Rhys M. Goodhead

Uptake and retention of microplastics by the shore crab Carcinus maenas.

Microplastics, plastics particles <5 mm in length, are a widespread pollutant of the marine environment. Oral ingestion of microplastics has been reported for a wide range of marine biota, but uptake into the body by other routes has received less attention. Here, we test the hypothesis that the shore crab (Carcinus maenas) can take up microplastics through inspiration across the gills as well as ingestion of pre-exposed food (common mussel Mytilus edulis). We used fluorescently labeled polystyrene microspheres (8-10 μm) to show that ingested microspheres were retained within the body tissues of the crabs for up to 14 days following ingestion and up to 21 days following inspiration across the gill, with uptake significantly higher into the posterior versus anterior gills. Multiphoton imaging suggested that most microspheres were retained in the foregut after dietary exposure due to adherence to the hairlike setae and were found on the external surface of gills following aqueous exposure. Results were used to construct a simple conceptual model of particle flow for the gills and the gut. These results identify ventilation as a route of uptake of microplastics into a common marine nonfilter feeding species.

Tracing Bioavailability of ZnO Nanoparticles Using Stable Isotope Labeling

Zinc oxide nanoparticles (ZnO NPs) are widely used in commercial products and knowledge of their environmental fate is a priority for ecological protection. Here we synthesized model ZnO NPs that were made from and thus labeled with the stable isotope (68)Zn and this enables highly sensitive and selective detection of labeled components against high natural Zn background levels. We combine high precision stable isotope measurements and novel bioimaging techniques to characterize parallel water-borne exposures of the common mudshrimp Corophium volutator to (68)ZnO NPs, bulk (68)ZnO, and soluble (68)ZnCl(2) in the presence of sediment. C. volutator is an important component of coastal ecosystems where river-borne NPs will accumulate and is used on a routine basis for toxicity assessments. Our results demonstrate that ionic Zn from ZnO NPs is bioavailable to C. volutator and that Zn uptake is active. Bioavailability appears to be governed primarily by the dissolved Zn content of the water, whereby Zn uptake occurs via the aqueous phase and/or the ingestion of sediment particles with adsorbed Zn from dissolution of ZnO particles. The high sorption capacity of sediments for Zn thus enhances the potential for trophic transfer of Zn derived from readily soluble ZnO NPs. The uncertainties of our isotopic data are too large, however, to conclusively rule out any additional direct uptake route of ZnO NPs by C. volutator.

Effect of Microplastic on the Gills of the Shore Crab Carcinus maenas.

Microscopic plastic debris (microplastics, <5 mm in diameter) is ubiquitous in the marine environment. Previous work has shown that microplastics may be ingested and inhaled by the shore crab Carcinus maenas, although the biological consequences are unknown. Here, we show that acute aqueous exposure to polystyrene microspheres (8 μm) with different surface coatings had significant but transient effects on branchial function. Microspheres inhaled into the gill chamber had a small but significant dose-dependent effect on oxygen consumption after 1 h of exposure, returning to normal levels after 16 h. Ion exchange was also affected, with a small but significant decrease in hemolymph sodium ions and an increase in calcium ions after 24 h post-exposure. To further asses the effects on osmoregulation, we challenged crabs with reduced salinity after microplastic exposure. Neither microspheres nor natural sediments altered the crabs response to osmotic stress regardless of plastic concentration added. Carboxylated (COOH) and aminated (NH2) polystyrene microspheres were distributed differently across the gill surface, although neither had a significant adverse impact on gill function. These results illustrate the extent of the physiological effects of microplastics compared to the physiological resilience of shore crabs in maintaining osmoregulatory and respiratory function after acute exposure to both anthropogenic plastics and natural particles.

Assessment of cultured fish hepatocytes for studying cellular uptake and (eco)toxicity of nanoparticles

Environmental context. The production and application of engineered nanoparticles is rapidly increasing, and development of suitable models for screening nanoparticles for possible toxic effects is essential to protect aquatic organisms and support the sustainable development of the nanotechnology industry. Here, the suitability of isolated rainbow trout hepatocytes was assessed for high through-put toxicity screening of nanoparticles and for studying uptake of nanoparticles into cells. Abstract. Relatively little is known regarding the fate and possible toxic effects of engineered nanoparticles (ENPs) in the aquatic environment. We assessed the suitability of isolated trout hepatocytes for high throughput toxicity screening of ENPs, exposing them to a variety of metal and metal oxide nanoparticles and their bulk counterparts. We found no effects of the ENPs on cell viability, or on lipid peroxidation, with the exception of exposure to ZnO nanoparticles, or on glutathione-S-transferase (GST) levels, for exposure concentrations up to 500 μg mL–1. All ENPs, however, were internalised in the cultured hepatocytes, as shown by coherent anti-Stokes Raman scattering (CARS) as an imaging technique. Our findings suggest that fish hepatocyte cultures are suitable for studies investigating the cellular uptake of ENPs, but they do not appear to be sensitive to ENP exposure and thus not a good in vitro model for nanoparticle toxicity screening.

Uptake and elimination kinetics of silver nanoparticles and silver nitrate by Raphidocelis subcapitata: The influence of silver behaviour in solution

Abstract Raphidocelis subcapitata is a freshwater algae species that constitutes the basis of many aquatic trophic chains. In this study, R. subcapitata was used as a model species to investigate the kinetics of uptake and elimination of silver nanoparticles (AgNP) in comparison to silver nitrate (AgNO3) with particular focus on the Ag sized-fractions in solution. AgNP used in this study were provided in a suspension of 1 mg Ag/l, with an initial size of 3–8 nm and coated with an alkane material. Algae was exposed for 48 h to both AgNP and AgNO3 and sampled at different time points to determine their internal Ag concentration over time. Samples were collected and separated into different sized fractions: total (Agtot), water column Ag (Agwater), small particulate Ag (Agsmall.part.) and dissolved Ag (Agdis). At AgNO3 exposures algae reached higher bioconcentration factor (BCF) and lower elimination rate constants than at AgNP exposures, meaning that Ag is more readily taken up by algae in its dissolved form than in its small particulate form, however slowly eliminated. When modelling the kinetics based on the Agdis fraction, a higher BCF was found. This supports our hypothesis that Ag would be internalised by algae only in its dissolved form. In addition, algae images obtained by Coherent Anti-stokes Raman Scattering (CARS) microscopy demonstrated large aggregates of nanoparticles external to the algae cells with no evidence of its internalisation, thus providing a strong suggestion that these AgNP were not able to penetrate the cells and Ag accumulation happens through the uptake of Ag ions.

Tracing engineered nanomaterials in biological tissues using coherent anti-Stokes Raman scattering (CARS) microscopy – A critical review

Abstract Nanomaterials (NMs) are used in an extremely diverse range of products and are increasingly entering the environment, driving a need to better understand their potential health effects in both humans and wildlife. A major challenge in nanoparticle (eco)toxicology is the ability to localise NMs post exposure, to enable more targeted biological effects analyses. A range of imaging techniques have been applied to do so, but they are limited, requiring either extensive processing of the material, staining or use of high intensity illumination that can lead to photo damage and/or have limited tissue penetration. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free imaging technique, providing contrast based on the intrinsic molecular vibrations of a specimen, circumventing the need for chemical perturbation by exogenous labels. CARS uses near infra-red excitation wavelengths which allow microscopy at depths of several hundred microns in intact tissues and minimises photo-damage to live and delicate samples. Here we provide an overview of the CARS process and present a series of illustrative examples demonstrating its application for detecting NMs within biological tissues, ranging from isolated cells to whole organisms and including materials spanning metals to polymers. We highlight the advantages of this technique which include chemically selective live imaging and substantial depth penetration, but we also discuss its limitations when applied to nanotoxicology, which most notably include the lack of resolution for studies on single nanoparticles.

Chapter 1 – Ecotoxicology of Nanomaterials in Aquatic Systems

Abstract The unique properties of manufactured nanomaterials (NMs), conveyed by their small size that are exploited for novel uses, may also confer different toxicological effects compared with their larger (bulk) counterparts. In this first chapter we provide a critical review on current toxicity data for NMs in aquatic organisms and illustrate thorough characterisation of NMs, which is essential for making comparisons on effects data. Adverse effects of NMs have been shown for a wide range of NMs and in diverse aquatic organisms but, with a few exceptions, only for concentrations that exceed current levels in the natural environment predicted by modelling studies. For some NMs, there appear to be particle-induced biological effectsthat include oxidative stress. For some metal NMs most evidence indicates that biological effects derive principally from metal ions due to dissolution. For carbon-based NMs some effects have been shown to derive from materials associated with the preparation of those NMs. The physicochemistry of the aquatic environment fundamentally effects the form of NMs altering their toxicity, and any standardised tests for assessing ‘nano’-related toxicity in aquatic wildlife will need comprehensive characterisation on the dynamics of NMs in relevant test media. Some nanomedicines are being designed specifically to penetrate cell membranes and induce altered biological function and they may warrant particular attention for risk analysis in the near future.

Does natural organic matter increase the bioavailability of cerium dioxide nanoparticles to fish

Environmental context Nanoparticles are present in growing volumes of consumer products and are suspected to be released into the environment at detectable levels. We focus on cerium dioxide nanoparticles and investigate their availability to fish from the water column, where we found increasing concentrations of natural organic material increased the ceria measured in the fish gills. This complex interaction between nanoparticle behaviour and uptake from environmentally relevant test systems is significantly understudied. Abstract Natural organic colloids affect the fate and behaviour of nanoparticles in the aquatic environment but how these interactions affect the bioavailability of nanoparticles to organisms is a major knowledge gap in risk-assessment analysis. Here, we investigated interactions of citrate-coated cerium dioxide (CeO2) nanoparticles with fulvic acids, representing natural organic matter, and assessed their bioavailability to fish (common carp, Cyprinus carpio) exposed chronically (32days) via the water. We show a fulvic acid concentration-related enhancement in the uptake of cerium (Ce) into gill tissues, with some evidence for an enhanced Ce uptake also into kidney and brain tissues in the presence of fulvic acids, but with more variable responses. We present evidence for differences in the aggregation behaviour for CeO2 nanoparticles in the different exposure scenarios, with reduced CeO2 particle aggregate size with citrate coating and fulvic acids, as determined from dynamic light scattering. We highlight that multiple analytical approaches are essential for understanding the dynamic nature of the particles and also that interpretations on measured particle sizes and characteristics may differ depending on the technique(s) employed. We conclude that conditions in natural waters are likely to play a fundamental role in affecting bioavailability and thus potential biological effects of CeO2 particles.