Introduction
Nanoplastics (NPs), characterized as measuring below 1000 nm, represent a major part of plastic pollution, and are now considered ubiquitous in aquatic ecosystems ( Sharma et al., 2021) . NPs have been detected and quantified in most environmental and urbanised matrices , with polystyrene (PS) being a commonly detected polymer ( Dong et al., 2021 ). Their nanoparticle properties translate into the ability to travel with blood through an organism, and to cross biological barriers, such as the blood/brain barrier ( Ma et al., 2021) . Although plastic contamination has been given increasing consideration over the past decades, little is still known on the effects of prolonged exposures to such pollutants in living organisms. The present study aimed to investigate the response of the commonly farmed gilthead seabream (Sparus aurata) to a waterborne exposure to PS-NPs of 42 nm diameter over a period of 28 days by investigating health and welfare parameters such as haematology and behaviour. In addition, accumulation of PS-NPs in different organs w as investigated.
Material and methods
Juvenile seabream (9.15 ± 0.75 cm total length and 9.09 ± 1.73 g total weight ) were randomly allocated into 9 experimental aquaria, each of which represented a replicate of either of 3 experimental conditions: Control (0µg/L ); low concentration of NPs (100 µg/L) and high concentration of NPs (1000µg/L) . Each aquarium contained 5 fish , and following an acclimation period in the experimental aquaria, the treatments were applied . Behaviour was recorded over a period of 10 minutes (2 minutes before feeding, while feeding and up to 8 minutes after feeding) on the first day of the challenge, and subsequently every 7 days. Following the 28-day exposure period, fish were randomly selected, and blood was extracted through caudal puncture using heparinized syringes. Samples were stored at 4 ºC and analysed using the automated haematological analyser SYSMEX XN-1000V adjusted for fish blood. Following blood extraction, fish were euthanised by spinal rupture, and gills, liver, gut, muscle, and brain were excised and immediately snap-frozen in liquid nitrogen. The video recordings were analysed using ImageJ (Mattson et al., 2015), taking into consideration feeding time, distance travelled during swimming, and exploratory behaviour after feeding. Quantification of PS-NPs in tissue was performed by size exclusion chromatography (SEC) coupled to high-resolution mass spectrometry (HRMS), equipped with an atmospheric pressure photoionization (APPI) working under negative conditions.
Results and discussion
The haematological parameters considered were white blood cell count (WBC), red blood cell count (RBC), haematocrit (HCT), haemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and platelet count (PLT), amongst others. No significant differences were found in any of these parameters, which is in accordance with a previously published study investigating the effects of this polymer in the model organisms Carassius auratus . (Brandts et al ., 2022). Gills, gut and liver were selected for PS-NPs quantification as they may represent a principal portal of entry for fish during waterborne exposure. Brain was also selected for this analysis, as NPs have been shown to cross the blood-brain barrier, and accumulation in this organ is likely to have strong deleterious effects on the health and welfare of fish, which will be potentially reflected by changes in behaviour. On the other hand, muscle was sampled for PS-NPs quantification as it represents a potential source of exposure to this contaminant for humans. Results will include both quantification of PS-NPs and behavioural analyses. At the overall functional level results reveal that variables are not initially affected by NPs, but at molecular and genetic level NP do induce alterations.
References
Brandts, I., Cánovas, M., Tvarijonaviciute , A., Llorca, M., Vega, A., Farré, M., Pastor, J., Roher , N. and Teles, M., 2022. Nanoplastics are bioaccumulated in fish liver and muscle and cause DNA damage after a chronic exposure. Environmental research, 212, p.113433.
Dong, X., Liu, X., Hou, Q. and Wang, Z., 2023. From natural environment to animal tissues: A review of microplastics (nanoplastics) translocation and hazards studies. Science of The Total Environment, 855, p.158686.
Ma, C., Chen, Q., Li, J., Li, B., Liang, W., Su, L. and Shi, H., 2021. Distribution and translocation of micro-and nanoplastics in fish. Critical Reviews in Toxicology, 51(9), pp.740-753.
Mattsson , K., Ekvall , M.T., Hansson, L.A., Linse , S., Malmendal , A. and Cedervall , T., 2015. Altered behavior , physiology, and metabolism in fish exposed to polystyrene nanoparticles. Environmental Science & Technology , 49(1), pp.553-561.
Sharma, V.K., Ma, X., Guo, B. and Zhang, K., 2021. Environmental factors-mediated behavior of microplastics and nanoplastics in water: a review. Chemosphere, 271, p.129597.