*4.4. The Effect of Single and Combined Stressors on the Taxonomic Diversity of Gut Microbiota*

Finally, the results also confirmed the fourth hypothesis, as the taxonomic diversity of bacteria was affected by the presence of each of the two stressors on its own, which was apparent in the relative increase in the Firmicutes (mainly the Bacillaceae family, representatives of which occur in the digestive tracts of many aquatic animals, e.g., in [95]), in relation to Actinobacteria and Proteobacteria at the phylum level. Despite this, in most of the experimental variants, the dominant taxa at the phylum level were Proteobacteria, which is consistent with the previous studies on the microbiome of other animals, including the soil fauna [88,96,97]. On the one hand, the relative increase in Firmicutes may have a positive effect on *Daphnia*, as it increases the diversity of the microbial composition, which is often equated with the improvement of the host's health [98]. It has been disclosed that Firmicutes produce short-chain fatty acids, which could be used for the de novo synthesis of lipids or glucose and an additional energy source for the host [23,99,100]. Therefore, from the perspective of the host energy input, Firmicutes in the gut may play a positive role. On the other hand, it has been shown that an increased proportion of Firmicutes to other bacteria phyla is an indicator of metabolic disorders in animals [101,102], which may explain the reduction in the body size and reproductive potential of *Daphnia* by each of the stressors on its own in our study.

In the case of NPs, the relative increase in Firmicutes in the gut microbiota may be due to the fact that NPs can provide them with a better matrix for biofilm growth compared to other bacteria taxa. The results are consistent with several recent studies, which found that NPs may affect a community of free-living bacteria [103,104] and the microbiome of *E. crypticus* [67]. This also includes a marine mollusk *Mytilus galloprovincialis* [105] and various fish species, including *O. melastigma* [106] and *D. rerio*, e.g., [107,108], which can induce dysbiosis and inflammation in their intestine. For example, it has been discovered that the dietary NPs (1 mg × <sup>g</sup>−1) affected the relative proportions of Microbacteriaceae, Streptococcaceae, Enterobacteriaceae, and Rhodocyclaceae in the whole body microbial community of *E. crypticus* [87]. It has been recognised that all of these taxa potentially negatively (with Enterobacteriaceae [109]) or positively (with the remaining taxa [110,111]) affect the host. However, our study contradicts the results of He et al. [23], who found a decrease in the relative abundance of Firmicutes in males exposed to (3.45 mg × <sup>g</sup>−1) dietary polystyrene NPs. It is worth noting that, in our study, the taxonomic diversity of the bacteria increased in the presence of NPs, as the relative abundance of different taxa was more even compared to the controls, which is consistent with the earlier studies for the diversity of the microbial communities in the gut of *D. rerio*, e.g., [107].

The increase in the relative abundance of Firmicutes in the presence of enrofloxacin is consistent with several previous studies, which revealed that antibiotics might change the taxonomic diversity of the gut microbiota of animals, including mice [112] soil organisms [87,102], fish [23], and *Daphnia* [76]. For example, it has been revealed that an exposure to tetracycline (1 <sup>μ</sup><sup>g</sup> × <sup>L</sup>−1) resulted in an increase in the relative abundance of

Pseudomonaceae in the intestinal microbial community of *D. magna* [75]. Other studies have shown that dietary tetracycline (0.01 mg × <sup>g</sup>−1) affected the relative proportions of Microbacteriaceae, Streptococcaceae, Enterobacteriaceae, and Rhodocyclaceae in the whole body microbial community of *E. crypticus* [87]. In another study, Motiei et al. [88] showed that G + bacteria, mostly Actinobacteria and Firmicutes, was better equipped to withstand an exposure to ciprofloxacin (of a similar mechanism of action as enrofloxacin) as their relative abundance increased with the antibiotic concentration.

Moreover, the results also confirmed the fourth hypothesis as we observed an interaction between the two stressors on the bacteria community structure, which was apparent in the weaker effect of each of the stressors on its own than combined. To our knowledge, only two earlier studies investigated the combined effect of NPs and antibiotics [23,88]. The first study showed that the combined exposure of tetracycline and polystyrene NPs negatively affected the abundance of bacteria belonging to several families, including Microbacteriaceae, Streptococcaceae, and Enterobacteriaceae in the whole body microbiome of *E. crypticus*, and additionally, significantly higher ratios of Planococcaceae/Chitinophagaceae and Bacillaceae/Chitinophagaceae were observed after a tetracycline and polystyrene exposure, especially when the two pollutants were combined [87]. The second study revealed that a parental exposure to polystyrene NPs and sulfamethazine had a weaker effect on the gut microbial communities in the offspring of marine fish (*O. melastigma*) than each of the stressors on its own [23]. Moreover, it has been released that after terminating the exposure, the microbiome was not permanently changed but impacted reversibly [87].

Summing up, the NPs and enrofloxacin altered the taxonomic (and also metabolic) structure of the bacterial communities living in the gut of *Daphnia*. The interaction of the enrofloxacin and NPs resulted in changes in the taxonomic composition of the bacterial communities slightly different from those induced by each of the stressors acting separately. This fact may result from the system's complexity, where NPs constitute the sorption surface for the dissolved organic compounds and antibiotics, causing both their local concentration to increase and a change in the conformation of sorbed molecules that can change their physical properties. The formation of biofilms on aggregated plastic particles may also change the dominant bacteria's taxonomic structure. No clear relationship has been found between the bacterial taxonomic composition and metabolic fingerprints. Thus, this may indicate the occurrence of the phenomenon of redundancy and the high plasticity of microorganism communities which, when changing the taxonomic composition, can replace their environmental functions.

#### **5. Conclusions**

In conclusion, despite the growing interest in the effects of NPs and antibiotics on the biology of organisms, knowledge on the subject is still fragmentary and is often based on contradictory results. Our study seems to be the first one to investigate the combined effect of NPs and antibiotics on the life history parameters of freshwater organisms and the metabolomic and taxonomic diversity of their intestinal microbial community. Among the most important results that we obtained is that both stressors: (i) negatively affected most of the *Daphnia* life history parameters, (ii) modified the use of different carbon sources by the intestinal microbiota, (iii) increased the Firmicute phyla participation in the microbiota taxonomic composition, and (iv) interacted with each other, affecting all the measured parameters. Moreover, we also observed that the NPs increased and enrofloxacin decreased the metabolic rate of the gut microbiota. The results of our study suggest a possible link between the observed effects. Future studies concerning the issue should primarily focus on integrating the results from different levels of the biological organisation on the combined effect of both stressors on different taxa.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/w14213403/s1, Tables S1–S8: The results of the analysis of ART two-way ANOVA.

**Author Contributions:** Conceptualization: P.M. and B.K.; methodology: P.M. and B.K.; investigation: E.B., S.G., P.M., C.J.-M. and B.K.; resources: P.M.; data curation: S.G., B.K., P.M., G.K. and M.L.Z.; writing—original draft preparation: P.M.; writing—review and editing: J.P., J.S., J.J.-L., B.K., P.M., E.B. and C.J.-M.; visualization: B.K. and P.M.; supervision: B.K. and P.M.; project administration: P.M.; funding acquisition: P.M. and E.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research described here was supported by the grants no. 2018/31/N/NZ8/03269 and 2019/35/B/NZ8/04523 from the National Science Center, Poland.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
