*2.3. Experimental System*

The system was installed in a room with a constant photoperiod (16 light:8 dark) and comprised of 12 glass containers (L = 25 cm, W = 25 cm, H = 40 cm, large enough to minimise the scale-effects) filled with a 9 L media placed in a water bath (L = 150 cm, W = 50 cm, H = 50 cm, and V = 200 L) with a submersible water-heater (Aquael Neoheather 150 W, Warsaw, Poland) and water pumps (Aquael Circulator 500, Warsaw, Poland) to maintain a stable temperature. The water bath had opaque walls with mounted warm white

(3000 K) LED lamps (5.76 Watts, manufacturer ID: FSLEDWW1200-EF, Green Lighting®, Worcester, UK) inside.

#### *2.4. Experimental Design*

2.4.1. Experimental Protocol

We performed the experiments at the Hydrobiological field station of the University of Warsaw in Pilchy (https://pilchy.biol.uw.edu.pl/, accessed on 1 June 2020). The samples obtained during the experiments were analysed at the station during and after the end of the experiments to assess the community-level metabolic fingerprinting and the life history parameters of *Daphnia*. Some analyses (e.g., the assessment of the taxonomic diversity of bacterial communities) were performed in the laboratories of the Department of Hydrobiology, Faculty of Biology, University of Warsaw. We completed the experimental part between May and July 2021 in 12 variants that represent the combination of 3 enrofloxacin concentrations (0, El = 10 and Eh = 100 ng × <sup>L</sup>−1) and 4 densities of PS-NPs (0, Nl = 1 × 103, Nm = 1 × 106, and Nh = 1 × 109 particles × <sup>L</sup>−1). The concentrations used in our study were within the environmental concentration range [41,42,44–46]. The concentration <sup>1</sup> × 109 × <sup>L</sup>−<sup>1</sup> of NPs corresponds to the range of bacteria abundance in the lake samples. We fixed the temperature at 23 ± 0.3 ◦C, which is close to the thermal optimum of *D. magna* [57], and we supplied the media daily with the same amount of algal food (*Ch. klinobasis*) set close to the limiting concentration of 0.6 mg Corg × <sup>L</sup><sup>−</sup>1. We calculated the organic carbon content from the calibration curve relating the organic carbon concentration to the absorbance level at 800 nm. We chose a high temperature and meagre food for the experiment to increase the *Daphnia* filtration rate. The LED lamps inside the water bath provided homogeneity throughout the water column and a low light intensity (1.0 ± 0.4 <sup>μ</sup>mol × <sup>m</sup>−<sup>2</sup> × <sup>s</sup><sup>−</sup>1) measured by a Li-Cor 189 quantum sensor that measures the radiance (LiCor Biosciences), and it was used at a low light intensity. According to the literature, photodegradation is the main cause of the deactivation of fluoroquinolones, including enrofloxacin, in the environment [58].

At the beginning of each experiment, we added tap water to each of the 12 containers (one container per variant), filtered through 0.45 μm pore membrane filters and aerated for 24 h to reach an oxygen concentration up to 8.00 ± 0.08 mg × <sup>L</sup>−<sup>1</sup> (pH = 7.4, <sup>μ</sup>S cm−<sup>1</sup> = 373 ± 0.7). The physicochemical parameters (the temperature, conductivity, and oxygen concentration) were determined using a multiparametric YSI 6000 probe (Yellow Spring, YSI Inc., Yellow Springs, OH, USA/Xylem Inc., Washington, DC, USA). We added NPs, enrofloxacin, and algal food in the following order. After, 90 newborn (0–24 h) *Daphnia* were collected and randomly distributed into all containers to a final density of 10 ind. × <sup>L</sup>−<sup>1</sup> in each variant. Every six hours, the media was gently mixed. The new media was prepared and replaced every 24 h. During the media preparation, we removed the *Daphnia* from each container using a strainer with a plankton net and placed them temporarily in 250 mL glass containers with the respective media. We prepared the new media in the same order as the initial one. The experiments lasted five days when at least 50% of individuals produced eggs in each variant. At the end of each of the replicates of the experiments, we placed *Daphnia* temporarily in 250 mL glass containers with the respective media. Then, we photographed all of the individuals in order to determine the life history traits. The photographed individuals were transferred to sterile 100 mL plastic containers with milliQ water to remove non-symbiotic bacteria from their guts. Then, we used randomly selected individuals from each variant to determine the diversity of the bacterial community in *Daphnia* guts, both metabolic (25 individuals) and taxonomic (10 individuals).
