1. Fate of Chemical and Biological Emerging Contaminants in Biosolids and After Land Application Ian L. Pepper The University of Arizona The Northwest Biosolids Management Association’s 23 rd Annual Biosolids Management Conference September 19-21, 2010 Campbell’s Conference Center Chelan, WA

2. EMERGING CONTAMINANTS ● Include chemical and biological entities ● By definition are newly discovered contaminants ● Little information on incidence, fate and transport, and potential adverse impacts on human and/or ecological health

3. CHEMICAL EMERGING CONTAMINANTS Endocrine Disrupting Compounds (EDCs ) ● Pharmaceuticals and personal care products (PPLP) ● Steroidal hormones (estrogenic activity) ● Flame retardants (polybrominated diphenyl ethers (PBDEs)

4. Representative Estrogenic Compounds

5. Polybrominated Diphenyl ethers....... Br m Br n - Flame retardants - 229 possible congeners

6. Characteristics of EDCs. ● EDCs interfere with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body that are responsible for the maintenance of homeostasis (normal cell metabolism), reproduction, development, and/or behavior. ● EDCs can be hormone mimics, with hormone-like structures and activities. That is EDCs sometimes have chemical properties similar to hormones and bind to hormone specific receptors in or on the cells of target organs. ● EDCs frequently have lower potency than the hormones they mimic (i.e., require higher dose to elicit an equivalent response), but may be present in water at high concentrations relative to natural hormones. Furthermore, EDCs may not be subject to normal (internal) regulations mechanisms. ● For all known EDCs, there is some dose below which there is no observable response.

7. Most potential adverse effects of EDCs focus on wildlife or ecosystems. ● Eggshell thinning and subsequent reproductive failure of waterfowl ● Reduced populations of Baltic seals ● Reproductive failure in alligators ● Development of male sex organs in female marine animals such as whelks and snails ● Reduced or malformed frog populations ● Disruption of normal sex ratios among exposed populations of fish

8. THE LANDMARK USGS 1999-2000 STUDY ● Barnes et al. (2002) water-quality data for phamaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999-2000. USGS Open-File Report

9. Locations of stream sampling sites in the 1999–2000 reconnaissance of United States surface water quality by USGS.

10. CompoundDescription Detection limit (µg L -1 ) Frequency of detection (%) Max. (µg L -1 ) Median (µg L -1 ) progesterone testosterone 17ß-estadiol 17ß-estradiol estriol estrone mestranol 19-norethisterone 17  -ethinyl estradiol cis-androsterone 4-nonylphenol 4-nonylphenol monoethoxylate 4-nonlyphenol diethoxylate 4-octyphenol monoethyoxylate 4-octyphenoldiethoxylate bisphenol A reproductive hormone reproductive hormone reproductive hormone reproductive hormone reproductive hormone reproductive hormone ovulation inhibitor ovulation inhibitor ovulation inhibitor urinary steroid detergent metabolite detergent metabolite detergent metabolite detergent metabolite detergent plasticizer 0.005 0.005 0.05 0.005 0.005 0.005 0.005 0.005 0.005 0.005 1.0 1.0 1.1 0.1 0.2 0.9 4.1 4.1 9.5 5.4 20.3 6.8 4.3 12.2 5.7 13.5 51.6 45.1 34.1 41.8 23.1 39.6 0.199 0.214 0.093 0.074 0.043 0.027 0.407 0.872 0.273 0.214 40 20 9 2 1 12 0.11 0.017 0.009 0.030 0.019 0.112 0.017 0.048 0.094 0.017 0.7 1 1 0.15 0.095 0.13 Hormones and hormone mimics observed in U.S. surface waters.

11. Representative pharmaceuticals measured in the 1999–2000 UGS reconnaissance of U.S. streams. A comparison of drinking water levels with medicinal doses. Chemical/use Percentage of samples with compound Maximum concentration (µg L -1 ) Medicinal dosage Caffeine/stimulant 716130 mg a Ibuprofen/anti-inflammatory 9.51400 mg b Cimetidine/antacid 9.5 0.58800 mg day 1c 17  -ethinyl estradiol/oral contraceptive 16 0.83120–35 µg d Testosterone/hormone replacement 2.8 0.214150–450 mg e Erythromycin/anti-bacterial21.5 1.71000 mg day 1f Ciprofloxacin/anti-bacterial 2.6 0.03400–800 day 1f a The mass of caffeine in two Excedrin tablets. There is 135 mg of caffeine in an 8-oz. cup of coffee. b The mass of ibuprofen in two tablets of Advil c The lowest adult daily dose of cimetidine d Range of 17  -ethinyl estradiol masses in birth control pills e Range of testosterone masses provided over 3–6 months when used for hormone replacement f Recommended adult dosages

12. Physical, chemical and biochemical properties of selected estrogenic chemicals. Chemical name Molecular weight Water solubility (mg L -1 at 20 °C) Log K ow (L kg -1 ) Relative Estrogenic Activity Nonylphenol (NP)220 5.434.481.2 × 10 -4 Nonylphenol monoethyoxylae 264 3.024.17~10 -5 Octylphenol (OP)206 12.64.12×10 -4 17 ß-estradiol (E 2 )272133.941.0 17  -ethinyl estradiol (EE 2 )296 4.84.151.4 Estrone (E 1 )270133.430.5

13. SUSTAINABILITY OF LAND APPLICATION Huruy Zerzghi University of Arizona

14. STUDY SITE Marana Study begun in 1986 at the U of A (Marana Agricultural Center) Study begun in 1986 at the U of A (Marana Agricultural Center) Biosolids has been continually applied for 20 years (1986 -2005) Biosolids has been continually applied for 20 years (1986 -2005) Crop grown - cotton Crop grown - cotton

15. DESIGN OF THE EXPERIMENT Control (# 1) Control (# 1) Inorganic fertilizer (# 3) Inorganic fertilizer (# 3) Low biosolids rate (1x, # 2) Low biosolids rate (1x, # 2) High biosolids rate (3x, # 4) High biosolids rate (3x, # 4)

16. The twentieth year of biosolids land application (March 1-15, 2005) 8% solids

17. Biosolids land application

18. FATE OF LAND APPLIED ENDOCRINE DISRUPTORS IN SOIL

19. Mean congener (BDE-47, -99, and -209) concentrations in soil in the 0X, 1X, and 3X biosolid loading rate plots.

20. Total mean concentration of PBDE congeners* in soils at the 0-30 cm soil depth compared to household indoor exposure. SourcePBDE Concentration (ng/g) No biosolids29.74 1X biosolids66.99 3X biosolids104.32 Range for indoor dust in U.S. households*1,064 to 3,450 (mean 2,534) From Fromme et al., 2009. *For BDE-47, -99, -153 and -209

21. Soil depth 0-1’ Nonylphenol

22. Dermal Adsorption Daily Exposure Dose Dermal Intake = [(C)(BSA)(SAS)(AF)(OEF)] / [(BS)(1,000)] Where BSA = body surface area (cm 2 /day), SAS = soil adhered to the skin (mg/cm 2 ), AF = fraction of PBDE adsorbed to the skin, and BW = body weight. Total exposure is the sum of ingestion and dermal intake. RISK ASSESSMENT FOR EXPOSURE TO PBDE Chuck Gerba Exposure RouteFactorValue Dermal IntakeWeight (Kg)65 Body surface area (cm 2 /day)4,615 Soil adhered to skin ( µ/cm 2 ) 0.096 PBDE fraction adsorbed through the skin0.03 Outdoor exposure factor (fraction of day spent outdoors) (assumed to be 8 hours) 0.33 Mean concentration of PBDE in soilvaries

23. Ingestion Daily Exposure Dose Daily ingestion exposure dose = [(C) (SIR) (OEF)] / BW Where C = PBDE concentration (ng/g dry weight), SIR = soil ingestion rate (m 3 /day), BW = body weight (kg), and OEF = outdoor exposure fraction (hours spent over a day in the field to which biosolids have been added). RISK ASSESSMENT FOR EXPOSURE TO PBDE Chuck Gerba Exposure RouteFactorValue Ingestion IntakeWeight (Kg)65 Body surface rate (g/day)0.05 Outdoor exposure factor (fraction of day spent outdoors) (assumed to be 8 hours) 0.33 Mean concentration of PBDE in soil (ng/g)varies

24. Evaluation of hazard indices for BDE 47, 99, 153 and 209. Congene r Ingestion dose (ng/day) Dermal dose (ng/day) Ingestion + dermal (ng/day) RfD µg/Kg/day Hazard Quotient* 470.002160.000570.002730.10.0000273 990.00750.001990.008490.10.0000849 1530.001220.000320.001540.20.0000016 2090.01560.01974 7.0.000000282 Hazard index is summation of Hazard Quotient values.

25. Non Cancer Risk ● Hazard Index is several orders of magnitude < 1 suggesting very low risk RISK Cancer Risk ● BDE 209 is only congener where information available to evaluate carcinogenicity - Mean concentration for 3X plot = 61.4 ng/g - Dermal and ingestion exposure = 0.0197 ng/kg/day6 Life Time Risk of Cancer = 9.11 x 10 -14

26. SURVIVAL OF PRIONS IN CLASS B BIOSOLIDS Kazue Takizawa, Chuck Gerba, and Ian Pepper The University of Arizona

27. Kazue Takizawa ● M.S. degree in SWES ● Optimized extraction of prion proteins from Class B biosolids ● Evaluated survival of prions in Class B biosolids at mesophilic and thermophilic temperatures

28. Prion Characteristics ●A normal prion (PrP c ) is composed of mostly alpha- helicies ●An infectious prion (PrP sc) is dominated by beta-sheets ●Resistant to inactivation by proteolytic enzymes, conventional disinfectants, and standard sterilization methods http://www.le.ac.uk/biology/research/phyto/prions.jpg

29. Infectious Prion Conversion ●When a infectious prion PrP sc comes in contact with a normal prion PrP c, it causes PrP c to convert to PrP sc ●Disease occurs when PrP sc concentration threshold is reached PrP c PrP sc

30. Prion Pathogenesis ●Infectious prion proteins (PrP sc ) cause Transmissible Spongiform Encephalopathy (TSE) diseases which affects humans and animals ●Most common Creutzfeldt-Jakob disease (CJD) Soto et al. 2004

31. TSEs in Animals ● Affects sheep, goats, minks, mules, deer, cows, cats, exotic felines, and ungulates ● Most common TSE in animals is called scrapie which affects sheep and goats ● Chronic Wasting Disease (CWD)- affecting deer and elk -Affecting deer population in U.S. :Colorado, Wyoming, Nebraska, New Mexico, South Dakota, Wisconsin

32. FATE OF PRIONS IN THE ENVIRONMENT ● PrP sc adsorbs to clay and organic colloids ● Sorbed prions reported to remain infective ● Prions reported to survive mesophilic anaerobic digestion during wastewater treatment ● Detection via Western Blot technology—did not assess infectivity

33. POTENTIAL ROUTE OF EXPOSURE TO PRIONS VIA LAND APPLICATION Animal Slaughterhouse Wastewater Wastewater Treatment Biosolids HumansCowsLand Application

34. METHODOLOGY ● Class B biosolids—7% solids ● CAD 1A2DS (CAD5) cell line ● Source of prions: Rocky Mountain Laboratory (RML)—infected mouse brain ● Enzyme-linked immunosorbent spot (ELISPOT) assay

35. The original plan was to use ScN2a cells which yielded 0.001% infectivity rate ● Currently, ELISPOT assays performed with CAD5 cells has dramatically increased the yield to a 50% infectivity rate. ● Decreased assay by 8 days Approximately 1,000 prion infected CAD5 cells

36. EVALUATION OF EXTRACTANTS OF PRION PROTEINS FROM BIOSOLID S ● Phosphate buffer saline (PBS) ● 3% beef extract ● Sodium dodecyl sulfate (SDS) ● 8M urea

37. RECOVERY OF PRIONS FROM BIOSOLIDS Extractant% Recovery PBS0.01 3% beef extract<0.001 SDS4 8M urea (room temperature)< 0.002 *4M urea at 80 ◦ C17.2% with no toxicity to cells

38. REDUCTION OF INFECTIOUS PRIONS AT MESOPHILIC (37 ◦ C) AND THERMOPHILIC (60 ◦ C) TEMPERATURES A)In PBS Mesophilic: log 10 N/N 0 (15 days) = ‐1.13 Thermophilic: log 10 N/N 0 (10 days) = ‐1.80 B)Class B biosolids Mesophilic: log 10 N/N 0 (15 days) = ‐2.43 Thermophilic: log 10 N/N 0 (10 days) = ‐3.41

39. INACTIVATION OF PRIONS IN BIOSOLIDS > PBS ● Mechanism: protolytic enzymes that denature proteins ● Ammonia or other substances in biosolids

40. COMPARISON OF STUDIES ● Maluquer de Motes et al. (2008) - prion survival in raw sewage - Western blot technology - 90% reduction of prions after 32.6 days at 20 ◦ C ● Our study - 99.1% reduction after 15 days at 37 ◦ C - 99.9% reduction after 10 days at 60 ◦ C Reason: infectivity assay

41. SUMMARY ● 4M urea an effective extractant of prions from biosolids ● Significant inactivation in biosolids at mesophilic and thermophilic temperatures ● Data needed on inactivation during actual wastewater treatment

42. ONGOING PRION WORK ● Evaluating wastewater treatment effects on prion inactivation - miniature anaerobic digesters (test tubes) - mesophilic anaerobic digestion 37°C - thermophilic anaerobic digestion 60°C - composting

43. FATE OF EMERGING CONTAMINANTS: CONCLUSIONS ● Estrogenic compounds added to soil via land application of biosolids degrade quickly within a matter of weeks ● PBDEs are strongly hydrophobic and sorbed to soil colloids with low solubility and bioavailability ● Overall risk from PBDE via land application is very low. ● Prions are inactivated in biosolids at mesophilic and thermophilic temperatures ● Overall more research is needed in both areas of concern