*4.1. General Effect*

The results of our study revealed that each of the stressors (the NPs and enrofloxacin) on their own influenced most of the measured life history parameters of *D. magna* (except the body length of 5-day-old individuals) and the metabolic and taxonomic diversity of the *Daphnia* gastrointestinal microbiota, and there was an interaction in the effect of both stressors on all of the measured parameters. On the one hand, since we used in our study temperature and food conditions very close to the optimal ones (23 ◦C and 0.6 mg Corg × <sup>L</sup>−1), it is rather unlikely that the stress caused by the suboptimal experimental conditions had any impact on the observed effects of NPs and enrofloxacin. On the other hand, it can be expected that the use of a lower temperature and a higher food concentration could reduce the *Daphnia* filtration rate and, consequently, could reduce the observed effects of the NPs and enrofloxacin, although they would not change the direction of the effects.

#### *4.2. The Effect of Single and Combined Stressors on the Life History Parameters*

The results confirmed our first hypothesis, as both stressors resulted in a decrease in the *Daphnia* body size and their reproductive parameters, including an average egg volume in the brood cavity and the number of eggs in the clutch of individuals during the first reproduction, which also resulted in decreasing the clutch volume being a common currency of the reproductive investment, which suggests that each of the stressors has a negative effect on the fitness of an individual. Although we did not assess the age of the first reproduction of *Daphnia* in the experiments, the greatest number of ovigerous females (in relation to the females without eggs) during the fifth day of the experiment in the control, moderate in the presence of each of the stressors on its own, and the lowest in the treatments with the combined stressors, suggests that each of the stressors delayed the start of reproduction and that there was an interaction in the effect of both stressors.

In the case of the NPs, these results may have been caused by the clogging of the filtration appendages and the gut which, in turn, results in decreasing the filtration and assimilation rate, as demonstrated in several earlier studies, e.g., in [76]. These findings are consistent with a great number of earlier studies in which the presence of NPs suspended in water alters the life history traits of various animals, and that these alterations are manifold [13], including a reduction in the body size of adult and juvenile individuals, reproduction (i.e., decreased numbers and body size of neonates), the individual growth rate and survival of freshwater, e.g., in [77], and saline lakes, e.g., in [78], planktonic animals, cnidarians [79], and fishes, e.g., in [23,80], although other studies did not find any effect of an acute exposure on the life history parameters, including negligible effects on the survival rate and development of *Danio rerio*, e.g., in [81], on the survival and individual growth rate of *Gammarus pulex* [82].

Several studies revealed the significance of antimicrobial drugs on aquatic organisms [83,84]. For example, the parental exposure of marine fish (*Oryzias melastigma*) to sulfamethazine (4.62 mg × <sup>g</sup><sup>−</sup>1) may negatively affect the growth performance in adults [14,85]. In the case of enrofloxacin, the decrease in all the measured life history parameters is consistent with the numerous earlier studies performed on fish [23,85] and *Daphnia* [75]. Another study revealed the negative effect of tetracycline (1 <sup>μ</sup><sup>g</sup> × <sup>L</sup>−1) on the reproduction and survival of *D. magna* [75]. However, it should be pointed out that not all studies reported the effect of antibiotics on the life history parameters. For example, Nunes et al. [86] showed that ecologically relevant ciprofloxacin concentrations (0.005–0.195 mg × <sup>L</sup>−1), did not cause significant impacts on the growth rate and reproductive parameters of *D. magna*, and Ma et al. [87] did not find the effect of tetracycline (10 mg × <sup>L</sup><sup>−</sup>1) on the growth rate of the soil annelid *Enchytraeus crypticus*. Moreover, in a single study, it was revealed that at very

low ciprofloxacin concentrations (10 ng L<sup>−</sup>1), that is, at a level of a lower concentration that the enrofloxacin used in our study, the growth and fecundity of *D. magna* were even higher than that in the control of animals [88].

We are aware of only two earlier studies in which the combined effect of the NPs and antibiotics on the life history parameters was determined [23,87]. While the first study revealed that the effect of tetracycline on the dry weight of *E. crypticus* after a seven-day exposure was stronger in the presence of polystyrene [87], the second study revealed that the adverse impact of the mixture of polystyrene NPs and sulfamethazine on the dry weight in the *O. melastigma* was weaker than the sole effect of the NPs [23]. In our study, the negative effect of each of the stressors (the NPs and enrofloxacin) on the body size was stronger, and on the reproductive parameters was lower in the presence of another factor, which may suggest that in the presence of cumulative stress, *Daphnia* redirects more resources to reproduction at the expense of somatic growth.
