Interspecific differences in oxidative DNA damage after hydrogen peroxide exposure of sea urchin coelomocytes.

Interspecific comparison of DNA damage can provide information on the relative vulnerability of marine organisms to toxicants that induce oxidative genotoxicity. Hydrogen peroxide (H2O2) is an oxidative toxicant that causes DNA strand breaks and nucleotide oxidation and is used in multiple industries including Atlantic salmon aquaculture to treat infestations of ectoparasitic sea lice. H2O2 (up to 100 mM) can be released into the water after sea lice treatment, with potential consequences of exposure in nontarget marine organisms. The objective of the current study was to measure and compare differences in levels of H2O2-induced oxidative DNA damage in coelomocytes from Scottish sea urchins Echinus esculentus, Paracentrotus lividus, and Psammechinus miliaris. Coelomocytes were exposed to H2O2 (0-50 mM) for 10 min, cell concentration and viability were quantified, and DNA damage was measured by the fast micromethod, an alkaline unwinding DNA method, and the modified fast micromethod with nucleotide-specific enzymes. Cell viability was >92% in all exposures and did not differ from controls. Psammechinus miliaris coelomocytes had the highest oxidative DNA damage with 0.07 ± 0.01, 0.08 ± 0.01, and 0.07 ± 0.01 strand scission factors (mean ± SD) after incubation with phosphate-buffered saline, formamidopyrimidine-DNA glycosylase, and endonuclease-III, respectively, at 50 mM H2O2. Exposures to 0.5 mM H2O2 (100-fold dilution from recommended lice treatment concentration) induced oxidative DNA damage in all three species of sea urchins, suggesting interspecific differences in vulnerabilities to DNA damage and/or DNA repair mechanisms. Understanding impacts of environmental genotoxicants requires understanding species-specific susceptibilities to DNA damage, which can impact long-term stability in sea urchin populations in proximity to aquaculture farms.

Potential Artifacts and Control Experiments in Toxicity Tests of Nanoplastic and Microplastic Particles.

To fully understand the potential ecological and human health risks from nanoplastics and microplastics (NMPs) in the environment, it is critical to make accurate measurements. Similar to past research on the toxicology of engineered nanomaterials, a broad range of measurement artifacts and biases are possible when testing their potential toxicity. For example, antimicrobials and surfactants may be present in commercially available NMP dispersions, and these compounds may account for toxicity observed instead of being caused by exposure to the NMP particles. Therefore, control measurements are needed to assess potential artifacts, and revisions to the protocol may be needed to eliminate or reduce the artifacts. In this paper, we comprehensively review and suggest a next generation of control experiments to identify measurement artifacts and biases that can occur while performing NMP toxicity experiments. This review covers the broad range of potential NMP toxicological experiments, such as in vitro studies with a single cell type or complex 3-D tissue constructs, in vivo mammalian studies, and ecotoxicity experiments testing pelagic, sediment, and soil organisms. Incorporation of these control experiments can reduce the likelihood of false positive and false negative results and more accurately elucidate the potential ecological and human health risks of NMPs.

Marine Sponges in a Snowstorm - Extreme Sensitivity of a Sponge Holobiont to Marine Oil Snow and Chemically Dispersed Oil Pollution.

Holobionts formed by a host organism and associated symbionts are key biological units in marine ecosystems where they are responsible for fundamental ecosystem services. Therefore, understanding anthropogenic impacts on holobionts is essential. Sponges (Phylum Porifera) are ideal holobiont models. They host a complex microbial community and provide ecosystem services including nutrient cycling. At bathyal depths, sponges can accumulate forming dense sponge ground habitats supporting biodiverse associated communities. However, the impacts of spilled oil and dispersants on sponge grounds cannot be understood without considering exposures mediated through sponge filtration of marine snow particles. To examine this, we exposed the model sponge Halichondria panicea to oil, dispersant and "marine oil snow" contaminated seawater and elucidate the complex molecular response of the holobiont through metatranscriptomics. While the host response included detoxification and immune response pathways, the bacterial symbiotic response differed and was at least partially the result of a change in the host environment rather than a direct response to hydrocarbon exposure. As the sponge host reduced its pumping activity and internal tissue oxygen levels declined, the symbionts changed their metabolism from aerobic to anaerobic pathways possibly via quorum sensing. Furthermore, we found evidence of hydrocarbon degradation by sponge symbionts, but sponge mortality (even when exposed to low concentrations of hydrocarbons) implied this may not provide the holobiont with sufficient resilience against contaminants. Given the continued proposed expansion of hydrocarbon production into deep continental shelf and slope settings where sponge grounds form significant habitats it is important that dispersant use is minimised and that environmental impact assessments carefully consider the vulnerability of sponge holobionts.

Time-Related Alteration of Aqueous-Phase Anthracene and Phenanthrene Photoproducts in the Presence of TiO2 Nanoparticles.

Polycyclic aromatic hydrocarbons (PAHs) and titanium dioxide (TiO2) nanoparticles (NPs) are photoactive environmental pollutants that can contaminate aquatic environments. Aqueous-phase interactions between PAHs and TiO2-NPs are of interest due to their emerging environmental relevance, particularly with the deliberate application of TiO2-NPs to remediate pollution events (e.g., oil spills). Our objective was to investigate anthracene (ANT) and phenanthrene (PHE) photoproduct formation and transformation following ultraviolet A (UVA) irradiation in the presence and absence of TiO2-NPs. ANT and PHE solutions were prepared alone or in combination with TiO2-NPs, UVA-irradiated, and either exposed to larval zebrafish or collected for chemical analyses of diverse hydroxylated PAHs (OHPAHs) and oxygenated PAHs (OPAHs). The expression profiles of genes encoding for enzymes involved in PAH metabolism showed PAH-specific and time-dependent inductions that demonstrated changes in PAH and photoproduct bioavailability in the presence of TiO2-NPs. Chemical analyses of PAH/NP solutions in the absence of zebrafish larvae identified diverse photoproducts of differing size and ring arrangements, which suggested photodissociation, recombination, and ring re-arrangements of PAHs occurred either during or following UVA irradiation. Both ANT and PHE solutions showed heightened oxidative potential following irradiation, but TiO2-NP-related increases in oxidative potential were PAH-specific. The exploitation of multiple analytical methods provided novel insights into distinct PAH photoactivity, TiO2-NP influence on photoproduct formation in a PAH-specific manner, and the significant role time plays in photochemical processes.

Antibacterial Activities of Ga(III) against E. coli Are Substantially Impacted by Fe(III) Uptake Systems and Multidrug Resistance in Combination with Oxygen Levels.

The continued emergence and spread of antimicrobial resistance (AMR), particularly multidrug resistant (MDR) bacteria, are increasing threats driving the search for additional and alternative antimicrobial agents. The World Health Organization (WHO) has categorized bacterial risk levels and includes Escherichia coli among the highest priority, making this both a convenient model bacterium and a clinically highly relevant species on which to base investigations of antimicrobials. Among many compounds examined for use as antimicrobials, Ga(III) complexes have shown promise. Nonetheless, the spectrum of activities, susceptibility of bacterial species, mechanisms of antimicrobial action, and bacterial characteristics influencing antibacterial actions are far from being completely understood; these are important considerations for any implementation of an effective antibacterial agent. In this investigation, we show that an alteration in growth conditions to physiologically relevant lowered oxygen (anaerobic) conditions substantially increases the minimum inhibitory concentrations (MICs) of Ga(III) required to inhibit growth for 46 wild-type E. coli strains. Several studies have implicated a Trojan horse hypothesis wherein bacterial Fe uptake systems have been linked to the promotion of Ga(III) uptake and result in enhanced antibacterial activity. Our studies show that, conversely, the carriage of accessory Fe uptake systems (Fe_acc) significantly increased the concentrations of Ga(III) required for antibacterial action. Similarly, it is shown that MDR strains are more resistant to Ga(III). The increased tolerance of Fe_acc/MDR strains was apparent under anaerobic conditions. This phenomenon of heightened tolerance has not previously been shown although the mechanisms remain to be defined. Nonetheless, this further highlights the significant contributions of bacterial metabolism, fitness, and AMR characteristics and their implications in evaluating novel antimicrobials.

Polyvinyl chloride (PVC) plastic fragments release Pb additives that are bioavailable in zebrafish.

Plastic polymers such as polyvinyl chloride (PVC) may contain chemical additives, such as lead (Pb), that are leachable in aqueous solution. The fragmentation into microplastics (MPs) of plastics such as PVC may facilitate desorption of chemical additives and increase exposure of aquatic animals. In this study, the role of chemical additives in the aqueous toxicity of PVC, high-density polyethylene (HDPE) and polyethylene terephthalate (PET) MPs were investigated in early-life stage zebrafish (Danio rerio) by assessment of changes in expression of biomarkers. Exposure of zebrafish larvae to PVC for 24 h increased expression of metallothionein 2 (mt2), a metal-binding protein, but no changes in expression of biomarkers of estrogenic (vtg1) or organic (cyp1a) contaminants were observed. HDPE and PET caused no changes in expression of any biomarkers. A filtered leachate of the PVC also caused a significant increase in expression of mt2 and indicated that a desorbed metal additive likely elicited the response in zebrafish. Metal release was confirmed by acid-washing the MPs which mitigated the response in mt2. Metal analysis showed Pb leached from PVC into water during exposures; at 500 mg PVC L-1 in water, 84.3 ± 8.7 µg Pb L-1 was measured after 24 h. Exposure to a Pb-salt at this concentration caused a comparable mt2 increase in zebrafish as observed in exposures to PVC. These data indicated that PVC MPs elicited a response in zebrafish but the effect was indirect and mediated through desorption of Pb from PVC into the exposure water. Data also indicated that PVC MPs may act as longer-term environmental reservoirs of Pb for exposure of aquatic animals; the Pb leached from PVC in 24 h in freshwater equated to 2.52% of total Pb in MPs leachable by the acid-wash. Studies of MPs should consider the potential role of chemical additives in toxicity observed.

Exposure to Pb-halide perovskite nanoparticles can deliver bioavailable Pb but does not alter endogenous gut microbiota in zebrafish.

Lead-halide perovskite nanoparticles (NPs) are a new technology, and investigation of toxicity is of considerable importance due to the potential lead (Pb) release into the environment. The aim of the study was to investigate aqueous and dietary toxicity of Pb-halide perovskite NP and Pb in zebrafish Danio rerio. Perovskite NP toxicity was evaluated in zebrafish by mortality, gene expression, histopathology, and phylogenetic analysis of gut microbiota. Zebrafish larvae were exposed to five Pb-halide perovskite NPs in parallel with Pb(NO3)2 exposures, and zebrafish adults were exposed to the three perovskite NPs that caused the strongest effect and Pb(NO3)2. No median lethal concentration (LC50) was observed for zebrafish larvae exposed to up to 200 mg/L of perovskite NPs for 96 h. Mortality, metallothionein 2 (mt2) and δ-aminolevulinic acid dehydratase (ala-d) gene expression (24-h exposure) in zebrafish larvae after aqueous perovskite NPs exposures did not differ from total Pb concentration - response curves. The lack of differences in mortality and gene expression between perovskite NPs and soluble Pb after aqueous exposure suggest that toxicity from perovskite NPs can be attributed to bioavailable Pb rather than nano-specific effects. Induction of mt2 and reduction of ala-d expression levels in liver tissues showed Pb bioavailability after 2-d and 4-d dietary exposure to perovskite-spiked feeds. Changes in gut microbiota of adult zebrafish were detected after 14-d exposure to Pb-spiked food, but no changes were detected from perovskite-NP spiked food. The phylogenetic analysis identified different microbiome profiles of Pb-fed fish compared to perovskite-fed fish suggesting a different mechanism of toxicity. Exposure to Pb-halide perovskite NPs led to absorption of Pb likely from release of Pb ions rather than absorption of NPs. Pb-halide perovskite NPs can release bioavailable Pb and this needs to be considered during the development of this technology.

Use of fluorescent-labelled nanoplastics (NPs) to demonstrate NP absorption is inconclusive without adequate controls.

Whether nanoplastics (NPs) are able to be absorbed across epithelial membranes and accumulate within internal tissues of organisms is an important determinant of their potential toxicity. Evidence of absorption and accumulation requires detection of NPs within internal tissues, and investigations with fluorescently labelled NPs have attempted to provide this information. We hypothesize that studies that do not control for the fluorescent dye leachate and/or cellular autofluorescence are inconclusive and can be misinterpreted. Our goal was to analyse previous investigations critically and conduct further research to determine if fluorescent-labelled polystyrene NPs (nanoPS) can provide conclusive evidence of absorption and internal accumulation of NPs. We exposed zebrafish embryos and larvae to NPs (500 and 1000 nm) labelled with a green or an orange fluorescent dye, to solutions resulting from nanoPS dialysis, and to Nile-Red (a fluorescent dye used as a positive control). Previous studies have claimed that NPs cross epithelia without accounting for dye leachates and/or cellular autofluorescence. Our results demonstrate that commercial fluorescent-labelled nanoPS can leach their fluorophores, and the fluorophore alone can accumulate within internal tissues of zebrafish larvae. We further observed green autofluorescence in fish larvae not exposed to any particles. Previous claims of NP absorption based on observations of fluorescence in zebrafish tissues should thus be considered inconclusive. Although the addition of purification steps and inclusion of controls for leaching of dyes are methodological improvements, the use of fluorescent nanoPS should not be considered to provide absolute conclusive evidence of particle absorption.

Differences in Engineered Nanoparticle Surface Physicochemistry Revealed by Investigation of Changes in Copper Bioavailability During Sorption to Nanoparticles in the Aqueous Phase.

Sorption of chemical substances to nanoparticles (NPs) in the aqueous phase strongly influences NP physicochemisty, and investigations of these complex interactions can provide important insights into the environmental fate of NPs. The objective of the present study was to use differences in copper (Cu) bioavailability to investigate aqueous-phase sorption with NPs that had different physicochemical characteristics (silicon [Si], perovskite, and titanium dioxide NPs [TiO2 NPs]). Sorption of Cu with NPs was assessed by the presence of adsorbent in water and onto the NP surface after ultracentrifugation, and by changes in Cu bioavailability under static conditions during exposure of larval zebrafish, as well as under conditions of continuous agitation during exposure of the alga Chlorella vulgaris. The presence of TiO2 NPs reduced total Cu in the water column and Cu bioavailability (measured by growth inhibition, mortality, and metallothionein 2 gene expression), confirming Cu sorption to TiO2 NPs. Nanoparticle surface area was the most important factor that affected Cu sorption, as indicated by less bioavailable Cu in the presence of smaller TiO2 NPs. The surface area effect was consistent regardless of exposure conditions (alga, continuous agitation; zebrafish, static water) and was further supported by the fact that the lowest total Cu concentration in the water column was found in the presence of the smallest NP. The results differed with other NP types, for example, silicon NPs, in which Cu sorption was indicated by analytical chemistry, but sorption was not sufficient to significantly alter Cu bioavailability. The bioavailability tests did not indicate Cu sorption with perovskite NPs. The results demonstrate that surface area critically influences sorption, that Cu sorption as measured by bioavailability is not affected by agitation or static conditions, and that Cu sorption differs among types of NPs, indicating differences in their surface physicochemistry. Environ Toxicol Chem 2019;9999:1-11. © 2019 SETAC.

Higher silver bioavailability after nanoparticle dietary exposure in marine amphipods.

On release into surface waters, engineered silver nanoparticles (AgNPs) tend to settle to sediments and, consequently, epibenthic fauna will be exposed to them through diet. We established Ag uptake and accumulation profiles over time in the hemolymph of a marine amphipod fed with a formulated feed containing AgNPs or AgCl. Silver bioavailability was higher in organisms exposed to AgNPs, indicating that the nanoparticles pose a higher risk of toxicity compared to similar concentrations of AgCl. Environ Toxicol Chem 2019;38:806-810. © 2019 SETAC.

Microplastic contamination in surface waters in Guanabara Bay, Rio de Janeiro, Brazil.

Microplastics (MPs) are contaminants of environmental concern that represent a threat to marine systems. Here we report data on the abundance and characteristics of MPs collected from surface waters of the urban Guanabara Bay. Samples were collected, by horizontal trawling of a plankton net on two occasions (summer of 2016). The MPs were obtained from samples by sieving and particles were manually sorted with microscope. Characterization of MPs was accomplished by gravimetry and digital image processing (for quantification and morphology categorization), and chemical composition identified by infrared spectroscopy and elemental analyses. Total MPs ranged from 1.40 to 21.3 particles/m3, which places Guanabara Bay amongst the most contaminated coastal systems worldwide by microplastics. Polyethylene and polypropylene polymers ≤1 mm were the most abundant particles. Therefore, the occurrence of MPs in Guanabara Bay is relevant to understand ecological hazards of exposition to marine biota and merits further investigation.

Conservation and restoration of a keystone species: Understanding the settlement preferences of the European oyster (Ostrea edulis).

The European oyster Ostrea edulis is a keystone species that is internationally recognised as 'threatened and declining' in the NE Atlantic by OSPAR and several nations have consequently adopted strategies for its conservation and restoration. Understanding the settlement behaviour of O. edulis larvae is crucial to inform these strategies. We compared the efficiency of several treatments in triggering settlement. The most effective settlement occurred with the presence of conspecifics: 100% settled in <23 h. Marine stones with habitat-associated biofilms induced 81% settlement that started after a 45 h delay. Sterile shells and terrestrial stones did not induce more settlement than control treatments. These results indicate that O. edulis larvae are gregarious and finely-tuned to settle in response to cues which are indicative of their adult habitat requirements. The role of chemical cues in mediating settlement, and the importance of this to restoration, are discussed.

Uptake, Whole-Body Distribution, and Depuration of Nanoplastics by the Scallop Pecten maximus at Environmentally Realistic Concentrations.

Previous studies of uptake and effects of nanoplastics by marine organisms have been conducted at what may be unrealistically high concentrations. This is a consequence of the analytical challenges in tracking plastic particles in organisms at environmentally relevant concentrations and highlights the need for new approaches. Here, we present pulse exposures of 14C-radiolabeled nanopolystyrene to a commercially important mollusk, Pecten maximus, at what have been predicted to be environmentally relevant concentrations (<15 µg L-1). Uptake was rapid and was greater for 24 nm than for 250 nm particles. After 6 h, autoradiography showed accumulation of 250 nm nanoplastics in the intestine, while 24 nm particles were dispersed throughout the whole-body, possibly indicating some translocation across epithelial membranes. However, depuration was also relatively rapid for both sizes; 24 nm particles were no longer detectable after 14 days, although some 250 nm particles were still detectable after 48 days. Particle size thus apparently influenced the biokinetics and suggests a need for chronic exposure studies. Modeling extrapolations indicated that it could take 300 days of continued environmental exposure for uptake to reach equilibrium in scallop body tissues although the concentrations would still below 2.7 mg g-1. Comparison with previous work in which scallops were exposed to nonplastic (silver) nanomaterials of similar size (20 nm), suggests that nanoparticle composition may also influence the uptake tissue distributions somewhat.

Potential Impacts of Offshore Oil and Gas Activities on Deep-Sea Sponges and the Habitats They Form.

Sponges form an important component of benthic ecosystems from shallow littoral to hadal depths. In the deep ocean, beyond the continental shelf, sponges can form high-density fields, constituting important habitats supporting rich benthic communities. Yet these habitats remain relatively unexplored. The oil and gas industry has played an important role in advancing our knowledge of deep-sea environments. Since its inception in the 1960s, offshore oil and gas industry has moved into deeper waters. However, the impacts of these activities on deep-sea sponges and other ecosystems are only starting to become the subject of active research. Throughout the development, operation and closure of an oil or gas field many activities take place, ranging from the seismic exploration of subseafloor geological features to the installation of infrastructure at the seabed to the drilling process itself. These routine activities and accidental releases of hydrocarbons during spills can significantly impact the local marine environment. Each phase of a field development or an accidental oil spill will therefore have different impacts on sponges at community, individual and cellular levels. Legacy issues regarding the future decommissioning of infrastructure and the abandonment of wells are also important environmental management considerations. This chapter reviews our understanding of impacts from hydrocarbon exploration and exploitation activities on deep-sea sponges and the habitats they form. These impacts include those (1) at community level, decreasing the diversity and density of benthic communities associated with deep-sea sponges owing to physical disturbance of the seabed; (2) at individual level, interrupting filtration owing to exposure to increased sedimentation; and (3) at cellular level, decreasing cellular membrane stability owing to exposure to drill muds. However, many potential effects not yet tested in deep-sea sponges but observed in shallow-water sponges or other model organisms should also be taken into account. Furthermore, to the best of our knowledge, no studies have shown impact of oil or dispersed oil on deep-sea sponges. To highlight these significant knowledge gaps, a summary table of potential and known impacts of hydrocarbon extraction and production activities combined with a simple "traffic light" scheme is also provided.

Low levels of microplastics (MP) in wild mussels indicate that MP ingestion by humans is minimal compared to exposure via household fibres fallout during a meal.

Microplastics (MPs) are the most numerous debris reported in marine environments and assessment of the amounts of MPs that accumulate in wild organisms is necessary for risk assessment. Our objective was to assess MP contamination in mussels collected around the coast of Scotland (UK) to identify characteristics of MPs and to evaluate risk of human exposure to MPs via ingestion of mussels. We deployed caged mussels (Mytilus edulis) in an urbanised estuary (Edinburgh, UK) to assess seasonal changes in plastic pollution, and collected mussels (Mytilus spp and subtidal Modiolus modiolus) from eight sampling stations around Scotland to enumerate MP types at different locations. We determined the potential exposure of humans to household dust fibres during a meal to compare with amounts of MPs present in edible mussels. The mean number of MPs in M. modiolus was 0.086 ±â€¯0.031 (SE, n = 6)/g ww (3.5 ±â€¯1.29 (SE) per mussel). In Mytilus spp, the mean number of MPs/g ww was 3.0 ±â€¯0.9 (SE, n = 36) (3.2 ±â€¯0.52 (SE) per mussel), but weight dependent. The visual accuracy of plastic fibres identification was estimated to be between 48 and 50%, using Nile Red staining and FT-IR methodologies, respectively, halving the observed amounts of MPs in wild mussels. We observed an allometric relationship between the number of MPs and the mussels wet weight. Our predictions of MPs ingestion by humans via consumption of mussels is 123 MP particles/y/capita in the UK and can go up to 4620 particles/y/capita in countries with a higher shellfish consumption. By comparison, the risk of plastic ingestion via mussel consumption is minimal when compared to fibre exposure during a meal via dust fallout in a household (13,731-68,415 particles/Y/capita).

Intravenous injection of unfunctionalized carbon-based nanomaterials confirms the minimal toxicity observed in aqueous and dietary exposures in juvenile rainbow trout (Oncorhynchus mykiss).

Numerous ecotoxicology studies of carbon-based nanomaterials (CNMs) have been conducted in fishes; however, different approaches have been used to make CNM dispersions and dose tanks for aqueous exposures, and to prepare food containing CNMs for dietary studies. This diversity of experimental methods has led to conflicting results and difficulties in comparing studies. The objective of the present study was to evaluate intravenous injection of unfunctionalized CNMs in rainbow trout (Oncorhynchus mykiss), as a means of delivering a known internal dose, on tissue biochemistry and histopathological lesions; then, subsequently, to compare the results with our previous work on aqueous and dietary exposures of rainbow trout to CNMs. Rainbow trout were injected in the caudal vein with corn oil dispersions of 200 µg (approximately 1 µg g-1) of either the fullerene C60, single-walled carbon nanotubes (SWCNTs), or amorphous carbon black. After 96 h, injected fish were euthanized and tissue samples collected for biochemistry and histology. Histological examination of the kidney of fish injected intravenously indicated the presence of black material consistent with the injected carbon treatments. However, there were no additional lesions associated with CNM exposure compared to controls. There were also no significant changes in haematology, or ionoregulatory disturbance in blood plasma among the intravenously injected fish. Significant elevation in lipid peroxidation (thiobarbituric acid reactive substances TBARS) was detected only in kidney and spleen of fish injected with SWCNTs, but not the other carbon treatments. The elevated TBARS following injection contrasted with CNMs delivered via aqueous or dietary routes in our previous studies, suggesting that the latter exposure routes may not lead to absorption and toxicity in the internal tissues. Comparison of the effects of injected CNMs with aqueous and dietary CNMs exposures indicates that these materials are of minimal environmentally-relevant toxicity in rainbow trout.

Adoption of in vitro systems and zebrafish embryos as alternative models for reducing rodent use in assessments of immunological and oxidative stress responses to nanomaterials.

Assessing the safety of engineered nanomaterials (NMs) is paramount to the responsible and sustainable development of nanotechnology, which provides huge societal benefits. Currently, there is no evidence that engineered NMs cause detrimental health effects in humans. However, investigation of NM toxicity using in vivo, in vitro, in chemico, and in silico models has demonstrated that some NMs stimulate oxidative stress and inflammation, which may lead to adverse health effects. Accordingly, investigation of these responses currently dominates NM safety assessments. There is a need to reduce reliance on rodent testing in nanotoxicology for ethical, financial and legislative reasons, and due to evidence that rodent models do not always predict the human response. We advocate that in vitro models and zebrafish embryos should have greater prominence in screening for NM safety, to better align nanotoxicology with the 3Rs principles. Zebrafish are accepted for use by regulatory agencies in chemical safety assessments (e.g. developmental biology) and there is growing acceptance of their use in biomedical research, providing strong foundations for their use in nanotoxicology. We suggest that investigation of the response of phagocytic cells (e.g. neutrophils, macrophages) in vitro should also form a key part of NM safety assessments, due to their prominent role in the first line of defense. The development of a tiered testing strategy for NM hazard assessment that promotes the more widespread adoption of non-rodent, alternative models and focuses on investigation of inflammation and oxidative stress could make nanotoxicology testing more ethical, relevant, and cost and time efficient.

Response of gene expression in zebrafish exposed to pharmaceutical mixtures: Implications for environmental risk.

Complex mixtures of pharmaceutical chemicals in surface waters indicate potential for mixture effects in aquatic organisms. The objective of the present study was to evaluate whether effects on target gene expression and enzymatic activity of individual substances at environmentally relevant concentrations were additive when mixed. Expression of zebrafish cytochrome P4501A (cyp1a) and vitellogenin (vtg) genes as well as activity of ethoxyresorufin-O-deethylase (EROD) were analyzed after exposure (96h) to caffeine-Caf, ibuprofen-Ibu, and carbamazepine-Cbz (0.05 and 5µM), tamoxifen-Tmx (0.003 and 0.3µM), and after exposure to pharmaceutical mixtures (low mix: 0.05µM of Caf, Ibu, Cbz and 0.003µM of Tmx, and high mix: 5µM of Caf, Ibu, Cbz and 0.3µM of Tmx). Pharmaceuticals tested individually caused significant down regulation of both cyp1a and vtg, but EROD activity was not affected. Exposure to low mix did not cause a significant change in gene expression; however, the high mix caused significant up-regulation of cyp1a but did not affect vtg expression. Up-regulation of cyp1a was consistent with induction of EROD activity in larvae exposed to high mix. The complex mixture induced different responses than those observed by the individual substances. Additive toxicity was not supported, and results indicate the need to evaluate complex mixtures rather than models based on individual effects, since in environment drugs are not found in isolation and the effects of their mixtures is poorly understood.

Assessment of microplastic-sorbed contaminant bioavailability through analysis of biomarker gene expression in larval zebrafish.

Microplastics (MPs) are prevalent in marine ecosystems. Because toxicants (termed here "co-contaminants") can sorb to MPs, there is potential for MPs to alter co-contaminant bioavailability. Our objective was to demonstrate sorption of two co-contaminants with different physicochemistries [phenanthrene (Phe), log10Kow=4.57; and 17α-ethinylestradiol (EE2), log10Kow=3.67] to MPs; and assess whether co-contaminant bioavailability was increased after MP settlement. Bioavailability was indicated by gene expression in larval zebrafish. Both Phe and EE2 sorbed to MPs, which reduced bioavailability by a maximum of 33% and 48% respectively. Sorption occurred, but was not consistent with predictions based on co-contaminant physicochemistry (Phe having higher log10Kow was expected to have higher sorption). Contaminated MPs settled to the bottom of the exposures did not lead to increased bioavailability of Phe or EE2. Phe was 48% more bioavailable than predicted by a linear sorption model, organism-based measurements therefore contribute unique insight into MP co-contaminant bioavailability.