On the importance of longer-term exposure to stressors - A critical review and proposal for multigenerational testing in standard soil invertebrates.

Standard laboratory tests to describe the impact of stressors (most notably: chemicals) on organisms offer a good compromise between feasibility and outcome, i.e., they should be reproducible and provide robust results. However, these tests may underestimate the potential effects of prolonged exposures, particularly for persistent contaminants. Within the last years, we have observed an increase in studies aiming to target prolonged exposure, e.g., via an extended test duration or by multigenerational (MG) exposure. Seemingly, both reduced and increased impacts have been observed in these studies, but it is also clear that no unique test setup was used, and test designs vary widely among studies. To better describe long term effects, MG is a highly relevant aspect which deserves more consideration at various testing and assessment levels. Therefore, we conducted a literature review focusing on available studies performed with soil invertebrates, exposed to stressors for periods longer than in standard laboratory tests, i.e., full life cycle tests, as well as extensions to standard and MG tests. So far, it has been recommended that such studies should cover more than one generation, but this statement is probably too vague. In this contribution, we summarize and critically discuss the information provided in the literature, and we provide suggestions for future research. The currently available test results from long-term studies have produced clear evidence to recommend the implementation of long-term tests in existing regulatory testing requirements (e.g., for pesticides), in particular for persistent substances and also for delayed effects. Consequently, we recommend the inclusion of such longer exposure test designs (e.g., as annexes) in current OECD and ISO guidelines. However, when doing so, the long-term test designs proposed so far have to be critically adapted for a selected set of representative soil invertebrate test species.

On virus and nanomaterials - Lessons learned from the innate immune system - ACE activation in the invertebrate model Enchytraeus crypticus.

Current human research on COVID-19 - SARS-CoV-2 (Severe Acute Respiratory Syndrome-Corona Virus) showed that ACE2 (Angiotensin Converting Enzyme 2) is a functional receptor to which the spike proteins attach. Invertebrates have been exposed to a wide array of threats for millennia and their immune system has evolved to deal with these efficiently. The annelid Enchytraeus crypticus, a standard ecotoxicological species, is an invertebrate species where extensive mechanisms of response studies are available, covering all levels from gene to population responses. Nanomaterials (NMs) are often perceived as invaders (e.g. virus) and can enter the cell covered by a corona, triggering similar responses. We created a database on E. crypticus ACE gene expression, aiming to analyse the potential knowledge transfer between invertebrates and vertebrates. Total exposure experiments sum 87 stress conditions for 18 different nanomaterials (NMs). ACE expression following TiO2 NM exposure was clearly different from other NMs showing a clear (6-7 fold) ACE down-regulation, not observed for any other NMs. Other NMs, notably Ag NMs, and to some extent Cu NMs, caused ACE up-regulation (up to 4 fold). The extensive knowledge from response to NMs can support the immuno-research community, especially to develop therapies for virus that trigger the innate immune system.

Full life cycle test with Eisenia fetida - copper oxide NM toxicity assessment.

Copper oxide nanomaterials (CuONM) are widely used, e.g. as antimicrobial coatings, wood preservatives,fertilizers, etc. Life cycle aspects of Copper Nanomaterials (CuONM) toxicity have been scarcely studied in earthworms, as the focus has been on standard survival and reproduction toxicity tests. Standard tests do not allow for an understanding of which life cycle stage is the most sensitive, and how this can be impacted by CuONMs toxicity to cause longer term population level effects. Since CuONM may release free Cu ions (Cu2+) it is relevant to compare CuONM toxicity with CuCl2 salt. Therefore, the aim of the present study was to assess the effects of CuONM and CuCl2 throughout the different stages of the full life cycle (FLC) of Eisenia fetida while comparing it with the OECD standard test. Additional endpoints included juvenile survival, growth, maturation, besides survival and reproduction. The FLC test showed that e.g. juvenile survival was especially susceptible during the first 28 days post-hatching, neither juvenile growth nor time to maturity were related to exposure concentration. Both CuONM and CuCl2 caused a concentration-dependent decrease in population growth, while a hormesis effect was observed at low concentrations of CuCl2. A reduction in instantaneous growth rate was observed at higher concentrations. The FLC test and the OECD test had different exposure history therefore the ECx values are not comparable across the test systems. Hence, the FLC test enabled the detection of the most vulnerable developmental stages and elucidate different life stage sensitivities between the two Cu exposures.

Nano-pesticides: the lunch-box principle-deadly goodies (semio-chemical functionalised nanoparticles that deliver pesticide only to target species).

Nature contains many examples of "fake promises" to attract "prey", e.g., predatory spiders that emit the same sex-attractant-signals as moths to catch them at close range and male spiders that make empty silk-wrapped gifts in order to mate with a female. Nano-pesticides should ideally mimic nature by luring a target and killing it without harming other organisms/species. Here, we present such an approach, called the lunch-box or deadly-goodies approach. The lunch-box consists of three main elements (1) the lure (semio-chemicals anchored on the box), (2) the box (palatable nano-carrier), and (3) the kill (advanced targeted pesticide). To implement this approach, one needs to draw on the vast amount of chemical ecological knowledge available, combine this with recent nanomaterial techniques, and use novel advanced pesticides. Precision nano-pesticides can increase crop protection and food production whilst lowering environmental impacts.

Impact of chromium on the soil invertebrate model Enchytraeus crypticus (Oligochaeta) in standard reproduction and full life cycle tests.

Chromium (Cr) has many applications in industry and day-to-day life, entering the terrestrial environment mostly from anthropogenic sources. Despite the fact that Cr is essential, it can be harmful in biota at high concentrations. Full life cycle tests (FLCt) are scarce, representing a gap for soil ecotoxicology. Hence, the effects of Cr were studied using the soil organism Enchytraeus crypticus in the 46-day FLCt and the standard 21-day enchytraeid reproduction test (ERT). FLCt hatching (day-11) and time to reach maturity (day 22-25) were the most sensitive endpoints, representing a delay, partly recovered with time. Reproduction was reduced to similar levels in both the ERT and the FLCt (EC50∼320 mg Cr/kg), but survival was more affected in the ERT (LC50 = 377 mg Cr/kg) than in the FLC (LC50 = 467 mg Cr/kg). This could be due to the induction of stress response mechanisms at earlier life stages (cocoons/juveniles), providing protection to toxicity in a later stage (adults). FLCt results provided considerably more details and data points, and the additional endpoints allowed to explain the source of observed effects, e.g. a direct impact on fecundity and not only due to adult mortality. The estimated population growth curves confirmed the significant effect of 320 mg Cr/kg and onwards, reflecting the impact of Cr on both cocoon production and juvenile performance, with follow-up consequences for reproduction. An Adverse Outcome Pathway was drafted. The FLCt design is recommended as an upgrade of the current standard ERT, which could be integrated into the existing OECD ERT test guideline.

Assessing the toxicity of safer by design CuO surface-modifications using terrestrial multispecies assays.

Safer by design (SBD) modifications of nanomaterials (NMs) have been pursued, aiming to maintain functionality and yet reduce hazard and support sustainable nanotechnology. The present case study involves copper oxide nanomaterials (CuO NMs) used in paint that have been surface modified by a SBD approach to particles coated with citrate (CIT-), ascorbate (ASC-), polyethylenimine (PEI+), and polyvinylpyrrolidone (PVP). We assessed the effect of the 4 different surface modified (CIT, ASC, PVP and PEI) NMs plus the pristine non-coated (PRI NM) and a Cu salt (CuCl2), using the soil multispecies test system (samples at 28-56-84 days). Further, the species were tested individually, and Cu was measured in the test media (soil and soil solution) and organisms. There was a potential relationship with zeta potential, and toxicity of CuO NMs was as follows: -PEI (+28 mV) caused the least impact, -ASC and -CIT (-17 mV, -18 mV) the most, while PVP and PRI (-8 mV, -9 mV) caused an intermediate response. Differences were not explained by the contribution of soluble Cu. Coating interfered with the release of Cu2+ and/or the activation of copper regulators and detoxification mechanisms in the organisms, i.e. time to reach some kind of stability in organisms' uptake was shorter for -ASC and longer for -PVP during prolonged time. Thus, one of the main findings is that NMs hazard assessment requires long term testing to understand predicted effects across materials. Further, the coverage using a multispecies approach offers increased relevance and a more ecosystem qualified response.

Novel egg life-stage test with Folsomia candida - A case study with Cadmium (Cd).

Toxicity of pollutants is known to have a different impact depending on the organisms' life stage. Standard tests are often based on one life stage, i.e. effects could be underestimated. We aimed here to develop and optimize a test system using eggs of Folsomia candida (4-5 days) instead of the juveniles (10-12 days old) required by the OECD standard test guideline No. 232 (2009). Accordingly, the exposure time and thus the test duration was extended. Tests with "standard" juveniles (10-12 days old) and, adults (21 and 28 days old) were also performed. Cadmium (Cd) was used as test substance. The extension to the test guideline starts as follows: 1) synchronization of eggs in a thin soil layer on plaster of Paris, 2) selection of viable eggs, 3) burying these eggs in groups of 5 in soil. Afterwards, the test procedure will follow the standard procedure as described in the OECD standard test. Cadmium caused ca. 50% effects on reproduction at 60 mg Cd/kg soil dry weight (DW) when exposing juveniles or adults. There was no significant impact of Cd on the eggs, the hatching process or the latter life stages until ca. 250 mg Cd/kg DW (Cd is stable during this exposure period). Hence, Cd seems to affect reproduction before egg laying, i.e., during egg formation or during juvenile-adult stages. In order to clarify whether other chemicals do act in a similar way testing with different chemicals is highly recommended. Testing of different life stages does provide insight on the mechanisms and effects of contaminants and offers important insight.

Mixture toxicity assessment of a biocidal product based on reproduction and avoidance behaviour of the collembolan Folsomia candida.

Biocidal products represent mixtures that might be released into the environment at application and continuously during service life. Concentration addition (CA) has been proposed as default model to calculate theoretical mixture toxicity. However, the suitability of CA for chronic toxicity towards soil organisms has so far rarely been evaluated and therefore needs further experimental evidence. The present study investigated the toxicity of a wood preservative product and the individual active substances (tebuconazole and IPBC) therein with the aim to evaluate the compliance with the CA prediction for the product. Folsomia candida was selected as test organism for this purpose using the endpoints reproduction and avoidance behaviour. Both endpoints were increasingly impacted by increasing concentrations of the wood preservative product as well as its active substances tested individually. The chronic effects of the product could be predicted by CA with less than 4-fold deviation, while the assessment for avoidance behaviour indicated a strong underestimation. This underestimation could not be attributed to the one known formulation additive, an organic solvent. Overall, the present study provides some more evidence that CA could be applied as default model for standard endpoints of soil organisms, but warns against using CA for behavioural responses.

Hazard assessment of the veterinary pharmaceuticals monensin and nicarbazin using a soil test battery.

Veterinary pharmaceuticals are widely used as food additives in the poultry industry, and the unknown consequences of releasing these compounds into the environment are of concern. The purpose of the present study was to determine the direct impact of 2 veterinary pharmaceuticals (nicarbazin and monensin), commonly used in the poultry industry, on nontarget invertebrates and plant species. Ecotoxicological tests were used to evaluate the acute and chronic toxicity in earthworms (Eisenia andrei), collembolans (Folsomia candida), and 2 plant species (Brassica rapa and Triticum aestivum). Chemical analytical measurements were in good agreement with the nominal concentrations used, although some variability was seen. The results obtained showed no effects of nicarbazin at the highest nominal tested concentration of 1000 mg a.i./kg soil dry weight on any of the organisms, whereas exposure to monensin caused a concentration-specific response pattern. Species sensitivity to monensin decreased in the following rank order: B. rapa > T. aestivum > E. andrei > F. candida, with measured median effect concentrations (based on soil exposure) ranging between approximately 10 and 120 mg/kg. Our results emphasize the importance of using a test battery when assessing ecotoxicological effects by using different ecophysiological endpoints and species from different trophic levels. Environ Toxicol Chem 2018;37:3145-3153. © 2018 SETAC.

Environmental fate and effect of biodegradable electro-spun scaffolds (biomaterial)-a case study.

Poly-ε-caprolactone (PCL) based medical devices are increasingly produced and thus, their presence in the environment is likely to increase. The present study analysed the biodegradation of PCL electro-spun scaffolds (alone) and PCL electro-spun scaffolds coated with human recombinant (hR) collagen and Bovine Achilles tendon (BAT) collagen in sewage sludge and in soil. Additionally, an eco-toxicological test with the model organism Enchytraeus crypticus was performed to assess environmental hazard of the produced materials in soils. The electro-spun scaffolds were exposed to activated sludge and three different soils for various time periods (0-7-14-21-28-56-180 days); subsequently the degradation was determined by weight loss and microscopical analysis. Although no toxicity occurred in terms of Enchytraeus crypticus reproduction, our data indicate that biodegradation was dependent on the coating of the material and exposure condition. Further, only partial PCL decomposition was possible in sewage treatment plants. Collectively, these data indicate that electro-spun PCL scaffolds are transferred to amended soils.

Multigenerational exposure of Folsomia candida to silver: Effect of different contamination scenarios (continuous versus pulsed and recovery).

Effects of pollutants are mostly assessed using standard testing procedures, which cover a fraction of the animals' life cycle. Although, in nature species are exposed during multiple generations of sub-lethal doses of persistent chemicals. In the present study, we focused on the multigenerational (MG) effects of silver in Folsomia candida during 6 generations using the EC50 for reproduction as exposure concentration. We tested 9 different exposure scenarios, going from continuous 6 generations Ag exposure over pulse exposure (i.e. one generation clean, next contaminated, next clean etc.) to gradually increasing the number of exposure generations, with a final transfer to clean media. The biological endpoints assessed included survival, reproduction and size, with reproduction being the most sensitive. The biological response depended on the specific MG scenario, e.g. the 6 Ag MG caused a decreased number of juveniles from F4, whereas the pulse exposure experienced an increase in reproductive output when in clean soil. It is uncertain whether Ag causes transgenerational effects, but the reproduction levels in both pulse exposures are lower than in continuous control over the 6 generations which could be due to transference of Ag by the maternal generation. Overall, population size distribution seemed to indicate a delay in time for egg laying, with close relationship between adult survival, organisms size and reproduction output. Size monitoring allowed significant added interpretation possibilities and we strongly recommend the addition of this endpoint to the standard guideline. The smaller observed size range can have implications in terms of adaptation potential, carrying associated increased risk.

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.

Effects of ivermectin on Danio rerio: a multiple endpoint approach: behaviour, weight and subcellular markers.

Ivermectin (IVM) is a broad acting antihelmintic used in various veterinary pharmaceuticals. It has been shown that IVM enters the aquatic compartment and adversely affects organisms including fish. This study is based on the hypothesis that long term exposure to IVM affects fish and thus, the main objective was to assess the chronic effects of 0.25 and 25 µg IVM/L to zebrafish using multiple endpoints representative of several levels of biological organization: weight, behaviour (swimming and feeding) and subcellular markers including biomarkers for oestrogenicity (vitellogenin-VTG), oxidative stress (catalase-CAT and glutathione-S-transferase-GST) and neurotransmission (cholinesterase-ChE). Concentrations as low as 0.25 µg IVM/L disrupted the swimming behaviour, causing fish to spend more time at the bottom of aquaria. Such reduction of the swimming performance affected the feeding ability which is likely responsible for the weight loss. The effects on weight were gender differentiated, being more pronounced in males (0.25 µg IVM/L) than in females (25 µg IVM/L). Fish exposed to 25 µg/L exhibited darker coloration and mild curvature of the spine. No effects on VTG and AChE were observed, but a reduction on CAT and GST levels was observed in fish exposed to 25 µg IVM/L, although these alterations probably only reflect the general condition of the fish which was significantly compromised at this concentration. Despite that predicted environmental concentrations of IVM are below 0.25 µg/L, the behavioural effects may be translated into important ecological impacts, e.g. at predator-prey interactions where fish competitive advantage can be decreased. Future work should address the link between behaviour disruption and population fitness. The current study was based on a one experiment and multiple endpoint (anchored) approach, allowing the results to be integrated and linked towards a mechanistic understanding.

Uptake and Elimination of 4-Nonylphenol in the Enchytraeid Enchytraeus albidus.

We determined the uptake and elimination kinetics of 4-nonylphenol (4-NP) in Enchytraeus albidus. A relatively fast degradation of 4-NP in test soil occurred at 20°C (λ = 0.11 day(-1)). The concentration of 4-NP in worm tissue followed a three-phase kinetics model, with a short phase of fast 4-NP accumulation shortly after exposure start (k u = 0.97 mg kg(-1) day(-1)), followed by partial elimination (K e1 = 1.47 day(-1)) until reaching the equilibrium phase (A = 44.7 mg kg(-1) fresh tissue), and finally the elimination upon transfer to uncontaminated soil (K e2 = 0.67 day(-1)). During uptake, the internal concentration was similar to the concentration found in the soil, with a BAF ~ 1. In un-spiked soil, elimination took place within the first 24 h (elimination t1/2 ~ 1 day).

Worms from the Arctic are better adapted to freezing and high salinity than worms from temperate regions: oxidative stress responses in Enchytraeus albidus.

Enchytraeus albidus is a freeze-tolerant enchytraeid found in diverse habitats, from supra-littoral to terrestrial, and spanning temperate to arctic regions. Thus, this worm is often exposed to sub-zero temperatures and fluctuating salinity regimes that can lead to physiological stress. We therefore studied the oxidative stress by measuring lipid peroxidation, anti-oxidant defenses and neurotransmission activity in E. albidus from arctic (Greenland) and temperate (Germany) regions during a short-term exposure to saline conditions (0, 15, 35 and 50‰ NaCl) and low temperatures (+2, -2 and -5 °C). Various enzymatic and non-enzymatic oxidative stress markers were analyzed. Results have shown that both salt and freezing caused oxidative stress in E. albidus, particularly from Germany, as confirmed by catalase, glutathione-S-transferase and superoxide dismutase activities and lipid peroxidation levels. Neurotransmission (as judged from acetylcholinesterase activity) was reduced by saline conditions at +2 °C, but stimulated at -2 and -5 °C. Worms from Greenland had relatively higher and more stable levels of antioxidants than worms from Germany, reflecting their higher tolerance of freezing and saline conditions.

Interaction between density and Cu toxicity for Enchytraeus crypticus--comparing first and second generation effects.

Density of organisms varies considerably in nature depending e.g. on seasonality or food availability. A recent investigation on interaction between Cu and density using Enchytraeus crypticus showed that density itself (5-50 per 20 gr dry soil) had an impact on population and individual growth [up to 3000 individuals per test vessel], but the interaction between density and Cu toxicity was not significant. Here, a follow-up study was performed, in which the interactions between density and Cu-exposure were investigated along a two-generation exposure using E. crypticus (three factorial: 1. density (5-50), 2. Cu (0-300 mg/kg) and generation (G1-G2)). After G1, the juveniles were retrieved and further exposed under the same conditions along a G2 (using a refined density set - 10 and 50). Results showed an interaction between density and Cu in the reproduction of E. crypticus, this being significant in G2, showing lower toxicity for higher density of organisms whereas in the 1st generation the opposite occurred. Hence, there was an interaction seen along G1 to G2, i.e. animals from density 50 in G1 when further exposed at density 50 in G2 had lower Cu toxicity compared to when further exposed at density 10. Possible explanations include the hydra effect (overcompensation in G2 due to stress in G1) or that for density 50 the organisms are exposed to less Cu than at lower densities, this by e.g. (1) organisms avoid exposure by lumping/clustering which would limit exposure to Cu and (2) there would be less available Cu contaminated soil per individual at high density hence less exposure.

Soil salinity increases survival of freezing in the enchytraeid Enchytraeus albidus.

Enchytraeus albidus is a freeze-tolerant enchytraeid found in diverse habitats, ranging from supralittoral to terrestrial and spanning temperate to arctic regions. Its freeze tolerance is well known but the effect of salinity in this strategy is still poorly understood. We therefore studied the combined effect of salinity (0, 15, 35, 50‰ NaCl) and sub-zero temperatures (-5, -14, -20°C) on the freeze tolerance of E. albidus collected from two distinct geographical regions (Greenland and Germany). A full factorial design was used to study survival, and physiological and biochemical end points. The effect of salinity on the reproduction of German E. albidus was also assessed. Exposure for 48 h to saline soils prior to cold exposure triggered an increase in osmolality and decrease in water content. Worms exposed to saline soils had an improved survival of freezing compared to worms frozen in non-saline soils, particularly at -20°C (survival more than doubled). Differential scanning calorimetry measurements showed that the fraction of water frozen at -5 and -14°C was lower in worms exposed to 35‰ NaCl than in control worms. The lowering of ice content by exposure to saline soils was probably the main explanation for the better freeze survival in saline-exposed worms. Glucose increased with decreasing temperature, but was lower in saline than in non-saline soils. Thus, glucose accumulation patterns did not explain differences in freeze survival. Overall, the physiological responses to freezing of E. albidus from Greenland and Germany were similar after exposure to saline soils. Soil salinity up to 30‰ improved reproduction by a factor of ca. 10.

Boric acid as reference substance: pros, cons and standardization.

Boric acid (BA) has been successfully used as reference substance in some standard test guidelines. Due to the fact that previously selected reference substances present a significant risk to human health and/or are banned for environmental reasons, BA is being discussed for broader adoption in OECD or ISO guidelines. To provide input on BA data and contribute to the discussion on its suitability as a reference substance, in the present study BA was tested with two standard soil organisms, Enchytraeus albidus and Folsomia candida, in terms of survival, reproduction and avoidance. Additionally, published data on other organisms was analysed to derive the most sensitive soil dwelling invertebrate (hazard concentration-HC5). Results showed that BA affected the tested organisms, being two times more toxic for collembolans (LC50 = 96; EC50 = 54 mg/kg) than for enchytraeids (LC50 = 325; EC50 = 104 mg/kg). No avoidance behaviour occurred despite the fact that BA affects earthworms. Actually, it is the recommended reference substance for the earthworm avoidance test. Clearly, the suitable performance of BA in one species should not be generalized to other species. Absolute toxicity is not an important criterion for the selection of a reference substance, but it has been proposed that effects should occur within a reasonable range, i.e. <1,000 mg/kg. We could confirm, compiling previous data that for most soil invertebrates, the EC50 is expected to be below 1,000 mg/kg. From these data it could be derived that the most sensitive soil dwelling invertebrate (HC5, 50%) is likely to be affected (EC10) at 28 (8-53) mg H(3)BO(3)/Kg, equivalent to 4.6 (1.4-8.7) mg boron/kg.

Assessing single and joint effects of chemicals on the survival and reproduction of Folsomia candida (Collembola) in soil.

Chemicals are often found in the environment as complex mixtures. There has been a large effort in the last decade to assess the combined effect of chemicals, using the conceptual models of Concentration Addition and Independent Action, but also including synergistic, antagonistic, dose-level and dose-ratio dependent deviations from these models. In the present study, single and mixture toxicity of atrazine, dimethoate, lindane, zinc and cadmium were studied in Folsomia candida, assessing survival and reproduction. Different response patterns were observed for the different endpoints and synergistic patterns were observed when pesticides were present. Compared with the previously tested Enchytraeus albidus and Porcellionides pruinosus, the mixture toxicity pattern for F. candida was species specific. The present study highlights the importance of studying toxicity of chemicals mixtures due to the observed potentiation of effects and confirms that for an adequate ecologically relevant risk assessment different organisms and endpoints should be included.