The high prevalence of synthetic or modified microfibres (e.g., cellulosic microfibres) has been reported in aquatic environments, including those supporting aquaculture. However, their potential adverse effects on far med fish remain poorly explored. T his study investigated the impact of dietary exposure to viscose-rayon microfibres (RFs) on the performance and tissue-specific transcriptomics of European sea bass ( Dicentrarchus labrax) . A commercial- based diet (CTRL) was formulated without the addition of RFs. Additionally, three diets with RFs (from a 100% viscose-rayon blouse) were prepared using the same formulation : RF1 (0.001 g/kg ), RF2 (0.01 g/kg ), RF3 (0.1 g/kg). G roups of 60 fish (~5.8 g) were grown up for 68 days (May-July ) in triplicate 500 L tanks in a flow-through marine system, f ollowing the natural rise in day-length and temperature (20-27 ºC) . Fish were fed three times per day with automatic feeders near to visual satiety. Water [O2 ] was always kept above 80% saturation. By the end of the trial , all fish were individually weighed and measured . Additionally, samples of liver, white skeletal muscle, head kidney, and anterior intestine were taken for the simultaneous gene expression profiling with customized qPCR-arrays of selected tissue markers (29 per tissue , 80 92
in total) , covering Gh/Igf growth regulation, lipid and energy metabolism, muscle cell proliferation and differentiation, protein turnover, and humoral and innate immune defence.
All fish in the study grew fast (SGR , 2.40-2.43) and efficiently (FCR, 0.95 -0.96), and any impact on growth performance was found with the addition of RFs in the diet., RFs were only detected with only detectable RF crossover in the water and fillets of RF2 and RF3 fed fish . L iver weight increased progressively with the RF addition, varying the hepatosomatic index from 1.98 in RF3 and 1.8 in RF2 to 1.49 in CTRL&RF1 fed fish. In line with this, hepatic lipogenic markers (scd1b ) and components of the negative feedback loop of the de novo lipogenesis (elovl5 ) were only markedly upregulated in RF2 and RF3 fish , while the opposite was found for hepatic lipolytic markers (cpt1a, atgl) . According to this, both in liver and the other analysed tissues, the number of differentially expressed genes w ith a discriminant value (VIP ≥1) in a Partial Least Square-Discriminant Analysis (PLS-DA) was limited to extreme groups (CTRL-& RF1 vs RF2-&RF3) . However, when all data and tissues were put together, the PLS-DA was able to differentiate three main groups (CTRL&RF1, RF2 , RF3) . Such separation was driven by 43 discriminant markers , and its c lustering h eatmap disclosed a clear over-representation of downregulated genes by RF exposure in head kidney, while the upregulation was the dominant response in liver, muscle, and intestine. All this suggests that the impact of RFs on fish performance could be initially considered slight, a lthough the transcriptomic multi- tissue target approach served to envisage the occurrence of a wide range of potential long-term alterations.
This work was supported by the TNA programme (PID: 22708 and 25209) within H2020 AQUAEXCEL3.0 project (871108) to R. Matias for accessing to IATS-CSIC facilities . Financial support from FCT to R. Matias, through a PhD fellowship (2022.10421.BD) is acknowledged.