*2.5. Statistical Analysis*

To assess the effect of the NPs and enrofloxacin on the life history parameters the (body length, body volume, clutch size, egg volume, and clutch volume) and on the respiration rate of the gut microbiota of *Daphnia*, we used an Aligned Rank Transform for a nonparametric factorial two-way ANOVA (ART ANOVA; ARTool package v.0.11.1 [71], with the "art" function (ARTool package 0.11.1 [72] Washington, USA), which allowed us to fit the model despite the non-normality and heteroscedasticity of the initial data distribution. To verify that the ART procedure was correctly applied and is appropriate for this dataset, the "summary()" function was used. To test the differences in the pairwise combinations of levels between the factors in the interactions, the ART-C method (multifactor contrast test) was conducted by using the "art.con" function (ARTool, [73]). The statistical analysis was performed using the R platform (v.4.2.0, R Team, Vienna, Austria) by setting the level of significance at *α* = 0.05 for all of the statistics.

A Bray–Curtis-based NMDS (non-metric multidimensional scaling) was applied (PAST3 software, [74]), aiming to group the experimental variants according to the microorganism metabolic and phylogenetic differences. The Bray–Curtis dissimilarity was used because, unlike many other common statistical tools (e.g., Jaccard and unweighted UniFrac), it takes into account not only the number of observed ASVs, but also their relative abundance. For the analyses of community-level physiological fingerprinting, we used the relative values of the respiration rate for each carbon source, calculated as the percentage share of each carbon source in the sum of the Vmax for the whole plate. For taxonomic NMDS, we managed the relative abundance of ASV at the family level. The NMDS analysis and data visualisation were performed in Statistica 13 software (StatSoft, TIBCO Software Inc., Palo Alto, CA, USA).

Additionally, a Mantel correlation between the taxonomic and metabolic profiles was performed (PAST3 software) to reveal the correlation between two Bray–Curtis similaritybased matrices of the relative metabolic and relative phylogenetic data.

#### **3. Results**
