Realistic Exposure Scenarios Research Articles

Potentials and pitfalls of transient in vitro reporter bioassays: interference by vector geometry and cytotoxicity in recombinant zebrafish cell lines

The water framework directive re-evaluation proposes the integration of effect-based tools, increasing the need for alternative methods. Especially within aquatic toxicology, coverage of specific toxicity pathways is scarce, and most applications are based on mammalian or bacterial models, not reflecting realistic exposure scenarios. The use of transient reporter gene assays in cells from organisms of interest could be a quick and inexpensive solution. However, interference with cellular homeostasis may impact the system beyond the function of the manipulated gene and thus lead to non-specific results. We describe how varying vector geometry and different regulatory gene elements on plasmids used for transfection in zebrafish hepatocytes and embryonic fibroblasts may lead up to a tenfold difference in potency. Cells were transiently co-transfected with an Nrf2-responsive Firefly luciferase reporter plasmid and eight different Renilla luciferase normalization plasmids. Transfected cells were exposed to two different regimes (0.1–100 µM and 7.8–250 µM) of the oxidative stress-inducing compounds, sulforaphane, tertbutylhydroquinone, and metazachlor. Nrf2 activity was measured in dual-luciferase assays. In parallel, cytotoxicity was assessed for different endpoints (energy metabolism, protein amount, membrane stability, and cell proliferation) in non-transfected cells and cells co-transfected with constructs of increasing size, to be used for normalization. Transfected cells were more susceptible to cytotoxicity in a vector size-dependent manner. Conclusively, we report that vector geometries (size, backbones, gene-regulatory units), cell line (tissue origin), applied transfection methods, and signal normalization may alter the sensitivity of reporter bioassays in a synergistic manner. Further, we propose that thorough bioassay design is needed to ensure reliability and regulatory acceptance.

Biological impacts of Ce nanoparticles with different surface coatings as revealed by RNA-Seq in Chlamydomonas reinhardtii

In order to better understand the risks of engineered nanoparticles (ENPs), it is necessary to determine their fate and biological effects under realistic exposure scenarios (e.g. low ENP concentrations). RNA-Seq was deployed to characterize the relative biological impacts of three small Ce ENPs (i.e. nominal size <20 nm, 70 μg L−1 Ce), with different coating properties (i.e. uncoated, citrate or poly-acrylic acid coated), towards a unicellular freshwater microalga, Chlamydomonas reinhardtii. After 2 h exposition at pH 7.0, distinct differences in transcriptomic effects were observed when comparing ionic Ce and Ce ENPs. Notably, Ce ENPs specifically modulated mRNA levels of genes related to the ubiquitin-proteasome system and to flagella structure. Compared to control conditions, transcriptomic effects induced by the citrate coated Ce ENPs were rather limited, as only 23 genes were differentially expressed by this treatment (Log2FC > |1.0|, padj < 0.001); compared to uncoated Ce ENPs (688); polyacrylic coated Ce ENPs (315) or a similar concentration of ionic Ce (138). Somewhat surprisingly, similar changes in the algal transcriptomes were observed for treatments with poly-acrylic acid coated Ce ENPs (mainly Ce(III), little dissolution) and uncoated Ce ENPs (mainly Ce(IV) atoms, largely agglomerated) (Log2FC > |1.0|, padj < 0.001). For the moderate exposure concentrations examined here, toxicity appeared to be minimal for both ionic Ce and Ce ENPs. Nonetheless, an important number of genes could not be assigned to a biological pathway. The study gives important insights with respect to the role of particle surface coatings on biological effects, the mechanisms of interaction of Ce ENP with a green alga, in addition to identifying several useful transcriptomic biomarkers of Ce ENP exposure.

Multigenerational Exposures of Daphnia Magna to Pristine and Aged Silver Nanoparticles: Epigenetic Changes and Phenotypical Ageing Related Effects.

Engineered nanoparticles (NPs) undergo physical, chemical, and biological transformation after environmental release, resulting in different properties of the "aged" versus "pristine" forms. While many studies have investigated the ecotoxicological effects of silver (Ag) NPs, the majority focus on "pristine" Ag NPs in simple exposure media, rather than investigating realistic environmental exposure scenarios with transformed NPs. Here, the effects of "pristine" and "aged" Ag NPs are systematically evaluated with different surface coatings on Daphnia magna over four generations, comparing continuous exposure versus parental only exposure to assess recovery potential for three generations. Biological endpoints including survival, growth and reproduction and genetic effects associated with Ag NP exposure are investigated. Parental exposure to "pristine" Ag NPs has an inhibitory effect on reproduction, inducing expression of antioxidant stress related genes and reducing survival. Pristine Ag NPs also induce morphological changes including tail losses and lipid accumulation associated with aging phenotypes in the heart, abdomen, and abdominal claw. These effects are epigenetic remaining two generations post-maternal exposure (F2 and F3). Exposure to identical Ag NPs (same concentrations) aged for 6 months in environmentally realistic water containing natural organic matter shows considerably reduced toxicological effects in continuously exposed generations and to the recovery generations.

In vitro cytotoxicity assessment of pristine and carboxyl-functionalized MWCNTs

The wide use of carbon nanotubes (CNTs) in consumer products, i.e. composites, coatings, food packaging, etc, raise concerns about the adverse effects that CNTs can induce in humans and environment. Yet, there is no global consensus regarding risks that CNTs may pose, while controversial evidence exists also on the toxic effects associated with chemical surface modification, a prerequisite for their incorporation in different matrices. Moreover, there is limited information available about the underlying mechanisms, especially when cells’ interactions with the nanomaterial is assessed by imaging techniques. The present study aims at evaluating the in vitro cytotoxicity of pristine and oxygen functionalized multi-walled CNTs (MWCNTs) by assessing cell viability and apoptosis in combination with scanning electron microscopy (SEM) observations of stabilised cells. Direct observation of adenocarcinoma human epithelial cells (A549) was performed after incubation with 12.5, 50 and 100 μg/ml MWCNTs, for 0.5, 1 and 3 h, simulating a real exposure scenario during an accident, taking into account industrial safety issues during the production and use of the nanomaterial. Functionalized MWCNTs induced higher time- and dose-dependent toxic effects as compared to pristine. The SEM observations revealed the damaging effect on the cell membrane, offering insights about the toxic mechanism that takes place.

Impact of temperature on the toxicity of Kraft 36 EC® (a.s. abamectin) and Score 250 EC® (a.s. difenoconazole) to soil organisms under realistic environmental exposure scenarios

Pesticides can affect all receiving compartments, especially soils, and their fate and effects may be enhanced by temperature, increasing their risk to ecological functions of soils. In Brazil, the most widely used pesticides are the insecticide Kraft 36EC® (a.s. abamectin) and the fungicide Score 250EC® (a.s. difenoconazole), which are commonly used in strawberry, often simultaneously as a mixture. The aim of this study was to evaluate the toxicity of realistic environmental applications, single and in mixtures, for both pesticides to the springtail Folsomia candida and the plant species Allium cepa (onion) and Lycopersicum esculentum (tomato). Mesocosms filled with Brazilian natural soil (lattosolo) were dosed with water (control), Kraft (10.8g a.s/ha), Score (20g.a.s/ha) and Kraft+Score (10.8+20g a.s./ha). The applications were repeated every 7 days, during 18 days of experiment, and simulating rainfall twice a week. Collembola reproduction tests were conducted with soils from the first (day 1) and last day (day 18) of experiment for each treatment. Plant toxicity tests were carried out in the experimental units. The experiments were run at 23°C and 33°C. Kraft, alone and in the binary mixture, showed high toxicity to the springtails in soils from both days 1 and 18, especially at 23°C where it caused 100% mortality. Score however, was not toxic to the springtails. Plant growth was reduced by Score, but responses varied depending on temperature. This study indicates a high environmental risk of the insecticide Kraft, particularly at lower temperatures (23°C), and an influence of temperature on pesticide fate and effects.

In vitro assessment of the combination of cylindrospermopsin and the organophosphate chlorpyrifos on the human neuroblastoma SH-SY5Y cell line.

Cylindrospermopsin (CYN) is a cyanotoxicant which occurrence is increasing due to climate change. Cylindrospermopsin is able to exert damage in the organism at several levels, among them, in the nervous system. Moreover, it is important to take into account that it is not usually present isolated in nature, but in combination with some other pollutants, being the case of the pesticide chlorpyrifos (CPF). Thus, the aim of the present work was to assess the effects of the interaction of CYN in combination with CPF in the human neuroblastoma cell line SH-SY5Y by evaluating cytotoxicity and mechanistic endpoints. The mixtures 0.25+21, 0.5+42, 1+84μg/mL of CYN+CPF based on cytotoxicity results, were evaluated, and the isobologram method detected an antagonistic effect after 24 and 48h of exposure. Moreover, although no alterations of reactive oxygen species were detected, a significant decrease of glutathione levels was observed after exposure to both, CPF alone and the combination, at all the concentrations and times of exposure assayed. In addition, CYN+CPF caused a marked decrease in the acetylcholinesterase activity, providing similar values to CPF alone. However, these effects were less severe than expected. All these findings, together with the morphological study results, point out that it is important to take into account the interaction of CYN with other pollutants. Further research is required to contribute to the risk assessment of CYN and other contaminants considering more realistic exposure scenarios.

Impact of wastewater-borne nanoparticles of silver and titanium dioxide on the swimming behaviour and biochemical markers of Daphnia magna: An integrated approach

Due to their widespread use, silver (Ag) and titanium dioxide (TiO2) nanoparticles (NPs) are commonly discharged into aquatic environments via wastewater treatment plants. The study was aimed to assess the effects of wastewater-borne AgNPs (NM-300 K; 15.5 ± 2.4 nm; 25−125 μg L−1) and TiO2NPs (NM-105; 23.1 ± 6.2 nm; 12.5−100 μg L−1), from a laboratory-scale wastewater treatment plant, on Daphnia magna, at individual and subcellular level. For effect comparison, animals were also exposed to ASTM-dispersed NPs at the same nominal concentrations.The behaviour of D. magna was evaluated through monitoring of swimming height and allocation time for preferred zones after 0 h and 96 h of exposure. Biochemical markers of neurotransmission, anaerobic metabolism, biotransformation, and oxidative stress were subsequently determined. No 96-h EC50 (immobilization ≤ 4 %) could be obtained with wastewater-borne NPs and ASTM-dispersed TiO2NPs, whereas the ASTM-dispersed AgNPs resulted in an immobilization 96-h EC50 of 113.8 μg L−1. However, both wastewater-borne and ASTM-dispersed TiO2NPs, at 12.5 μg L−1, caused immediate (0 h) alterations on the swimming height. Allocation time analyses showed that animals exposed to ASTM-dispersed AgNPs spent more time on the surface and bottom at 0 h, and in the middle and bottom at 96 h. This pattern was not observed with ASTM-dispersed TiO2NPs nor with wastewater-borne AgNPs and wastewater-borne TiO2NPs. At the biochemical level, the more pronounced effects were observed with wastewater-borne AgNPs (e.g. induction of lactate dehydrogenase and glutathione S-transferase activities, and inhibition of catalase activity).This integrative approach showed that: (i) the behavioural and biochemical response-patterns were distinct in D. magna exposed to environmentally relevant concentrations of wastewater-borne and ASTM-dispersed NPs; (ii) the most pronounced effects on allocation time were induced by ASTM-dispersed AgNPs; and (iii) at the subcellular level, wastewater-borne AgNPs were more toxic than wastewater-borne TiO2NPs. This study highlights the need for the assessment of the effects of wastewater-borne NPs under realistic exposure scenarios, since processes in wastewater treatment plants may influence their toxicity.

Assessment of the Personal Dose Received by School Children due to PM10 Air Pollution in Lisbon

ABSTRACT Investigation of the personal dose caused by air pollution in children is important due to their vulnerability. Exposure to PM10 and its components, particularly certain metals, may pose significant health risks therefore many studies have focused on measuring the ambient indoor/outdoor PM10 concentrations in school environments. However, little research has aimed at assessing the resultant personal dose. Hence, this study applied a dosimetry model (ExDoM2) to predict the personal dose received by students in five primary schools in Lisbon, Portugal. The calculations were performed for PM10 and PM10-bound metals, and the exposed subjects were assumed to be 10-year-old nose breathers. A realistic exposure scenario involving three different settings (the indoor home, indoor school and outdoor school microenvironments) was implemented for an exposure period of one week (Monday–Sunday). Although the students spent only 24% of their total time inside a school (vs. 73% at home), this environment contributed 44% on average to the weekly deposited dose of PM10, providing further evidence that indoor exposure at schools is a major contributor to the total dose. The modeling results showed that the cumulative deposited doses in the respiratory tract (RT) reached as high as 2,004 µg, 0.16 µg, 0.65 µg, 0.58 µg and 0.06 µg for PM10, Cr, Mn, Pb and Ni, respectively, after one week.

Microplastics but not natural particles induce multigenerational effects in Daphnia magna

Several studies have investigated the effects of nano- and microplastics on daphnids as key freshwater species. However, while information is abundant on the acute toxicity of plastic beads, little is known regarding the multigenerational effects of irregular microplastics. In addition, a comparison of microplastics to naturally occurring particles is missing. Therefore, we investigated the effects of irregular, secondary polystyrene microplastics (<63 μm) and kaolin as natural reference particle on the survival, reproduction, and growth of Daphnia magna over four generations under food-limited conditions. Additionally, we tested the sensitivity of the neonates in each generation to a reference compound as a proxy for offspring fitness. Exposure to high concentrations of microplastics (10,000 and 2000 particles mL−1) reduced daphnid survival, resulting in extinction within one and four generations, respectively. Microplastics also affected reproduction and growth. Importantly, an exposure to kaolin at similar concentrations did not induce negative effects. The sensitivity of neonates to potassium dichromate was not affected by maternal exposure to particles. Taken together, our study demonstrates that irregular PS particles are more toxic than natural kaolin in daphnids exposed over multiple generations under food limitation. Thus, our work builds towards more realistic exposure scenarios needed to better understand the impacts of microplastics on zooplankton.

Evaluation of Second-Hand Exposure to Electronic Cigarette Vaping under a Real Scenario: Measurements of Ultrafine Particle Number Concentration and Size Distribution and Comparison with Traditional Tobacco Smoke.

The present study aims to evaluate the impact of e-cig second-hand aerosol on indoor air quality in terms of ultrafine particles (UFPs) and potential inhalation exposure levels of passive bystanders. E-cig second-hand aerosol characteristics in terms of UFPs number concentration and size distribution exhaled by two volunteers vaping 15 different e-liquids inside a 49 m3 room and comparison with tobacco smoke are discussed. High temporal resolution measurements were performed under natural ventilation conditions to simulate a realistic exposure scenario. Results showed a systematic increase in UFPs number concentration (part cm−3) related to a 20-min vaping session (from 6.56 × 103 to 4.01 × 104 part cm−3), although this was one up to two order of magnitude lower than that produced by one tobacco cigarette consumption (from 1.12 × 105 to 1.46 × 105 part cm−3). E-cig second-hand aerosol size distribution exhibits a bimodal behavior with modes at 10.8 and 29.4 nm in contrast with the unimodal typical size distribution of tobacco smoke with peak mode at 100 nm. In the size range 6–26 nm, particles concentration in e-cig second-hand aerosol were from 2- (Dp = 25.5 nm) to 3800-fold (Dp = 9.31 nm) higher than in tobacco smoke highlighting that particles exhaled by users and potentially inhaled by bystanders are nano-sized with high penetration capacity into human airways.

Ecotoxicological responses of a reef calcifier exposed to copper, acidification and warming: A multiple biomarker approach

Multiple global and local stressors threat coral reefs worldwide, and symbiont-bearing foraminifera are bioindicators of reef health. The aim of this study was to investigate single and combined effects of copper (Cu) and climate change related stressors (ocean acidification and warming) on a symbiont-bearing foraminifer by means of an integrated biomarker analysis. Using a mesocosm approach, Amphistegina gibbosa were exposed for 25 days to acidification, warming and/or Cu contamination on a full orthogonal design (two levels each factor). Cu was the main factor increasing bleaching and respiration rates. Warming was the main cause of mortality and reduced growth. Calcification related enzymes were inhibited in response to Cu exposure and, in general, the inhibition was stronger under climate change. Multiple biological endpoints responded to realistic exposure scenarios in different ways, but evidenced general stress posed by climate change combined with Cu. These biological responses drove the high values found for the ‘stress index’ IBR (Integrated Biomarker Response) – indicating general organismal health impairment under the multiple stressor scenario. Our results provide insights for coral reef management by detecting potential monitoring tools. The ecotoxicological responses indicated that Cu reduces the tolerance of foraminifera to climate change (acidification + warming). Once the endpoints analysed have a high ecological relevance, and that responses were evaluated on a classical reef bioindicator species, these results highlight the high risk of climate change and metal pollution co-exposure to coral reefs. Integrated responses allowed a better effects comprehension and are pointed as a promising tool to monitor pollution effects on a changing ocean.

Release of Munitions Constituents in Aquatic Environments Under Realistic Scenarios and Validation of Polar Organic Chemical Integrative Samplers for Monitoring.

Munitions constituents (MC) may be released into aquatic environments as a result of underwater military munitions (UWMM) corrosion and breach. The present study investigated the release of 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) from Composition B fragments under 2 realistic exposure scenarios in a large flume with flow set at 15 cm/s: the first represented the release of MC from fully exposed Composition B, and the second represented release through a small hole, simulating a breached munition. Release of MC through a small hole was approximately 10 times lower than from exposed Composition B, demonstrating the strong influence of exposure to flow on release. The rate of release of MC into the flume was similar to that previously reported in a related field experiment, but a similar mass loss resulted in MC concentration in the field >300 times lower, likely by the dilution effect of hydrodynamic transport. The present study corroborates previous findings of release of MC at UWMM sites resulting in concentrations below the toxicity threshold to most species. In the flume water, MC was quantified using frequent grab sampling and polar organic chemical integrative samplers (POCIS). For TNT, POCIS-estimated time-weighted average concentrations were up to 40% higher than those derived from grab samples, whereas for RDX differences were 6% or less, demonstrating that POCIS provide reliable temporal integration of changing environmental concentrations for common MC. Environ Toxicol Chem 2019;38:2383-2391. Published 2019 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Cytotoxicity, Genotoxicity and Disturbance of Cell Cycle in HepG2 Cells Exposed to OTA and BEA: Single and Combined Actions.

Mycotoxins are produced by a number of fungal genera spp., for example, Aspergillus, Penicillium, Alternaria, Fusarium, and Claviceps. Beauvericin (BEA) and Ochratoxin A (OTA) are present in various cereal crops and processed grains. This goal of this study was to determine their combination effect in HepG2 cells, presented for the first time. In this study, the type of interaction among BEA and OTA through an isobologram method, cell cycle disturbance by flow cytometry, and genotoxic potential by in vitro micronucleus (MN) assay following the TG 487 (OECD, 2016) of BEA and OTA individually and combined in HepG2 cells are presented. Cytotoxic concentration ranges studied by the MTT assay over 24, 48, and 72 h were from 0 to 25 µM for BEA and from 0 to 100 µM for OTA, while BEA + OTA combinations were at a 1:10 ratio from 3.4 to 27.5 µM. The toxicity observed for BEA was higher than for OTA at all times assayed; additive and synergistic effects were detected for their mixtures. Cell cycle arrest in the G0/G1 phase was detected for OTA and BEA + OTA treatments in HepG2 cells. Genotoxicity revealed significant effects for BEA, OTA, and in combinations underlining the importance of studying real exposure scenarios of chronic exposure to mycotoxins.

Risk assessment of intake of pyrrolizidine alkaloids from herbal teas and medicines following realistic exposure scenarios

In this study five types of herbal teas were used to quantify the effect of comminution of the leaves on resulting PA exposure. Results show that PA levels extracted from intact leaves were consistently lower than from comminuted tea leaves. The Margin of Exposure (MOE) approach was applied to evaluate the consequences of this difference for the associated risks in the scenario of lifetime exposure. Furthermore, we considered medicinal use of these teas for shorter-than-lifetime exposure scenarios, and also analysed the risks of shorter-than-lifetime use of eight herbal medicines and 19 previously analysed plant food supplements. This analysis revealed that shorter-than-lifetime use resulted in MOE values < 10,000 upon use for 40–3450 weeks during a lifetime, with for only a limited number of herbal teas and medicines use of two weeks a year (150 weeks during a 75 year lifetime) would still raise a concern. It is concluded that taking more realistic conditions into account markedly reduces the concerns raised for these herbal preparations. These results also illustrate the need for development of a generally accepted method for taking short term exposure into account in risk assessment of compounds that are genotoxic and carcinogenic.

From laboratory to the field: Validating molecular markers of effect in Folsomia candida exposed to a fungicide-based formulation

Under controlled laboratory conditions, toxicity data tend to be less variable than in more realistic in-field studies and responses may thus differ from those in the natural environment, creating uncertainty. The validation of data under environmental conditions is therefore a major asset in environmental risk assessment of chemicals. The present study aimed to validate the mode of action of a commercial fungicide formulation in the soil invertebrate F. candida, under more realistic exposure scenarios (in-field bioassay), by targeting specific molecular biomarkers retrieved from laboratory experiments. Organisms were exposed in soil cores under minimally controlled field conditions for 4 days to a chlorothalonil fungicide dosage causing 75% reduction of reproduction in a previous laboratory experiment (127 mg a.i. kg−1) and half this concentration (60 mg a.i. kg−1). After exposure, organisms were retrieved and RNA was extracted from each pool of organisms. According to previous laboratorial omics results with the same formulation, ten genes were selected for gene expression analysis by qRT-PCR, corresponding to key genes of affected biological pathways including glutathione metabolism, oxidation-reduction, body morphogenesis, and reproduction. Six of these genes presented a dose-response trend with higher up- or down-regulation with increasing pesticide concentrations. Highly significant correlations between their expression patterns in laboratory and in-field experiments were observed. This work shows that effects of toxicants can be clearly demonstrated in more realistic conditions using validated biomarkers. Our work outlines a set of genes that can be used to assess the early effects of pesticides in a realistic agricultural scenario.

Added value of human biomonitoring in assessment of general population exposure to xenobiotics.

Human biomonitoring (HBM) has a wide range of applications and long tradition both in health care and the field of public health. Its major advantage is the integration of all exposure routes and sources. Since HBM information is an integrated exposure finding it offers the opportunity to trace and mimic a realistic exposure scenario. It reduces the number of assumptions that need to be made when estimating exposure and thus helps to reduce the uncertainties in exposure science. In spite of some challenges, such as further harmonization in the area of HBM, necessity of deriving equivalents of markers of external exposure and addressing the ethical and political aspects of its application, HBM is an efficient and cost-effective way to measure exposure levels of the human body to xenobiotics.

A contribution towards improving the applicability of the Myriophyllum aquaticum sediment contact test

BackgroundWhole sediment contact tests provide realistic exposure scenarios, but the inherent properties of sediments may play a significant role in organism’s response and interfere with result interpretation. Recently, a sediment contact test with a rooted aquatic macrophyte Myriophyllum aquaticum has been standardized. The present study aims to distinguish between effects of basic sediment properties and sediment-bound pollutants on M. aquaticum growth and evaluate the method as a tool in sediment quality assessment. Tests with artificial sediments with different organic matter, sand and clay content, as well as freshwater sediments with different levels of contamination were conducted.ResultsResults were based on comparison to the standard artificial control sediment. Organic matter content and grain size distribution in different variants of artificial sediments significantly affected M. aquaticum growth. Growth was impaired in formulations with low (1%) and high (10%) organic matter content, while better growth of plants compared to control was recorded in artificial samples with higher fine particles content. Because of the presence of unmeasured pollutants in freshwater sediments and inherent sediment properties, results of the M. aquaticum sediment contact tests were not always in accordance with the results of chemical analysis. Whorl, shoot and root parameters had different variability and showed a particular growth pattern in natural sediments. If the threshold of 20% for sediment toxicity is applied, then about 60% of tested natural sediments may be considered as toxic. As sediment structure may influence plant growth, this interpretation may be false as the physico–chemical properties of the control sediment used for comparison are considerably different from the properties of natural sediments.ConclusionsSince inherent properties showed a significant effect on plant growth, the use of suitable controls more similar to tested natural sediments is advisable. To avoid over-, but also under-estimations, the use of the strict toxicity threshold of 20% or even higher inhibition for this test can be recommended, provided the statistical power of the test is high. The results of this study might contribute to the ongoing process of including effect-based methods in water quality monitoring under the Water Framework Directive.

Combined effects of nanoplastics and copper on the freshwater alga Raphidocelis subcapitata

Nanoplastics are recognized as able to interact with other pollutants including heavy metals, and with natural organic matter, with implications for the potential risks to biota. We investigated the interaction of carboxylated polystyrene nanoparticles (PS–COOH NPs) with copper (Cu) and algal exudates (EPS) and how such interaction could affect Cu toxicity towards the freshwater microalga Raphidocelis subcapitata. PS–COOH NPs behavior in the presence of Cu and EPS was determined by dynamic light scattering (DLS), while PS–COOH NPs surface interaction with Cu ions and EPS was investigated by fluorimetric analysis. ICP-MS was used to test Cu ion adsorption to PS–COOH NPs in the presence and absence of algae. The interaction between PS–COOH NPs and the algal cell wall was assessed by fluorescence microscopy. Short- and long-term toxicity tests were carried out in parallel to assess the impact of PS–COOH NPs on algal growth. Results showed altered nanoparticle surface charge and hydrodynamic diameter following algal EPS exposure, supporting the hypothesis of a protein corona formation. In contrast, no absorption of Cu ions was observed on PS–COOH NPs, either in the presence or absence of algae. No differences on algal growth inhibition were observed between exposure to Cu only, and to Cu in combination with PS–COOH NPs, in short-term as well as long-term tests. However, after 72 h of exposure, the adsorption of PS-COOH NPs to algal cell walls appeared to correspond to morphological alterations, revealing potential disturbances in the mitotic cycle. Our findings confirm the ability of PS–COOH NPs to interact with EPS as shown for other nanomaterials. Environmentally realistic exposure scenarios are thus needed for evaluating nanoplastic toxicity, as nanoparticles will not maintain their pristine nature once released into natural media. Prolonged exposure and use of different end-points such as cell morphological changes and EPS production seem more reliable for the investigation of nanoplastic/algal cell interactions which can drive food chain transfer of nanoplastics and ultimately toxicity.

Comparative multi-generation study on long-term effects of pristine and wastewater-borne silver and titanium dioxide nanoparticles on key lifecycle parameters in Daphnia magna

The rising production volume of engineered nanoparticles (NPs) leads to an increasing risk of environmental pollution. After passing sewage treatment plants (STPs), a significant concentration of NPs may end up in the aquatic environment where NPs can accumulate in the aquatic food chain and may cause harmful effects on aquatic organisms. However, when passing STPs some NPs such as silver nanoparticles (AgNPs) are transformed and enter the aquatic environment mostly as sulphide species with lower bioavailability and reduced toxicity compared to pristine NPs. For the environmental risk assessment of NPs, it is thus crucial to consider the transformation processes of nanomaterials during STP processes. For other NPs, such as titanium dioxide nanoparticles (TiO2NPs), knowledge about the acute and chronic toxicity of NPs from STP effluents on aquatic organisms is still missing. Chronic studies, such as the Daphnia reproduction test following OECD TG 211, cover a period of only 21 days and hence allow only to evaluate the reproduction performance of a single generation. Multi-generation studies provide a more realistic exposure scenario and offer the opportunity to identify transgenerational effects which may possess a significant impact on the population dynamic. Hence, the aim of this study was to assess the impact of wastewater-borne AgNPs and TiO2NPs on the aquatic invertebrate Daphnia magna in a multi-generation approach covering six generations. The effects of long-term exposure to pristine AgNPs (NM-300K; 14.9 ± 2.4 nm) and TiO2 NPs (NM-105; 21 ± 9 nm) on the reproductive success (number of offspring), mortality, time to first brood and body size of adult Daphnia were measured and compared to those caused by wastewater-borne AgNP and TiO2NPs. In all six generations, the exposure to environmentally relevant concentrations (determined by inductively coupled plasma mass spectrometry, ICP-MS) of pristine AgNPs caused a significant reduction in the mean number of offspring compared to the control. However, wastewater-borne AgNPs had no effects on reproduction in any generation. STEM analysis shows, that the AgNPs particles were transformed to Ag2S while passing the STP. No effects could be detected following exposure to environmentally relevant concentrations (determined by inductively coupled plasma optical emission spectrometry, ICP-OES) of pristine TiO2NPs and wastewater-borne TiO2NPs. The present study is the first multi-generation study on long-term effects of pristine and wastewater-borne nanoparticles on Daphnia. No transgenerational effects of wastewater-borne AgNPs nor TiO2NPs were observed. The results confirm that realistic exposure conditions are required in order to allow for a reliable environmental risk assessment of NPs.

Cellular Responses of Industrially Relevant Silica Dust on Human Glial Cells In Vitro.

Despite the rigorous emission control measures in the ferroalloy industry, there are still emissions of dust during the production of various alloys. Dust particles were collected from laboratory scale processes where oxide particulate matter was formed from liquid silicon (metallurgical grade). The dust was produced in a dry air atmosphere to mimic industrial conditions. To investigate possible effects of ultrafine dust on the central nervous system, a human astrocytic cell line was employed to investigate inflammatory effects of particles as astrocytes play a number of active and neuron supporting roles in the brain. Toxicity on the astrocytes by amorphous silica generated in laboratory scale was compared to crystalline macro-sized silica using several doses to determine toxicological dose response curves. The cell viability experiments indicated that low particle doses of amorphous silica induced a small nonsignificant reduction in cell viability compared to crystalline silica which led to increased levels of toxicity. The gene expression of amyloid precursor protein (APP), a biomarker of neurodegenerative disease, was affected by particle exposure. Furthermore, particle exposure, in a dose-and time-dependent manner, affected the ability of the cells to communicate through gap junction channels. In conclusion, in vitro studies using low doses of particles are important to understand mechanisms of toxicity of occupational exposure to silica particles. However, these studies cannot be extrapolated to real exposure scenarios at work place, therefore, controlling and keeping the particle exposure levels low at the work place, would prevent potential negative health effects.