1. Antibiotics in the WWTP environment Heike Schmitt, Andrew C. Singer

2. Swine Flu: Netherlands

3. How to model antibiotics at a sewage treatment plant and watershed during a pandemic? Which antibiotics would be used during a pandemic? How much is excreted in the active form?

4. Amoxicillin Doxycycline Moxifloxacin Clarithromycin Levofloxacin Erythromycin Cefotaxime Clavulanic acid Cefuroxime β-lactam Cephalosporin Macrolide Tetracycline Quinolone

5. How much will be given to a patient? Lim (2007) Thorax Antivirals Severely sick Moderately sick 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 Cefuroxime Cefotaxime Amoxicillin Erythromycin Clarithromycin Levofloxacin Clavulanate Moxifloxacin Doxycycline Tamiflu Zanamivir Dose (mg d ) CURB 0-2 CURB 3-5

6. How to model the ecotoxicological effects? Bacteria form the functional unit of sewage works and are key for ecosystem services in the river (and greater environment). Bacteria are also the target organisms of antibiotics  bacterial toxicity investigated Very little information on sensitivity of sewage sludge bacteria  use of MIC values of human pathogens as surrogate (EUCAST database, sensitive wild-types)

7. How to deal with MIC values? – amoxicillin In total, 8 antibiotics, 21-100 species per antibiotic, >1 mio MIC values

8. Species sensitivity distributions Show percent of species that is affected at a given concentration Potentially affected fraction (PAF) N. van Straalen, T. Traas, L. Postuma, T. Aldenberg

9. Complications.. We have data on many different strains of one bacterium per antibiotic  Evaluate different ways of setting up the SSD Cautious: the most sensitive strain (5% percentile) Easy: median of all MICs per strain Most precise: use the whole distribution of values

10. Complications II.. All is easy if sensitivities are normally distributed Is this the case? No..  Evaluate different ways of curve fitting Normal distribution Weibull / logistic curve fit

11. Final model: PAF! Whole distribution of MIC per species curve fit

12. How to model the ecotoxicological effects of many antibiotics simultaneously? All 8 antibiotics are present at the same time Do they act independently or jointly? Independently: drinking alcohole and getting a flower pot on your head Jointly: drinking beer and whine  Apply models for mixture toxicity

13. Mixture toxicity models Calculate joint action based on either of two models Or of a combination of the models Erythromycin, clarithromycin: macrolides (joint action) → msPAF! msPAF = tox REF * Σ ( conc / tox substance )

14. Results: Sewage Works Toxicity Maximum toxicities: 20-30% PAF at R0=2.3

15. Results: Sewage Works Toxicity Maximum toxicities: 20-30% PAF at R0=2.3

16. Sewage Works Toxicity - parameter influence Toxicity model parameters: add 10% variation in toxicity

17. Sewage Works Toxicity – background antibiotics use Normal antibiotic use yields quite some predicted toxicity Reasons: difficult... Bioavailability Bacteria ‘used’ to antibiotics Sensitivity of WWTP bacteria lower

18. Sewage Works Toxicity – background antibiotics use Total toxicity of pandemic and background increases background toxicity by 0.1-16%

19. Toxicity to river stretches

21. Comparison with existing experimental data For shortest-term toxicity, PAF matches experimental data

22. Comparison with existing experimental data II For short-term toxicity, PAF matches experimental data Louvet 2010 Process Biochem, Env Poll

23. Comparison with existing experimental data III 100 ug/L erythromycin (PAF: 22%) in sequencing batch reactors fed synthetic wastewater for 180 days: no effects But: up to 80% decreased functional diversity (ammonium oxidizing bacteria, nitrite oxidizing bacteria) Also: still effects with acclimated sludge in short-term tests with higher concentrations Fan 2009 Appl Microbiol Biotechnol

24. Comparison with existing experimental data IV Limnic bacterial communities (protein synthesis) EC50: around PAF doxycycline of 8-19% Brosche 2010 ET&C

25. And what about antibiotic resistance? WWTP are already a hot-spot for antibiotic resistance (and its transfer) Antimicrobial treatment during pandemics will most likely lead to increased influx of resistant bacteria from human effluent Do antibiotic residues in WWTP also favour resistance maintenance or resistance transfer? Schlüter 2008 J Biotech

26. Experimental evidence Resistance in WWTP of pharmaceutical production plants Concentrations: comparable (penicillin G – PAF of amoxicillin: 34%) / much higher (oxytetracycline) Highest MICs observed for the class of antibiotics produced Also: resistance to unrelated groups Li 2009 Env Microbiol

27. General Conclusions: Ecotoxicity Low viral infectivity (Ro=1.65, R0=1.9): no ecotoxicity risk Medium viral infectivity (Ro=2.3) 20-30% inhibition of sewage works bacterial species, ~40% of river stretches with toxicities between 5 to 30%, when secondary infection rates is 15%. Effects under “shock conditions”? What if limnic communities are affected?

28. Resistance: General Conclusions Increase in resistance likely Human exposure to water-borne resistance?

29. What Next Experimental work to assess vulnerability of sewage works to pandemic quantities of pharmaceuticals needed Assess resistance development under shock concentrations of antibiotics