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.

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.

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.

Selection of an optimal culture medium and the most responsive viability assay to assess AgNPs toxicity with primary cultures of Eisenia fetida coelomocytes.

Earthworm immune cells (coelomocytes) have become a target system in ecotoxicology due to their sensitivity against a wide range of pollutants, including silver nanoparticles (AgNPs). Presently, in vitro approaches (viability assays in microplate, flow cytometry, cell sorting) with primary cultures of Eisenia fetida coelomocytes have been successfully used to test the toxicity and the dissimilar response of cell subpopulations (amoebocytes and eleocytes) after PVP-PEI coated AgNPs and AgNO3 exposures. In order to obtain reliable data and to accurately assess toxicity with coelomocytes, first an optimal culture medium and the most responsive assay were determined. AgNPs posed a gradual decrease in coelomocytes viability, establishing the LC50 value in RPMI-1640 medium at 6 mg/l and discarding that the observed cytotoxicity was attributable to its coating agent PVP-PEI. Exposure to AgNPs caused selective cytotoxicity in amoebocytes, which correlated with the Ag concentrations measured in sorted amoebocytes and reinforced the idea of dissimilar sensitivities among amoebocytes and eleocytes. Silver nano and ionic forms exerted similar toxicity in coelomocytes. The in vitro approaches with coelomocytes of E. fetida performed in this study have the capacity to predict impairments caused by pollutants at longer exposure levels and thus, provide rapid and valuable information for eco(nano)toxicology.

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.

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.

Parametrization of nanoparticles: development of full-particle nanodescriptors.

While metal oxide nanoparticles (NPs) are one of the most commonly used nanomaterials, the theoretical models used to analyze and predict their behavior have been mostly based on just the chemical composition or the extrapolation from small metal oxide clusters' calculations. In this study, a set of novel, theoretical full-particle descriptors for modeling, grouping or read-across of metal oxide NP properties and biological activity was developed based on the force-field calculation of the potential energies of whole NPs. The capability of these nanodescriptors to group the nanomaterials acoording to their biological activity was demonstrated by Principal Component Analysis (PCA). The grouping provided by the PCA approach was found to be in good accordance with the algal growth inhibition data of well characterized nanoparticles, synthesized and measured inside the consortia of the EU 7FP framework MODERN project.

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.

Interaction between density and Cu toxicity for Enchytraeus crypticus and Eisenia fetida reflecting field scenarios.

Effect assessment is usually based on responses obtained from standard tests, in which organisms are well fed and in an optimal population density. For a more thorough assessment of ecotoxicological risk, information is needed for chemical effects in systems that closer reflect the potential exposure in the field systems. Responses measured in standard density experiments do not fully reflect the field scenario, where populations' size fluctuate with environmental conditions, leading to very low organism number in certain season/conditions and high number in others. In the present study, the possible interaction between density and Cu-pollution was investigated in regard to population growth, using Enchytraeus crypticus, and for individual juvenile growth, using Eisenia fetida. The standardized ISO and OECD guidelines for enchytraeids and earthworms were adapted to test four densities and four Cu concentrations. The final population number was used to assess the effects and possible interaction between densities and Cu toxicity for population responses and the increase in individual organism wet weight was used as growth response. The study showed that although initial density itself had tremendous impact on population and individual growth, organisms under different densities had the same sensitivity to Cu.

The toxicity testing of double-walled nanotubes-contaminated food to Eisenia veneta earthworms.

Nanotechnology is playing an increasing role in the society, but very little is known about the environmental toxicity of nanotechnological products. Few previous studies have been published with regard to the effect of nanoparticles on soil organisms. The present paper deals with the toxicity of carbon double-walled nanotubes (DWNT) and C60 fullerenes to the soil-dwelling earthworm Eisenia veneta, with the aim of evaluating the lethal and sub-lethal toxicity. Reproduction of E. veneta was affected by DWNT administered through food at concentrations above 37 mg DWNT/kg food. The most sensitive toxicological parameter was reproduction (cocoon production), with no effect on hatchability, survival or mortality at up to 495 mg DWNT/kg and 1000 mg C60/kg.

Seasonal variation in heavy metal accumulation in subtropical population of the terrestrial isopod, Porcellio laevis.

The aim of the present study is to evaluate the seasonal fluctuation of heavy metals in the isopod Porcellio laevis at four uncontaminated subtropical locations. This study was carried out at four different field sites in Assiut, Egypt. The concentrations of cadmium, lead, copper, and zinc in animal, soil, and litter (mug/g dry weight) were monthly recorded during the period from June 2002 till May 2003. There was little difference in metal accumulation trends between the sites. In general, the isopod showed significant increased Pb and Zn concentration during summer and spring months, whereas this was not the case for Cd and Cu. The bioaccumulation (BAF) and bioconcentration factors (BCF) of the metals revealed marked seasonal changes throughout the year. Generally, BAF of metals were higher during summer and spring, and BCF were higher during summer and autumn. Comparing the metal accumulation with climatic fluctuations (measured) it was speculated that temperature was the main factor causing seasonal fluctuations of the internal metal concentration in the isopod.

Effects of eight polycyclic aromatic compounds on the survival and reproduction of the springtail Folsomia fimetaria L. (Collembola, isotomidae).

The effects of eight polycyclic aromatic compounds on the survival and reproduction of the collembolan Folsomia fimetaria L. were investigated in a well-characterized Danish agricultural soil. With the exception of acridine, polycyclic aromatic hydrocarbons (PAHs) and neutral N-, S-, and O-monosubstituted analogues showed similar toxicities to soil collembolans when the results were expressed in relation to total soil concentrations (mg/kg). The estimated concentrations resulting in a 10% reduction of reproductive output (EC10 values) were based on measured initial concentrations and were for acridine 290 mg/kg, carbazole 10 mg/kg, dibenzofuran 19 mg/kg, dibenzothiophene 7.8 mg/kg, fluoranthene 37 mg/kg, fluorene 7.7 mg/kg, phenantrene 23 mg/kg, and pyrene 10 mg/kg. When the EC10 values were converted to soil pore-water concentrations, they showed a highly significant correlation (r2 = 0.71, p < 0.01) to no-observed-effect concentrations for the freshwater crustacean Daphnia magna, as estimated by a quantitative structure activity relation (QSAR) for baseline toxicity (nonpolar narcosis). Only carbazole and acridine were more than two times more toxic (4.9 and 3.1, respectively) than expected from the Daphnia QSAR data. The latter result indicates that the toxicity of the tested substances is close to that expected for compounds with nonpolar narcosis as the mode of action. However, the relatively large uncertainties in the extrapolation method prevent final conclusions from being drawn.

Biomarkers in earthworms.

Earthworms are believed to be so-called key species within ecosystems and are often exposed to a wide range of anthropogenic compounds released to the terrestrial environment. As a consequence, they may suffer from the toxicity of these compounds. For these and other reasons, earthworms have been used extensively in ecotoxicological studies. In recent years the use of other biological responses (biomarkers) to estimate either exposure or resultant effects of chemicals has received increased attention. Biomarkers address the question of bioavailability by only responding to the bioactive fraction. They may incorporate effects following exposure to a mixture of chemicals. Biomarkers may also reduce extrapolation of results from the laboratory to the field, as they may be applicable under both conditions. The present review has drawn together current knowledge on potential biomarkers in earthworms and appraised them in relation to basic requirements needed for supplying information relevant to devising satisfactory risk assessment. A wide range of potential biomarkers have been measured in earthworms, including DNA alteration, induction of metal-binding proteins (MTs and MBP), depression of ChE activity and other enzymatic responses, energy reserve responses, responses in neural impulse conductivity, lysosomal membrane stability, immunological responses, changes in sperm numbers, histopathological changes, and behavioral changes. Both organic and inorganic compounds have been included; however, for each biomarker the main emphasis historically has been placed on only a few chemicals. Dose-response relationships were in some cases observed. Little information is available on the linkage of the biomarker response to effects at population or community levels. The influence of other factors, biotic and abiotic, on the biomarker responses and their temporal duration have been only sporadically reported.

Toxicity of nickel to a soil-dwelling springtail, Folsomia fimetaria (Collembola: Isotomidae).

Exposure of the collembolan Folsomia fimetaria L. to nickel via soil caused significant mortality and reduced growth and reproductive output. Nickel may be present in elevated concentrations due to anthropogenic discharge. Although collembolans are very numerous and important organisms in the soil ecosystem, the effect of nickel has not previously been studied on these organisms. The aim of this study was to investigate the toxic effects of high soil nickel concentrations on the collembolan F. fimetaria following a 3-week exposure in a loamy sand spiked with nickel up to 1000 mg Ni/kg. A 10% decrease in adult female numbers at 427 mg Ni/kg and at 645 mg Ni/kg for adult male numbers was observed for nickel-spiked soil. Juvenile numbers were reduced at 701 mg Ni/kg following a 3-week exposure. The corresponding EC50 values were 786 mg Ni/kg for females, 922 mg Ni/kg for males, and 859 mg Ni/kg for juveniles. The reproductive output seems to be the most sensitive parameter being reduced at soil nickel concentrations above 173 mg Ni/kg (EC10). Adult growth was not affected by soil nickel concentrations up to 1000 mg Ni/kg, but juvenile growth was reduced at concentrations above 480 mg Ni/kg (EC10).