No effect of selected engineered nanomaterials on reproduction and survival of the springtail Folsomia candida

Although the number of studies on engineered nanomaterial (NM) toxicity to soil invertebrates is increasing, only a few studies have reported toxicity of NMs to soil dwelling model species, such as the invertebrate Folsomia candida. The main objective of this study was to determine the toxicity of five different engineered NMs (WCCo, CuO, Fe2O3, organic pigment and MWCNTs) for the springtail F. candida. Copper(ii), cobalt and iron chlorides were taken as positive controls. A standardized OECD test was used to measure effects on reproduction and survival, and toxicity was related to metal concentrations in soil and pore water. None of the NMs exerted adverse effects on springtail reproduction and survival at concentrations up to 6400 mg per kg dry soil, whereas the Cu, Co and Fe chlorides resulted in 50% decline in springtail reproduction at 981, 469 and 569 mg metal ion per kg dry soil, respectively. The absence of toxicity of the NMs could partly be explained by the low porewater metal concentrations, suggesting low solubility or slow solubilisation. The fate of engineered NMs in soil is rather complex but needs better understanding to facilitate predicting exposure of soil organisms

Phenotypic and transcriptional responses associated with multi-generation exposure of Folsomia candida to engineered nanomaterials

Sublethal effects of toxicants may cumulate over time and become apparent only when test organisms are exposed for multiple generations. In this study we determined phenotypic effects and transcriptional responses in the parthenogenetic soil invertebrate Folsomia candida over four generations, followed by two generations of recovery. Animals were exposed to two metal-based nanomaterials (NMs): copper oxide (CuO) and tungsten carbide–cobalt (WCCo), both homogenously mixed in with the soil. Survival and reproduction were not affected in any of four consecutive generations of F. candida exposed to CuO-NM at concentrations as high as 6400 mg Cu per kg dry LUFA 2.2 soil. WCCo-NM affected reproduction and survival from the third generation onwards, with EC50 values between 2400 and 5600 mg NM per kg dry soil, but recovery was seen in recovery generations 1 and 2 when kept in clean soil. Histological investigations showed that WCCo-NM (3200 mg kg−1) induced tissue damage and loss of villi from the gut epithelial cells. Expression of four target genes known to be responsive to stress were investigated by quantitative PCR at different exposure levels and in different generations. Expression of all genes was significantly affected by NMs even though exposures were below toxic threshold concentrations. In addition, gene expression did not always return to control levels during consecutive recovery over two generations in clean soil. This shows that gene expression assays can detect physiological alterations cumulating from one generation to the next initially without visible effects on phenotypic variables such as reproduction. The possibility of multi-generation carry-over of sublethal toxicity needs more attention in environmental risk assessment

DNA typing of birch:Development of a forensic STR system for Betula pendula and Betula pubescens

Although botanical trace evidence is often encountered in case investigations, the utilization of such traces in forensic investigations is still limited. Development of a forensic STR system for the two species of Betula (birch) indigenous to and abundant in North West Europe is a step in enhancing the applicability of traces from these species. We describe six microsatellite markers developed for birch species in detail, including repeat structure, and we propose a nomenclature for the encountered alleles. To assess the population characteristics, the genetic composition of wild, planted and intermediate populations of Betula pendula (a diploid species) and Betula pubescens (a tetraploid species) were investigated. The genetic differences between these two species were larger than the differences between populations of one species, even when both species co-occurred at one location. Therefore allele frequencies were estimated for both species separately. General, conservative random match probabilities were estimated for wild trees based on these allele frequencies (5∙10−6 for the diploid B. pendula and 1∙10−13 for the tetraploid B. pubescens), illustrating the potential relevance if trace evidence secured from a suspect is found to match a birch tree growing on or near a crime scene. Apart from wild trees, planted Betula trees also occur that may not originate from seeds, but may have been propagated through cloning. Based on the studied Betula trees, the random match probability of a potentially planted profile might be as high as 1.4∙10−2

Exploring DNA methylation patterns in copper exposed Folsomia candida and Enchytraeus crypticus

Accumulating evidence shows that epigenetics-mediated phenotypic plasticity plays a role in an organism's ability to deal with environmental stress. However, to date, the role of epigenetic modifications in response to stress is hardly investigated in soil invertebrates. The main objective of this proof of principle study was to explore whether total cytosine and locus-specific CpG methylation are present in two important ecotoxicological model organisms, the springtail Folsomia candida and the potworm Enchytraeus crypticus, and if so, whether methylation patterns might change with increased toxicant exposure. LC–MS/MS analyses and bisulfite sequencing were performed to identify the CpG methylation state of the organisms. We show here, for the first time, a total level of 1.4% 5-methyl cytosine methylation in the genome of E. crypticus, and an absence of both total cytosine and locus-specific CpG methylation in F. candida. In E. crypticus, methylation of CpG sites was observed in the coding sequence (CDS) of the housekeeping gene Elongation Factor 1α while the CDS of the stress inducible Heat Shock Protein 70 gene almost lacked methylation. This confirms previous observations that DNA methylation differs between housekeeping and stress-inducible genes in invertebrates. DNA methylation patterns in E. crypticus were not affected by exposure to copper (II) sulfate pentahydrate (CuSO 4 ·5H 2 O) mixed in with LUFA 2.2 soil at sublethal effect concentrations that decreased reproduction by 10%, 20% and 50%. Although, differences in CpG methylation patterns between specific loci suggest a functional role for DNA methylation in E. crypticus, genome-wide bisulfite sequencing is needed to verify whether environmental stress affects this epigenetic hallmark

Exploring DNA methylation patterns in copper exposed Folsomia candida and Enchytraeus crypticus

Accumulating evidence shows that epigenetics-mediated phenotypic plasticity plays a role in an organism's ability to deal with environmental stress. However, to date, the role of epigenetic modifications in response to stress is hardly investigated in soil invertebrates. The main objective of this proof of principle study was to explore whether total cytosine and locus-specific CpG methylation are present in two important ecotoxicological model organisms, the springtail Folsomia candida and the potworm Enchytraeus crypticus, and if so, whether methylation patterns might change with increased toxicant exposure. LC–MS/MS analyses and bisulfite sequencing were performed to identify the CpG methylation state of the organisms. We show here, for the first time, a total level of 1.4% 5-methyl cytosine methylation in the genome of E. crypticus, and an absence of both total cytosine and locus-specific CpG methylation in F. candida. In E. crypticus, methylation of CpG sites was observed in the coding sequence (CDS) of the housekeeping gene Elongation Factor 1α while the CDS of the stress inducible Heat Shock Protein 70 gene almost lacked methylation. This confirms previous observations that DNA methylation differs between housekeeping and stress-inducible genes in invertebrates. DNA methylation patterns in E. crypticus were not affected by exposure to copper (II) sulfate pentahydrate (CuSO 4 ·5H 2 O) mixed in with LUFA 2.2 soil at sublethal effect concentrations that decreased reproduction by 10%, 20% and 50%. Although, differences in CpG methylation patterns between specific loci suggest a functional role for DNA methylation in E. crypticus, genome-wide bisulfite sequencing is needed to verify whether environmental stress affects this epigenetic hallmark