Applicability of nanomaterial-specific guidelines within long-term Daphnia magna toxicity assays: A case study on multigenerational effects of nTiO2 and nCeO2 exposure in the presence of artificial daylight.

In recent years, various ecotoxicological test guidelines and (technical) guidance documents have been evaluated and updated with regard to their applicability to nanomaterials (NMs). Several of these have currently reached official regulatory status. Ensuring their harmonized implementation with previously recognized methods for ecotoxicity testing of chemicals is a crucial next step towards effective and efficient regulation of NMs. In the present study, we evaluated the feasibility of assessing multigenerational effects in the first generation of offspring derived from exposed Daphnia magna whilst maintaining test conditions in accordance with regulatory test guidelines and guidance documents for NMs. To do so, we integrated the recommendations for ecotoxicological testing of NMs as defined in OECD Guidance Document 317 into an extended long-term D. magna reproduction test method (OECD Test Guideline 211) and assessed effects of two poorly soluble NMs (nTiO2 and nCeO2). Our results show adverse effects on life-history parameters of D. magna exposed to the selected nanomaterials within the range of reported environmental concentrations. We argue that conforming to OECD test guidelines and accompanying guidance for nanomaterials is feasible when performing D. magna reproduction tests and can minimize unnecessary duplication of similar experiments, even when extensions to the standardized test setup are added.

Correlation analysis of single- and multigenerational endpoints in Daphnia magna toxicity tests: A case-study using TiO2 nanoparticles.

Multigenerational toxicity tests provide more sensitive measures of population-level effects than conventional single-generation tests. Particularly for stressors which exhibit slow uptake rates (e.g. nanomaterials), multigenerational tests may also provide a more realistic representation of natural exposure scenarios. To date, the inherently high costs and labor intensity have however limited the use of multigenerational toxicity tests and thereby their incorporation in environmental risk assessment. The aim of the present study was therefore to determine to what extent short(er) term endpoints which are conventionally measured in Daphnia magna toxicity tests hold predictive capacity towards reproduction measured over longer timescales, including multiple generations. To assess this, a case-study was performed in which effects of TiO2 nanoparticles (0, 0.02, 0.2, 2 and 5 mg L-1) on D. magna life-history traits were assessed over five generations. Additionally, it was determined whether offspring derived from exposed parents exhibited sustained adverse effects when rearing them in clean (non-exposed) media after each generation of exposure. The present study showed that although various life-history traits correlate with the total reproductive output in the same- and subsequent generation under non-exposed conditions, these correlations were decoupled in presence of exposure to nTiO2. In addition, it was found that nTiO2 can induce adverse effects on population relevant endpoints at concentrations 1-2 orders of magnitude lower than previously found (i.e. 0.02 mg L-1), and close to the range of concentrations occurring in natural freshwater ecosystems.

Chronic toxicity of core-shell SiC/TiO2 (nano)-particles to Daphnia magna under environmentally relevant food rations in the presence of humic acid.

To date, research on the toxicity and potential environmental impacts of nanomaterials has predominantly focused on relatively simple and single-component materials, whilst more complex nanomaterials are currently entering commercial stages. The current study aimed to assess the long-term and size-dependent (60 and 500 nm) toxicity of a novel core-shell nanostructure consisting of a SiC core and TiO2 shell (SiC/TiO2, 5, 25, and 50 mg L-1) to the common model organism Daphnia magna. These novel core-shell nanostructures can be categorized as advanced materials. Experiments were conducted under environmentally realistic feeding rations and in the presence of a range of concentrations of humic acid (0.5, 2, 5, and 10 mg L-1 TOC). The findings show that although effect concentrations of SiC/TiO2 were several orders of magnitude lower than the current reported environmental concentrations of more abundantly used nanomaterials, humic acid can exacerbate the toxicity of SiC/TiO2 by reducing aggregation and sedimentation rates. The EC50 values (mean ± standard error) based on nominal SiC/TiO2 concentrations for the 60 nm particles were 28.0 ± 11.5 mg L-1 (TOC 0.5 mg L-1), 21.1 ± 3.7 mg L-1 (TOC 2 mg L-1), 18.3 ± 5.4 mg L-1 (TOC 5 mg L-1), and 17.8 ± 2.4 mg L-1 (TOC 10 mg L-1). For the 500 nm particles, the EC50 values were 34.9 ± 16.5 mg L-1 (TOC 0.5 mg L-1), 24.8 ± 5.6 mg L-1 (TOC 2 mg L-1), 28.0 ± 10.0 mg L-1 (TOC 5 mg L-1), and 23.2 ± 4.1 mg L-1 (TOC 10 mg L-1). We argue that fate-driven phenomena are often neglected in effect assessments, whilst environmental factors such as the presence of humic acid may significantly influence the toxicity of nanomaterials.

Accumulation kinetics of polystyrene nano- and microplastics in the waterflea Daphnia magna and trophic transfer to the mysid Limnomysis benedeni.

Despite the pervasive presence of nano- and microplastics (NMPs) in aquatic environments, their movement through food chains remains poorly understood. In this study, we explored the uptake of polystyrene plastics (PSPs) of varying sizes (26, 500, and 4800 nm) in Daphnia magna and their subsequent transfer to the freshwater mysid Limnomysis benedeni, shedding light on the intricate dynamics of NMP transfer in freshwater ecosystems. Our results show that in D. magna the internal concentration of 4800 nm PSPs was 4-10 times higher than that of 26 and 500 nm PSPs, respectively. The uptake rate constants in daphnids decreased in the following order: 4800 nm (2.4 ± 0.5 L/g·h) > 26 nm (1.7 ± 0.4 L/g·h) > 500 nm (0.6 ± 0.1 L/g·h) PSPs. Importantly, only a small fraction (1-5 %) of the PSPs ingested by D. magna was transferred to L. benedeni. Additionally, larger particle sizes were associated with a higher extent of transfer in the food chain. Elimination rate constants in L. benedeni were found to be 0.03 ± 0.03, 0.1 ± 0.2, and 0.2 ± 0.8 per hour for 26, 500, and 4800 nm PSPs, respectively. Fluorescence observations revealed that PSPs were predominantly located in the stomach and intestine of L. benedeni. Furthermore, the calculated trophic transfer factor, based on the mass of particles accumulated in the organisms, was <1 for all PSP treatments. Our results indicate that NMPs can be transferred along the daphnia-mysids food chain, and that there is no evidence of biomagnification along this chain. These findings underscore the importance of understanding particle size effects on NMP transfer and accumulation in aquatic food webs, offering valuable insights for assessing the ecological risks associated with NMP pollution in freshwater ecosystems.

The biodistribution of polystyrene nanoparticles administered intravenously in the chicken embryo.

Nanoplastics can cause severe malformations in chicken embryos. To improve our understanding of the toxicity of nanoplastics to embryos, we have studied their biodistribution in living chicken embryos. We injected the embryos in the vitelline vein at stages 18-19. We injected polystyrene nanoparticles (PS-NPs) tagged with europium- or fluorescence. Their biodistribution was tracked using inductively-coupled plasma mass spectrometry on tissue lysates, paraffin histology, and vibratome sections analysed by machine learning algorithms. PS-NPs were found at high levels in the heart, liver and kidneys. Furthermore, PS-NPs crossed the endocardium of the heart at sites of epithelial-mesenchymal transformation; they also crossed the liver endothelium. Finally, we detected PS-NPs in the allantoic fluid, consistent with their being excreted by the kidneys. Our study shows the power of the chicken embryo model for analysing the biodistribution of nanoplastics in embryos. Such experiments are difficult or impossible in mammalian embryos. These findings are a major advance in our understanding of the biodistribution and tissue-specific accumulation of PS-NPs in developing animals.

Zooplankton community turnover in response to a novel TiO2-coated nano-formulation of carbendazim and its constituents.

Novel nanomaterial-based pesticide formulations are increasingly perceived as promising aids in the transition to more efficient agricultural production systems. The current understanding of potential unintended (eco)toxicological impacts of nano-formulated pesticides is scarce, in particular with regard to (non-target) aquatic organisms and ecosystems. The present study reports the results of a long-term freshwater mesocosm experiment which assessed responses of individual zooplankton taxa and communities to a novel TiO2-coated nano-formulation of the fungicide carbendazim. Population- and community trends were assessed and compared in response to the nano-formulation and its constituents applied individually (i.e. nano-sized TiO2, carbendazim) and in combination (i.e. nano-sized TiO2 & carbendazim). Minimal differences were observed between effects induced by the nano-formulation and its active ingredient (i.e. carbendazim) when applied at equivalent nominal test concentrations (4 µg L-1). Nano-sized TiO2 was found to affect zooplankton community trends when applied separately at environmentally realistic concentrations (20 µg L-1 nominal test concentration). However, when nano-sized TiO2 was applied in combination with carbendazim, nano-sized TiO2 was found not to alter effects on community trends induced by carbendazim. The findings of the current study provide an extensive and timely addition to the current body of work available on non-target impacts of nano-formulated pesticides.

Impacts of a novel controlled-release TiO2-coated (nano-) formulation of carbendazim and its constituents on freshwater macroinvertebrate communities.

Recently, the delivery of pesticides through novel controlled-release (nano-)formulations has been proposed intending to reduce (incidental) pesticide translocation to non-target sites. Concerns have however been raised with regards to the potentially enhanced toxicity of controlled-release (nano-)formulations to non-target organisms and ecosystems. We evaluated long-term (i.e. 1 and 3 month-) impacts of a novel controlled-release pesticide formulation (nano-TiO2-coated carbendazim) and its individual and combined constituents (i.e. nano-sized TiO2 and carbendazim) on naturally established freshwater macroinvertebrate communities. In doing so, we simultaneously assessed impacts of nano-sized TiO2 (nTiO2), currently one of the most used and emitted engineered nanomaterials world-wide. We determined ecological impacts on diversity (i.e. ß-diversity), structure (i.e. rank abundance parameters), and functional composition (i.e. feeding guilds & trophic groups) of communities and underlying effects at lower organizational levels (i.e. population dynamics of individual taxa). Freshwater macroinvertebrate communities were negligibly impacted by nTiO2 at environmentally realistic concentrations. The controlled-release (nano-)formulation significantly delayed release of carbendazim to the water column. Nevertheless, conventional- (i.e. un-coated-) and nTiO2-coated carbendazim induced a similar set of adverse impacts at all investigated levels of ecological organization and time points. Our findings show fundamental restructuring of the taxonomic- and functional composition of macroinvertebrate communities as a result of low-level pesticide exposure, and thereby highlight the need for mitigating measures to reduce pesticide-induced stress on freshwater ecosystems.

Postregistration monitoring of pesticides is urgently required to protect ecosystems.

Current admission policies for pesticides follow a controlled experimental tiered risk assessment approach, giving results that are difficult to extrapolate to a real-world situation. Later analyses of compounds such as DDT and neonicotinoid pesticides clearly show that the actual chemical impacts frequently affect many more components of an ecosystem than a priori suggested by risk assessment. Therefore, to manage the actual risks for ecosystems imposed by manufactured compounds, it is proposed that current admission policies for chemicals be enriched by using postregistration monitoring. Such monitoring is essential to identify unexpected direct and indirect impacts on organisms by accounting for multiple propagation routes and exposures. Implementation of postregistration monitoring could build on existing monitoring networks. This approach would tackle the current policy impasse of compartment-based regulations versus exposure-based regulations, and, more importantly, would provide a safety lock for risk assessment across compartments and more likely ensure the protection of our natural environment. Environ Toxicol Chem 2017;36:860-865. © 2017 SETAC.