1. Biodegradation of trace contaminants – pharmaceuticals and personal care products Dr. Abbie Porter May 3, 2010

2. Outline Discuss abundance of these compounds in the environment Overview of bacterial degradation Specific pathways and how they relate to model aromatic degradation pathways Predicting biodegradability

3. What are PPCPs? PPCPs are Pharmaceutical and Personal Care Products. Personal care products include products used for personal hygiene Classified by use, not structure Antimicrobials, fragrances, surfactants

4. Entry into the environment PCPs enter wastewater treatment plants (WWTP) as components of gray water. Pharmaceuticals are also components of the waste stream, but may have been modified via conjugation after being ingested. While there is removal of these compounds during the wastewater treatment process, it may not be complete, which results in environmental release with the treated water Constant use = continued release

5. PPCP environmental abundance Top 5 contaminants included: – DEET (N, N- diethyltoluamide) – Caffeine – Triclosan – 4-nonylphenol Concentrations ranged parts per trillion to parts per billion Kolpin et al. 2002

6. Factors related to PPCP environmental influx Removal in the WWTP – Abiotic – photolysis/photodegradation – Biotic – microbial (bacterial and fungal) degradation – Sorption to biosolids

7. Factors related to PPCP environmental influx WWTP parameters that might have an effect on biodegradation 1.Temperature 2.Hydraulic retention time 3.Solids retention time – Some microbes have a slower growth rate – May require a period of adaptation before degradation – Gene induction – More easily utilized substrates must be removed first

8. Environmental parameters that might have an effect on biodegradation 1.Temperature 2.O 2 availability 3. Availability of alternate electron acceptors 4. Acclimation of indigenous microbial population Factors related to PPCP environmental presence

9. Toxicology concerns: endocrine disruption Definition: interference with endocrine system. – Mimic hormones – Block hormones – Cause hormone production at inappropriate times – Stimulate overproduction of hormones Focus today: estrogen mimicking compounds Estrogenic exposure: *not always visually obvious, usually expression of certain female biomarkers (proteins associated with egg production)

10. Environmental estrogens Effects resulting from estrogen exposure Feminization of male trout – intersexual individuals Changes in sex ratio to female dominant Reduced hatching rates (fish) Weak binding to estrogen receptor Environmentally relevant concentrations Synergistic effects - additive Estradiol Nonylphenol

11. Fate of antimicrobials Compounds: triclosan and triclocarban Toxicity: possible endocrine disruption activity Persistence: One study found triclosan in sediment cores dating back >30 years (Singer et al. 2002) Triclosan can be degraded aerobically (Hay et al., 2001), but not as readily anaerobically Triclocarban can be degraded anaerobically (Miller et al., 2008) but not as readily aerobically. OCl ClHO Cl Triclosan Triclocarban

12. Fate of synthetic musks Trade names Galaxolide (HHCB) and Tonalide (AHTN), HHCB is most commonly used Use: fragrance compounds Toxicity: have shown both estrogenic and estrogen-blocking effects O CH 3 H 3 C H 3 C CH 3 H 3 C CH 3 HHCB 33 CH 3 H 3 C CH 3 CHHC H 3 C O CH 3 AHTN

13. Estrogens Anoxic

14. Estrogens 17α-ethynylestradiol (EE2) is a component of birth control pills Some report EE2 as more recalcitrant than E1, E2, or E3, but there are isolates able to metablize it (strain JCR5) May be co-metabolized with E1, E2, or E3.

15. Model of aromatic degradation

16. Aromatic catabolism Common features: mono- or dioxygenation to activate the ring Formation of catechol or substituted catechols Ring cleavage: either ortho or meta Annu. Rev. Microbiol. 1996. 50:553-590

17. DEET Chemical name: N,N-diethyl-m-toluamide Use: insect repellent Strain: Pseudomonas putida DTB DEET 3-methylbenzoate 3-methylcatechol 2-hydroxy-6-oxo-hepta- 2,4-dienoate diethylamine

18. Ibuprofen Ibuprofen is the 3 rd most widely used pharmaceutical in the world. Chemical name: 2-(4-isobutylphenyl)-propionic acid Use: analgesic, anti-inflammatory frequently found in the environment, but readily degraded Strain: Sphingomonas sp. Ibu-2 Ibuprofen Ibuprofen-CoA Isobutylcatechol

19. Alkylphenol polyethoxylates (APE) Nonionic surfactants Mostly used in agricultural and industrial processes, but about 15% of the total production goes to household use (cleaners, PCPs) Have been banned in the EU

20. APE degradation - aerobic n Di-ethoxylateMono-ethoxylate OR Polyethoxylate n- 1 n- 2

21. APE degradation - anaerobic X nn- 1 n-2 Continued input of APE parent compounds and lack of alkylphenol removal leads to accumulation under anaerobic conditions

22. Alkylphenols Octylphenol (1 isomer) and nonylphenol (>22 isomers) Use: metabolites of alkylphenol polyethoxylates Toxicity: mimic estrogen Strains: Sphingomonas sp. TTNP3, Sphingobium xenophagum Bayram, and Sphingomonas sp. PWE1

23. Hypothesized pathway Meta Cleavage

24. Degradation is isomer dependent NP isomers with low amounts of branching were co-metabolically transformed

25. Degradation via ipso substitution OP Hydroquinone 1,2,4-benzenetriol

26. Examples of ipso substitution substrates OHOHOH a. b.c. OHOHOH a. b.c. OH OH d. e. OH OH OPNP

27. Bisphenol-A Kolvenbach et al. 2007 Chemical name: Bisphenol A Use: plasticizer Toxicity: estrogen mimicking compound Strain: Sphingomonas sp. TTNP3 Mechanism: ipso substitution BPA Hydroquinone 4-(2-hydroxypropan- 2-yl)phenol

28. Predicting biodegradability While PPCPs look different at first, there are structural elements that are frequently found in common, such as the aromatic ring. Based on the literature, it’s possible to make rational hypotheses as to how the chemicals could be metabolized without having done any experiments. There are programs that have compiled all of the known metabolic mechanisms in the literature and use that information to predict reasonable mechanisms for compounds that have not been published yet. This can be very useful. – Keep in mind, experimental data may be more useful that something from an untested model. – Bacteria continue to surprise us. The obvious pathway may not always be in use (OP pathway).

29. Predicting biodegradability Database of published pathways Also a feature to examine the probability that a compound might be degraded through a specific pathway.

30. Predicting DEET Biodegradation http://umbbd.msi.umn.edu/predict/index.html

31. Predicting DEET Biodegradation

33. Predicting OP biodegradation http://umbbd.msi.umn.edu/predict/

34. Predicting OP biodegradation This pathway is similar to what had been predicted earlier for OP biodegradation. However, this does not appear to be the case for OP biodegradation in the specific Sphingomonas strains studied.

35. Notes of caution The biodegradation prediction function is based on rules generated from pathways that are in the literature. There may be multiple pathways for degradation Not all pathways have been identified and are not in the database. While this is useful to provide a starting point for examining biodegradation, this does not outweigh experimental observations – Example: OP biodegradation pathway

36. Reasons to study PPCP biodegradation Environmental persistence Possible toxic or endocrine disrupting effects Widespread use and continual entry into the environment Unknown metabolites – need a way to track the fate of these compounds in the environment