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Abstract 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 NiNO 3. 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 NiNO 3 , including increase in proteolysis, apoptosis and inflammatory response, and interference with the nervous system. Mechanisms unique to NiNO 3 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 NiNO 3 and NiNPs, providing an important framework for further, more targeted, studies. Graphical abstract Image 1 Highlights • High-throughput gene expression results for NiNPs and NiNO 3 were obtained. • Gene expression was anchored to higher effect level (reproduction EC20/50). • Common mechanisms were observed between the two Ni forms. • Mechanisms unique to NiNO 3 were observed, but not for NiNPs. • Mechanistic scheme (Adverse Outcome Pathway like) was drafted for Ni materials. An Adverse Outcome Pathway for nickel based NM was built, providing an important framework for further, more targeted, studies. [ABSTRACT FROM AUTHOR]

Nanoagrochemicals have the potential to revolutionize agriculture towards a precision farming system, able to reduce application rates and consequently their environmental footprint, while keeping efficacy. Several nanoagrochemicals (including nanopesticides (Npes) and nanofertilizers (Nfer)) are already commercialized but the environmental risk assessment of these advanced materials is often lacking. In the present study, we studied the commercial fertilizer WELGRO® Cu + Zn and assessed its ecotoxicity to the soil invertebrate species Enchytraeus crypticus (Oligochaeta), further comparing it to its individual active substances CuO and ZnO. To get a comprehensive picture of possible effects, we used four types of highly relevant tests in LUFA 2.2 soil: 1) avoidance behaviour (2 days), 2) reproduction (OECD standard, 28 d), 3) its extension (56 d), and 4) the full life cycle (FLC) (46 d) – this high level of hazard screening allows for increased interpretation. The results confirmed the nano-features of WELGRO® and a higher toxicity than the mixture of the individual components CuO + ZnO. E. crypticus avoided the soil spiked with WELGRO® and CuO + ZnO, this being the most sensitive endpoint - avoidance behaviour. Both WELGRO® and the active substances were little to non-toxic based on the OECD standard test. However, the toxicity dramatically increased in the tests focussing on longer-term sustainability measures, i.e., 56 days, ca. 170 for WELGRO®. This seems related to the nano-features of WELGRO®, e.g., slow release of ions from the nanoparticles throughout time. The FLCt results showed WELGRO® affected hatching and juveniles' survival, being these the most sensitive life stages. Hence, under actual real world field usage scenarios, i.e., based on the recommended application rates, nanoenabled WELGRO® can affect oligochaete populations like enchytraeids, both via the immediate avoidance behaviour and also via prolonged exposure periods. [Display omitted] • WELGRO®Cu + Zn (commercial) represents a nanoenabled fertilizer (150 nm NPs). • Effects of WELGRO® and its active substances, a mixture of CuO and ZnO were assessed. • WELGRO® was more toxic than the mixture of its active substances. • Avoidance behaviour was the most sensitive endpoint. • The toxicity increased with prolonged exposure (from 28 to 56 d), ca. 170x for WELGRO®. [ABSTRACT FROM AUTHOR]

Gene expression studies provided a major step forward in ecotoxicology and ecological functional genomics, among other areas. How to classify changes in gene expression to be significant compared to its normal operating range (NOR) requires a number of biological and statistical criteria. Doubts have been casted about when a change in gene expression has ecologically relevant meaning and what is the normal biological variation or NOR. Because genes are highly sensitive and self-regulating pieces, how can we design our experiments to control normal variation? The main aim of the present paper is to discuss such questions, while providing suggestions for improvement and interpretation. We have analysed several gene control data sets of Enchytraeus albidus comparing standard control test conditions, i.e. (1) from experiments performed at different occasions in time, (2) to culture media, (3) to acetone control, (4) from exposure time series (2, 4 and 8 days) and (5) between replicates. We discriminated between some normal variation sources. For instance, the transference of organisms from cultures to standard testing condition suggests a lowered general metabolism. Chemical carriers like acetone seem to cause changes related with soil structure and hypoxia conditions. Organisms have a “time-like” transcription signature when sampled along a time series. The coefficient of variance (CV) between biological replicates was 0.1 < CV < 1.3 for 17% of the genes. Variability will partly be inherent to the species, but most important is to understand the ground for interpretations as discussed. Here, the first attempt for the establishment of a NOR for E. albidus gene expression was made which represents a naturally varying ecological window – this contributes for the discrimination between stress responses and natural ecological variations in soil studies. [ABSTRACT FROM AUTHOR]

Cellular energy allocation (CEA) is a methodology developed to evaluate the effects of toxic stress on the metabolic balance of organisms. It consists of the integration of the energy reserves available (Ea; total carbohydrate, protein and lipid content) and energy consumption (Ec) estimated by measuring electron transport system (ETS) activity. The main goal of the present study was to evaluate the effects on the energy budget of the soil invertebrate Enchytraeus albidus (Oligochaeta) after exposure to dimethoate, atrazine, and carbendazim (by testing the reproduction 10% effective concentration, 20% effective concentration, 50% effective concentration and 90% effective concentration) over periods of time from 0 d to 2 d, 4 d, and 8 d. Significant changes in energy reserves were observed with all pesticides, together with effects on energy consumption. Carbohydrates were the first energy source to be used, and clear depletions occurred with all pesticides. Energy consumption increased generally over longer exposures and with higher concentrations of the pesticides. Although clear changes were seen in the individual energy reserve budgets and on Ec, CEA was only significantly reduced with atrazine exposures longer than 4 d. The nearly absent effects on CEA at concentrations known to affect reproduction indicate that the reduction in reproduction is not likely to be caused by a reduction in the total energy budget during the first 8 d of exposure. The present study showed the importance of complementing CEA interpretation with the individual Ea and Ec parameters, in particular if these show opposite balances. The Ea and Ec results were in good agreement with gene transcription results from a parallel study, hence suggesting translation and showing the advantage of combining various effect levels to advance the understanding of mechanisms. Environ Toxicol Chem 2013;32:2800-2807. © 2013 SETAC [ABSTRACT FROM AUTHOR]

[Display omitted] • Aging and its interaction with toxicity is seldom assessed in standards. • Toxicity of Cd was assessed in aged E. crypticus : young (19d), old (92d) and oldest (128d). • Age influenced Cd toxicity: oldest > young > old. • Oldest and youngest were most sensitive to Cd. Aging is a natural process for all living beings and there is often a decline of overall performance after a certain age tipping point. The response to stressors, e.g. exposure to chemicals can differ depending on the individuals' age. Standard guidelines do not always include synchronized age animals and are often optimized based on healthy animals of homogeneous populations, not aiming to cover age aspects. Hence, in the present study we aimed to assess the effects of aging on Enchytraeus crypticus (Oligochaeta) response to cadmium (Cd) exposure. For that, cultures with 3 synchronized ages [young: 18–20 days old, old: 91–93 days old and oldest: 127–129 days old] plus the standard (non-synchronized adults), were exposed to CdCl 2 (0, 1, 3.2, 10, 32, 100, 320 mg Cd/kg) in LUFA 2.2 soil. Results showed that Cd toxicity varied depending on the animal's age, with the young and oldest being most sensitive to Cd (reproduction EC 50 _old = 138 > EC 50 _young = 69 > EC 50 _oldest = 46 mg Cd/kg soil). This highlights the importance of age when assessing toxicity. Testing animals with synchronized age should improve results comparability but will not necessarily capture the most sensitive age. Toxicity to Cd has been shown to be higher in younger animals compared to standard cultures, although this may not be a general pattern with other chemicals or stressors. When using standardized guidelines, if cultures are not synchronized results will suffer from variability due to various ages of the animals. For a standard context we recommend the start with young (18–20 days) animals instead of non-synchronized adults. On the other hand, the study of populations on the edge windo w is highly relevant and can be much more explored in an ecotoxicology frame, as this can also enlighten impacts of aging in a wider species context. [ABSTRACT FROM AUTHOR]

Abstract: Enchytraeids are important inhabitants of a wide variety of soil types and suitable test organisms for environmental risk assessment through the determination of effects on survival and reproduction. In this study, the effect of pesticides with different modes of action is assessed in the standard test species Enchytraeus albidus. Main goals were: (a) to test the toxicity of a range of pesticides to E. albidus in a natural soil (LUFA 2.2), (b) to assess whether avoidance gave a prediction of effects on the survival and reproduction, and (c) to investigate if the organisms’ response to different toxics can be grouped into the respective chemical classes. Pesticides selected were the: (1) herbicides: phenmedipham and atrazine; (2) fungicides: carbendazim and pentachlorophenol; and (3) insecticides: dimethoate and lindane. All tested pesticides caused effects in the survival and reproduction of E. albidus and the compounds that showed a higher toxicity were carbendazim, dimethoate and atrazine. The effect concentrations were not chemical class dependent. In general, survival and reproduction showed similar response patterns. Avoidance behaviour showed trends of response similar to these other measured endpoints, but was less sensitive and more variable (data from a previous study). It was not possible to establish a clear correlation between survival, reproduction and avoidance endpoints. From an ecological point of view, avoidance tests are relevant but due to lower sensitivity and higher variability it is recommended that the enchytraeid avoidance test should not be used for risk assessment purposes. [Copyright &y& Elsevier]

Abstract 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. Graphical abstract Image 1 Highlights • E. crypticus embryos are more sensitive to Cd than adults. • Embryos' mechanisms were studied using high-throughput gene expression. • Major modulators of Cd toxicity were identified in the embryos. • Cd competes with Ca inside the cells (novel), besides extracellular (known). • Adverse Outcome Pathway (AOP) was improved. [ABSTRACT FROM AUTHOR]

The nano-form of copper (Cu-NPs) is already extensively used. In this paper the toxic effect of Cu in the worm Enchytraeus crypticus (Oligochaeta: Enchytraeidae) was assessed following exposure to (1) Cu-salt: freshly spiked soil with copper-nitrate, (2) Cu-NPs: freshly spiked soil with Cu nanoparticles (80 nm), and (3) Cu-field: historically Cu contaminated soil (80 years ago). Our main aims were to compare the three different exposure regimes and respective toxicity, and to determine how the oxidation state of the Cu and dissolution state of the particles differed. Characterization of in situ -exposure included identification of oxidation states with synchrotron generated X-ray absorption near-edge spectroscopy (XANES) analysis, activity of free Cu 2+ in soil-solution (Ion Selective Electrode), and the relative distribution of the labile Cu-fractions (Sequential Extraction). Freshly spiked Cu-salt was the most toxic for reproductive output of the worms, followed by Cu-NPs and then Cu-field. XANES indicated only one oxidation state (II) in Cu-salt and Cu-field soil, whereas in Cu-NPs soil it was present in all oxidation states (0, I and II). The partial oxidation of the Cu-NPs (in soil) was evident and with limited dissolution. [ABSTRACT FROM AUTHOR]

Abstract: Cellular energy allocation (CEA) is a measure of the energy status of an organism. The effects of Cd and Zn (reproduction EC50s and EC90s) on the total energy budget of Enchytraeus albidus (Oligochaeta) were assessed through CEA determination, over periods of time from 0 to 8d. Results showed reduction on the energy reserves for both metals after 2d exposure. Lipids were the first reserves to be used and carbohydrates were reduced exclusively after Cd exposure. Electron transport system (ETS) activities were enhanced, suggesting increased metabolism and higher energy requirements for metal detoxification. This was supported by previous results at transcription level, where an up-regulation of genes involved in the mitochondrial oxidative phosphorylation was verified. Additionally, the reduction of CEA may be related with the decrease on the reproductive output. These results showed the relevance of integrating various endpoints, which enabled an overview of various processes and to unravel mechanisms of action of chemicals. [Copyright &y& Elsevier]