*3.1. The Effect of the Stressors on the Life History Parameters*

None of the stressors (enrofloxacin and NPs) had a significant effect on the *Daphnia* body length (Table S1, Supplementary Materials). **The interaction between the stressors** was also not significant (at *p* < 0.001, Table S1), that is, the effect of one stressor has not been modified by the effect of another stressor. Neither the effect of a single stressor nor the effect of combined stressors was significant (Figures 1a and 2a), neither for the combined data from all the concentrations of NPs and enrofloxacin (Table S2, Figure 2a), nor for the data from each of the concentrations assessed, separately (Table S3, Figure 1a).

Both stressors significantly affected the *Daphnia* body volume (at *p* ≤ 0.001, Table S1). The effect of the NPs was negative, which was apparent in the significant difference between the NEmean and Emean (Table S2, Figure 2b) and between NmEl and El treatments (Table S4, Figure 1b). The effect of enrofloxacin was also negative, which was apparent in the significant difference between the NEmean and Nmean treatments (Table S2, Figure 2b). **The interaction between the stressors** was also significant (at *p* = 0.008, Table S1); more specifically, the presence of one stressor resulted in increasing the negative effect of another one (Tables S2 and S4, Figures 1b and 2b). This was apparent: (1) in the significant difference between the NEmean and Emean treatments (at *p* = 0.031, Table S2, Figure 2b) in comparison to

the non-significant difference between the Nmean and control treatments (Table S2, Figure 2b), (2) in the significant difference between the NEmean and Nmean (at *p* = 0.001, Table S2, Figure 2b) in comparison to the non-significant difference between the Emean and control treatments (Table S2, Figure 2b), and (3) in the significant difference between the NmEl and El treatments (at *p* ≤ 0.017, Table S4, Figure 1b) in comparison to the non-significant difference between the Nm and control treatments (Table S4, Figure 1b).

**Figure 1.** Mean values (± 1 SE) of: (**a**) body length, (**b**) body volume, (**c**) clutch size (the number of eggs per ovigerous female), and (**d**) egg volume of 5-day-old *D. magna* from the control variant (Cont.) and from variants of a single or combined low, medium, and high density of polystyrene NPs (Nl = 103, Nm = 106, and Nh = 109 particles L−1, respectively) and low and high concentration of enrofloxacin (El = 10 and Eh = 100 ng L<sup>−</sup>1, respectively). Statistical significance is accepted at \* *p* < 0.05, \*\* *p* < 0.005, or \*\*\* *p* < 0.0005. The NPs effect is marked on blue, and the enrofloxacin effect on green.

**Figure 2.** Mean values (± 1 SE) of: (**a**) body length, (**b**) body volume, (**c**) clutch size (the number of eggs per ovigerous female), and (**d**) egg volume of 5-day-old *D. magna* from the control variant (Cont.) and from variants of a single or combined mean density of polystyrene NPs (Nmean for the combined data from Nl = 103, Nm = 106, and Nh = 10<sup>9</sup> particles L<sup>−</sup>1) and the mean concentration of enrofloxacin (Emean for the combined data from El = 10 and Eh = 100 ng L<sup>−</sup>1). Statistical significance is accepted at \* *p* < 0.05, \*\* *p* < 0.005, or \*\*\* *p* < 0.0005, ns stands for non-significant. The NPs effect is marked on blue, and the enrofloxacin effect on green.

The NPs and enrofloxacin significantly affected the clutch size (at *p* < 0.001, Table S1). The effect of the NPs was negative, which was apparent in the significant difference between: (1) the Nmean and control treatments (at *p* < 0.001, Table S2, Figure 2c), and (2) each of the concentrations of the NPs and the control, NmEl and El, as well as the NmEl and NlEl treatments (at *p* ≤ 0.005, Table S5, Figure 1c). The impact of enrofloxacin was also negative, which was apparent in the significant difference between: (1) the Emean and control treatments (at *p* < 0.001, Table S2, Figure 2c), and (2) each of the two concentrations in relation to the control (at *p* ≤ 0.005, Table S5, Figure 1c). **The interaction between the stressors** was also significant (at *p* < 0.001, Table S1); more specifically, the presence of one stressor resulted in decreasing the negative effect of another one (Tables S2 and S5, Figures 1c and 2c). This was apparent: (1) in the non-significant difference between the NEmean and Emean treatments in comparison to the significant difference between the Nmean and control treatments (at *p* < 0.001, Table S2, Figure 2c), (2) in the non-significant difference between the NEmean and Nmean treatments in comparison to the significant difference between the Emean and control treatments (at *p* < 0.001, Table S2, Figure 2c), and (3) in the non-significant differences in the majority of comparisons between the single and combined stressors in relation to the significant difference between the single stressors and control for the data from each of the concentrations of NPs and enrofloxacin, separately (Table S5, Figure 1c). The only exception was the significant differences between the NmEl and El, and between the NmEl and NlEl treatments (Table S5, Figure 1c). The number of ovigerous females in relation to the females without eggs was the greatest in the control (84%), moderate in the presence of NPs on their own (81–83%), and in the presence of enrofloxacin on its own (79–80%), and was the lowest in the NmEl (70%) and NhEh (75%) treatments.

The NPs and enrofloxacin also significantly affected the egg volume (at *p* < 0.001, Table S1). The effect of the NPs was negative, which was apparent in the significant difference between: (1) the Nmean and control treatments (at *p* < 0.001, Table S2, Figure 2d), and (2) each of the concentrations of NPs and the control (at *p* ≤ 0.001, Table S6, Figure 1d). The impact of enrofloxacin was also negative, which was apparent in the significant difference between: (1) the Emean and control treatments (at *p* < 0.001, Table S2, Figure 2d), and (2) the Eh and the control, NmEh and Nm, Eh and El, as well as the NmEh and NmEl treatments (at *p* ≤ 0.018, Table S6, Figure 1d). **The interaction between the stressors** was also significant (at *p* < 0.001, Table S1); more specifically, the presence of one stressor resulted in decreasing the negative effect of another one (Tables S2 and S6, Figures 1d and 2d). This was apparent: (1) in the non-significant difference between the NEmean and Emean treatments in comparison to the significant difference between the Nmean and control treatments (at *p* < 0.001, Table S2, Figure 2d), (2) in the non-significant difference between the NEmean and Nmean treatments in comparison to the significant difference between the Emean and control treatments (at *p* < 0.001, Table S2, Figure 2d), and (3) in the non-significant differences in the majority of comparisons between the single and combined stressors in relation to the significant difference between the single stressors and the control for the data from each of the concentrations of NPs and enrofloxacin, separately (Table S6, Figure 1d).

Additionally, the NPs and enrofloxacin significantly affected the clutch volume (at *p* ≤ 0.001, Table S1). The effect of the NPs was negative, which was apparent in the significant difference between: (1) the Nmean and control treatments (at *p* < 0.001, Table S2), and (2) each of the concentrations of the NPs and the control, NmEl and El, NhEl and El, as well as the NmEh and Eh treatments (at *p* ≤ 0.040, Table S7). The impact of enrofloxacin was also negative, which was apparent in the significant difference between: (1) the Emean and control treatments (at *p* < 0.001, Table S2), and (2) each of the two concentrations in relation to the control, the NlEh and Nl, NmEh and Nm, and the NhEh and Nh treatments (at *p* ≤ 0.031, Table S7). **The interaction between the stressors** was also significant (at *p* < 0.001, Table S1); more specifically, the presence of one stressor resulted in decreasing the negative effect of another one (Tables S2 and S7). This was apparent: (1) in the non-significant difference between the NEmean and Emean treatments in comparison to the significant difference between the Nmean and control treatments (at *p* < 0.001, Table S2), (2) in the non-significant

difference between the NEmean and Nmean treatments in comparison to the significant difference between the Emean and control treatments (at *p* < 0.001, Table S2), and (3) in the non-significant differences in the majority of comparisons between the single and combined stressors in relation to the significant difference between the single stressors and the control for the data from each of the concentrations of NPs and enrofloxacin, separately (Table S7).

#### *3.2. Metabolomic Diversity of Gut Microbiota*

The NPs and enrofloxacin significantly affected the mean respiration rate of the gut microbiota expressed as the Vmax values (at *p* < 0.001, Table S1). The effect of the NPs was positive (Tables S2 and S8, Figures 3 and 4), which was apparent in the significant difference between: (1) the Nmean and control treatments (at *p* < 0.001, Table S2, Figure 4), and (2) the Nl and control, Nm and control, NhEl and El, as well as the NhEl and NmEl treatments (at *p* ≤ 0.002, Table S8, Figure 3). The impact of enrofloxacin was negative, which was apparent in the significant difference between: (1) the NEmean and Nmean treatments (at *p* < 0.001, Table S2, Figure 4), and (2) the NlEl and Nl, NlEh and Nl, NmEl and Nm, NmEh and Nm, NhEh and Nh, as well as the NhEh and NhEl treatments (at *p* ≤ 0.001, Table S8, Figure 3). **The interaction between the stressors** was significant (at *p* < 0.001, Table S1), more specifically in the majority of comparisons, the presence of NPs increased the inhibitory effect of enrofloxacin, and the presence of enrofloxacin reduced the positive effect of the NPs (Tables S2 and S8, Figures 3 and 4). The former was apparent: (1) in the non-significant difference between the Emean and control in comparison to the significant difference between the NEmean and Nmean treatments (at *p* < 0.001, Table S2, Figure 4), and (2) in the non-significant difference between the El and control and the Eh and control in relation to the significant difference in the majority of comparisons (in six among nine) between the treatments in which enrofloxacin was combined with the NPs (at *p* < 0.001, Table S8, Figure 3). The latter was apparent (1) in the significant difference between the Nmean and the control (at *p* < 0.001, Table S2, Figure 4) in comparison to the non-significant difference between the NEmean and Emean treatments (Table S2, Figure 4), and (2) in the significant difference between the Nl and the control and the Nm and the control (at *p* < 0.001, Table S8, Figure 3) in relation to the non-significant difference in the majority of comparisons (in 10 among 12) between the treatments in which NPs were combined with enrofloxacin (Table S8, Figure 3).

**Figure 3.** Mean values (±1 SE) of respiration rate expressed as Vmax values of 31 carbon sources by *Daphnia* gut microbiota from the control variant (Cont.) and from variants of a single or combined low, medium, and high density of polystyrene NPs (Nl = 103, Nm = 106, and Nh = 109 particles L−1, respectively) and low and high concentration of enrofloxacin (El = 10 and Eh = 100 ng L<sup>−</sup>1, respectively). Statistical significance is accepted at \*\*\* *p* < 0.0005. The NPs effect is marked on blue, and the enrofloxacin effect on green.

**Figure 4.** Mean values (± 1 SE) of respiration rate expressed as Vmax values of 31 carbon sources by *Daphnia* gut microbiota from the control variant (Cont.) and from variants of a single or combined mean density of polystyrene NPs (Nmean for the combined data from Nl = 103, Nm = 106, and Nh = 10<sup>9</sup> particles L−1) and the mean concentration of enrofloxacin (Emean for the combined data from El = 10 and Eh = 100 ng L<sup>−</sup>1). Statistical significance is accepted at \*\*\* *p* < 0.0005, ns stands for non-significant. The NPs effect is marked on blue, and the enrofloxacin effect on green.

The analysis of the percentage share of the respiration rate of different carbon sources by the gut microbiota of *Daphnia* revealed that the presence of each of the stressors resulted in a relative increase in the usage of carboxylic acids, amino acids, and carbohydrates, as well as a relative decrease in the usage of phosphorylated carbons and complex carbon sources with the control (Figure 5a,b). The pattern was similar in the presence of single and combined stressors, which suggests a negative interaction between their effects.

**Figure 5.** Percentage share of respiration rate (Vmax) of different carbon sources by the gut microbiota of *Daphnia* (**a**) from the control variant (Cont.) and from variants of a single or combined low, medium and high density of polystyrene NPs (Nl = 103, Nm = 106, and Nh = 109 particles L<sup>−</sup>1, respectively) and low and high concentration of enrofloxacin (El = 10 and Eh = 100 ng L−1, respectively), and (**b**) from the control variant (Cont.) and from variants of a single or combined mean density of polystyrene NPs (Nmean for the combined data from Nl, Nm, and Nh) and the mean concentration of enrofloxacin (Emean for the combined data from El and Eh).

A Bray–Curtis-based NMDS analysis revealed two distinct groups of variants: variants with a high concentration of enrofloxacin (in the presence and absence of NPs) and variants with a low and medium density of NPs, which suggests a different effect of each of the stressors on the metabolic profile of the gut microbial community (Figure 6). In the first group, a relatively low usage of phosphorylated carbon and amines compared to the control and the majority of the remaining variants was observed (Figure 5a). In the second group, there was relatively even usage of the different carbon sources with a relatively low usage of the complex carbon sources, as well as a relatively high usage of amines concerning the majority of the remaining variants (Figure 5a).

**Figure 6.** Bray–Curtis-based NMDS analysis of the relative respiration of 31 different carbon sources by *D. magna* gut microbiota from the control (Cont.), and from variants of the combination of two variables: (1) NPs in low, medium, and high concentrations (Nl, Nm, Nh, respectively) and (2) enrofloxacin in low and high concentrations (El and Eh, respectively).
