in pore water and in organisms showed that the two homocyclic compounds caused narcotic effects during chronic exposure, but only one of the four tested heterocyclic PACs caused narcotic effects. The transformation product phenanthridone was not toxic at the tested concentrations (up to 4000 micromol/kg dry sediment), whereas EC50 values for the parent compound phenanthridine and the isomer acridone were below the estimated limit for narcosis, suggesting a specific mode of action. These results demonstrated the unpredictable (isomer) specific toxicity of azaarenes and their transformation products, emphasizing the need of chronic toxicity testing to gain insight into the long-term effects of heterocyclic PACs, which have been overlooked in risk assessment.

Influence of Temperature on the Regulation and Toxicity of Zinc in Folsomia candida(Collembola)

To generate reproducible and reliable results, most protocols for standardized toxicity tests prescribe strict limits for test conditions such as temperature and relative humidity. In the field, however, variability of climate can influence the functioning of organisms and induce changes in sensitivity. To evaluate the influence of temperature on the ecotoxicity of zinc, the springtail Folsomia candidawas exposed to zinc-contaminated soil at different temperatures. In one experiment, the toxicity of ZnCl 2was determined at a constant temperature and under alternating temperature conditions. Although control performance of F. candidawas influenced by the temperature treatment, effect concentrations for reproduction were not significantly different between treatments. Levels of internal zinc regulation were higher in animals exposed to alternating temperatures. Based on the observed influence of temperature on the accumulation of zinc, a second experiment was performed to determine the sensitivity of F. candidato zinc at four different exposure temperatures. A decrease in temperature led to an increase in sensitivity when sublethal parameters such as growth and reproduction were considered. However, animals were less sensitive at lower temperatures where mortality was concerned. Internal zinc concentrations in the animals increased with decreasing temperatures, and when the effect on reproduction was based on internal zinc levels, differences between temperature treatments were not significant. From the results of this study it is concluded that for F. candida,constant temperature conditions form a good basis for the extrapolation of toxicity levels to fluctuating temperature conditions. Since the average exposure temperature can alter the sensitivity of F. candidafor zinc, and changes in sensitivity are parameter dependent, exposure temperature should be chosen carefully.

Occurrence of non‐ortho‐, mono‐ortho‐ and di‐ortho‐substituted PCB congeners in different organs and tissues of polecats (Mustela putorius L.) from the Netherlands

AbstractThe presence and concentrations of non‐ortho‐, mono‐ortho‐, and di‐ortho‐substituted PCB congeners in the polecat (n = 7), a member of the mustelid family, were investigated. PCBs were extracted with a Soxhlet apparatus. After cleanup the non‐ortho‐substituted PCB congeners were separated from the other PCBs by HPLC. Determinations were accomplished with GC‐ECD or GC‐MSD. Patterns of PCBs were examined in different organs and tissues: liver, kidney, muscle, anal gland secretion, mesenteric fat, and subcutaneous fat. Using a multivariate statistical method for data analysis (SIMCA), a significant difference of PCB patterns between anal gland secretion and the other organs and tissues was revealed. Lesser concentrations of congeners with seven and eight chlorine atoms in anal gland secretion were mainly responsible for this phenomenon. A more or less organ‐ and tissue‐specific PCB pattern was observed in all animals. PCB patterns were not dependent on prey choice, which ranged from terrestrial (small rodents) to aquatic (amphibians). This finding implies that PCB patterns in the polecat seem to be controlled by metabolic processes rather than diet factors. The total concentration of PCBs (sum of 29 congeners) in polecats varies widely, two orders of magnitude, from 1 to 370 μg/g lipid. In some animals, PCBs exceeded the experimentally determined reproduction effect concentrations of mink and ferrets. Using the toxic equivalent approach, it was observed that planar PCB 126 accounts for 63 to 98% of the toxic equivalents. The results showed that juvenile animals contain greater PCB levels than adult males and females, which might be related to an increased elimination of PCBs in adult animals due to anal gland secretion. High concentrations of PCBs were observed in such secretion. A preliminary model for concentrations of PCBs in polecats including this effect is proposed.