Aquatic toxicity testing for hazard identification of engineered nanoparticles

Abstract

Within the last few decades, major advances in the field of nanotechnology have enabled production of engineered nanoparticles (ENPs) for various applications and consumer products already available on the market. ENPs may exhibit unique and novel properties compared to their bulk counterparts, which is often related to a high surface-to-volume ratio. These properties have also caused concern amongst scientists and regulators, who have called for timely identification of the potential adverse effects of ENPs to human health and the environment. Despite intensive research on the aquatic toxicity of ENPs, the applicability of the generated data for hazard identification purposes is generally impaired by poor reproducibility and reliability of data, and limited understanding of the underlying effect mechanisms. Consequently, it has been questioned whether the standardized aquatic toxicity tests, developed for testing soluble compounds, are equally applicable for ENPs. The preconditions for aquatic toxicity tests include aqueous solubility of the chemical test compound and stability during incubation. These criteria are not met for ENPs, as they are suspended rather than dissolved in aqueous media. Moreover, ENPs undergo time-dependent transformation processes of agglomeration, dissolution, sedimentation, and interactions with organisms and their exudates. Together, these processes challenge the establishment of traditional concentration-response data by affecting both the exposure and the response axes. The actual exposure experienced by organisms may not be reflected by the ENPconcentration in medium, commonly applied as the exposure metric, and the responses of organisms may result from various toxic and non-toxic mechanisms occurring simultaneously. In this thesis, the challenges related to exposure control and response mechanisms in aquatic toxicity tests with ENPs are addressed through: 1) Exposure timing measures to minimize the transformation processes of ENPs during test incubation, and 2) Multi-dimensional approaches including investigations of other organisms responses than the traditionally applied, and determination of different exposure fractions such as the concentration of dissolved ions from ENPs and body burdens. Although these approaches are scientifically exploratory by nature, the aim is to generate data applicable for regulatory hazard identification of ENPs. The focus has been on the algal growth rate inhibition test and acute and chronic toxicity tests with crustaceans, all commonly applied in a regulatory context. The exposure timing measures included aging of ENPs in test media prior to incubation, and/or shortened exposure duration. For algae, shorter exposure duration was obtained through the application of an acute 2h 14C-assimilation test. For daphnids, a short-term (1-3h) pulse exposure was applied, followed by transfer of the organisms to pure medium, where acute and chronic effects were monitored according to standard guidelines during 48h and 21 days respectively. These approaches assisted to minimize the ENP transformation during incubation, but also influenced the toxicity. While aging of ENPs may both increase and decrease toxicity, the shortened exposure mainly appeared to involve a risk of underestimating, or in worst case overlooking chronic effects in algae and daphnids. Thus, more sensitive endpoints may be relevant for algal tests with shortened exposure, such as oxidative stress, found to occur within few hours’ exposure to certain ENPs. The traditional endpoints of algal growth rate inhibition and daphnia immobility were found to be confounded by physical effects. As examples, the algal growth rate can be inhibited by ENPs physically obstructing the light available to the algae (shading), and the immobility of daphnids may partly result from ENPs adsorbed to these organisms’ exterior. In addition to different effect mechanisms, several exposure fractions are available to interact with the organisms, including ENPs adsorbed to or internalized into the organisms/cells, suspended ENPs and ions dissolved from the ENPs. Together, these various exposure fractions and the multiple effect mechanisms complicate the establishment of traditional concentration-response relationships that are based on a single response and exposure dose-metric. A multi-dimensional approach is therefore suggested for aquatic toxicity testing of ENPs for hazard identification purposes. This includes investigation of both physical and cellular effects in organisms in addition to the traditional endpoints. Also, the different exposure fractions of ENPs should be considered including the adsorbed and internalized fractions in organisms, as well as the dissolved and total concentrations in the medium. In practice it is neither unambiguous nor straightforward to determine the different exposure fractions and effect mechanisms, thus some consensus on the best available practices would be beneficial, as well as harmonization of testing approaches in a regulatory context. Ultimately, a multi-dimensional approach may assist to identify which organism responses and exposure fractions are related and improve our understanding of the concentration-response data generated from aquatic toxicity tests with ENPs.