*Article* **Combined Effects of Polystyrene Nanoplastics and Enrofloxacin on the Life Histories and Gut Microbiota of** *Daphnia magna*

**Piotr Maszczyk 1,\*, Bartosz Kiersztyn 1, Sebastiano Gozzo 2, Grzegorz Kowalczyk 1, Javier Jimenez-Lamana 2, Joanna Szpunar 2, Joanna Pijanowska 1, Cristina Jines-Muñoz 1, Marcin Lukasz Zebrowski <sup>1</sup> and Ewa Babkiewicz <sup>1</sup>**


**Abstract:** The effect of nanoplastics (NPs) has been shown to interact with the effect of pollutants, including antibiotics. However, little is known about studies performed on freshwater organisms. In this study, we aimed to test the hypothesis that both NPs and antibiotics affect the life history traits of freshwater planktonic *Daphnia magna*, a model organism in ecotoxicological research, as well as the metabolic and taxonomic fingerprint of their gut microbiota, and whether there is an interaction in the effect of both stressors. To assess this, we experimented with the effect of different spherical polystyrene nanoplastic concentrations and antibiotic enrofloxacin measured through (i) the *Daphnia* body size and their selected reproductive parameters (the clutch size, egg volume, and total reproductive investment), (ii) the metabolomic diversity of gut microbiota (the respiration rate and the relative use of different carbon sources), and (iii) the microbial taxonomic diversity in the *Daphnia* intestine. Our results supported the hypothesis as each of the stressors on its own significantly influenced most of the measured parameters, and because there was a significant interaction in the effect of both stressors on all of the measured parameters. Therefore, the results suggest an interactive negative effect of the stressors and a possible link between the observed effects at the different levels of a biological organisation.

**Keywords:** antibiotics; enrofloxacin; nanoplastics; polystyrene; stressors; *Daphnia magna*; gut microbiota; life history; Trojan horse effect
