**4. Conclusions**

In this study, 70% of the examined antimicrobials in both wastewater and sewage sludge collected from two Polish WWTPs were detected. To the best of our knowledge, this is the first report on antimicrobial occurrence in Polish WWTPs. The removal efficiency, antimicrobial resistance risk, and ecological threat (RQs) were examined according to the obtained data. The WWTPs removed ~50% of selected antimicrobials with good efficiency, above 50%. The level of antimicrobials in both untreated and treated wastewater, river as well as sewage sludge, poses a risk of resistance selection as shown by RQ calculations. Moreover, influents and river waters posed high and medium risk, particularly for cyanobacteria and eukaryotes due to the presence of ciprofloxacin, macrolides, and sulfamethoxazole. Following the obtained results, the watch list of substances for Union-wide monitoring in the field of water policy (already includes macrolides and ciprofloxacin) should be extended with sulfamethoxazole. Our study also indicates the need for evaluation of antimicrobials' concentrations not only in treated wastewater, but also in sewage sludge because of its usage in the fertilization process, which is environmentally sustainable options for re-use of the WWTP by-products. Several antimicrobials tested were present at levels that have been suggested to promote resistance development in sludge-amended soils (predicted concentrations).

The most important observation made is a possible pressure for the development of antimicrobial resistance in the WWTPs. WWTPs can be considered as potential hot spots for the dissemination of antimicrobial resistance. Leakage of antimicrobials can select for increased resistance among environmental bacteria and influence the virulence of antimicrobial-sensitive bacterial infections directly by reducing the infective dose and transmission [27,29]. Therefore, additional studies on the characterization of wastewater treatment plants' microbial communities and the profiles of antimicrobial-resistant genes are necessary. Our study also highlights the lack of sufficient data to evaluate or predict the risk of resistance development and environmental threat. In fact, data on risk assessment of wastewater and sludge in other European countries is also scarce.

**Author Contributions:** All authors contributed equally to the development of the manuscript in all aspects: Investigations and conceptualization, J.G., G.N.-J., D.K.; writing—original draft, J.G., G.N.-J., G.P.; writing—review and editing, J.G., G.N-J., E.K., M.H., G.P.; funding acquisition, G.P. and J.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was done under the project No 2017/26/M/NZ9/00071 funded by National Science Center, Poland. LC-MS/MS analyses were carried out with the use of the CePT infrastructure financed by the European Union, the European Regional Development Fund within the Operational Program "Innovative economy" for 2007–2013.

**Acknowledgments:** The authors would like to thank Marcin Giebułtowicz for drug sales data analysis and Ryszard Marszałek for his technical support during LC-MS/MS measurement. The authors are also very grateful to Bartosz Kózka and Bartłomiej Sankowski for their contribution in the conduct of the analytical study. We gratefully acknowledge the technical support provided by the WWTPs.

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



**Table A1.** The comparison of the predicted (PLoad) and measured load of antimicrobial into WWTP1.

1 PLoad, predicted load of a compound calculated on the basis of sales data; 2 LoadW, load of a compound calculated on the basis of measured concentration in a water phase of an

influent; 3 LoadS, load of a compound calculated on the basis of measured concentration in a primary sludge; 4 LoadW+S, total load of a compound (water phase and a primary sludge); 5 S summer; A, autumn; W, winter sampling. 6 The pharmaceuticals used only in hospitals and frequently used in veterinary medicine were not presented


**Table 2.** Mean values of sorption coe fficients (log Kd) calculated for sludge and the percent of the total mass of the compound sorbed in sludge to its total daily mass load to WWTP.

**Table 3.** Predicted antimicrobial concentration in sludge-amended soil (single sludge application).


1 WWTP-PS, sludge after mechanical treatment; WWTP-ES, sludge after gravity treatment; WWTP-FS, sludge after an open fermentation pool; WWTP-RS, sludge for management.


**Table 4.** NOEC/EC50 (μg/L) and PNEC (ng/L) used for the risk assessment; nd, no data available. Abbreviations in parentheses indicate the most sensitive taxa: MA, microalgae; MP, macrophytes; IN, invertebrates. The data based on the review of Le Page [27].


**Table 5.** Predicted no e ffect concentration values for resistance selection of microbial community in wastewater, sludge, and soil.

1 According to Bengtsson-Palme and Larsson [28]; 2 calculated based on Kd obtained for sludge; 3 calculated according to [26,40,42,62].
