Bioavailability and Effects of Polystyrene Nanoparticles in *Hydra circumcincta*

*Joëlle Auclair, Brian Quinn and François Gagné*

### **Abstract**

The release of nanoplastics (NPs) from the weathering and degradation of plastics in the environment is an important concern to aquatic ecosystems. The purpose of this study was to examine the bioavailability and toxicity of 50 and 100 nm fluorescently labeled polystyrene nanoplastics (NP) to the invertebrate *Hydra attenuata*. The hydrae were exposed to increasing concentrations of 50 and 100 nm NPs (1.25-80 mg/L) for 96 h at 20°C. A subgroup of hydra was depurated in media to determine the persistence of effects. The results revealed that the animals accumulated detectable amounts fluorescent NP and produced morphological changes at a threshold concentration between 5 and 10 mg/L. The hydrae were able to eliminate 76 and 78% of the 50 and 100 nm NPs, respectively. A characteristic tentacle detachment from the body was observed. Biochemical markers were also determined in exposed organisms and increased glutathione S-transferase (GST) activity, oxidative damage and neutral lipids levels that persisted after the 24 h. In conclusion, NPs are bioavailable to *Hydra*, produce morphological changes and increase oxidative stress and neutral lipids. The formation of neutral lipids could be the result of reduced food assimilation or a means for the elimination of NPs.

**Keywords:** nanoplastics, polystyrene, hydra, oxidative stress, neutral lipids

### **1. Introduction**

The occurrence of plastics in the environment represents a major contamination problem, particularly for the aquatic environment. Indeed, it is estimated that over 8 million tons of plastic are released in oceans per year including micro- and nanoplastic (NP) fragments [1, 2]. Microplastic and NP are operationally defined as particles in the size range of 5 mm to 1 μm and 100–1 nm respectively, but plastics between 0.1 and 1 μm are considered by some authors to be NP [2, 3]. Given their small size, NPs can permeate cells and interact with biological macromolecules, possibly leading to unexpected long-term effects. This is further emphasized by the fact that at present NPs are difficult to measure in organisms due to a lack of bioanalytical methods. Recently, a method based on a fluorescent molecular rotor probe was proposed for NP bioanalysis in the attempt to address this bottleneck [4]. Among plastic polymers found in the marine environment, polystyrene, composed of vinylarene repeats, is the most abundant type accounting for up to 8% of total plastic production worldwide, with an annual production of more than 23 million tons per year [5, 6]. Plastics are subject to both abiotic and biotic weathering leading to degradation into the micro- and nanoscale, dramatically increasing the number of plastic-based nanomaterials in the environment.

As mentioned above, the small size of NPs allows them to be internalized by cells which could lead to severe cell damage such as apoptosis, protein denaturation/ fibrillation and the formation of hydrophobic liquid crystals [7–9]. A number of laboratory studies have investigated the aquatic toxicity of NPs, primarily focusing on marine life, i.e. phytoplankton, zooplankton (crustacean, sea urchins) and bivalves (filter-feeders) [10–13]. NPs were shown to reduce algal growth, the development of sea urchin embryos and shrimp survival and produce immune-related effects in bivalves. These studies show that NPs could be harmful to marine organisms, but to date less information exists for freshwater invertebrates. For this reason, it is of value to include freshwater organisms when assessing the risks of nanomaterials given the potential for their behaviour to be influenced by surface water properties such as salinity, pH and organic matter [14, 15]. For example, in high-salinity environments, e.g. saltwater, low Zeta potential (surface charges), nanoparticles would tend to form aggregates, whereas aggregation would be less likely in low salt conditions [16].

The cnidarian *Hydra circumcincta Schulze*, *1914*, is ubiquitously found in freshwater ecosystems and is often used as sensitive test species for toxicity assessments [17]. Hydra is considered an immortal organism which can regenerate for decades and is composed of a tube-like body (foot and digestive tube) with a head surrounded by six or more antennae to catch food [18]. The severity of toxic effects can be easily observed through characteristic morphological changes [19] (Figure 1S, supplementary data). Moreover, the impacts of chemicals or other stressors can be conveniently followed at multiple levels of biological organization such as behaviour (feeding activity), change in antennae and foot morphology, growth, regeneration, asexual reproduction and various biochemical markers (gene expression, proteomics, oxidative stress, genotoxicity, etc.) [20]. Hydrae are easy to maintain in culture and show unique regeneration activity. Recently, hydrae were used to investigate the effects of microplastics [21]. The study revealed that hydrae were able to ingest small microplastic fragments from facewash soaps that compromised feeding activity and displayed toxicity by morphological changes (antenna regression). The purpose of the current study was therefore to determine the bioavailability and toxicity of fluorescently labeled polystyrene NPs to hydra. Hydrae were exposed to 50 and 100 nm polystyrene NP and analysed for toxicity using morphological changes and biomarkers of energy metabolism (neutral lipids) and oxidative stress to relate the presence of NPs in hydra with toxicity responses.
