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

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

CHEMICAL EMERGING CONTAMINANTS Endocrine Disrupting Compounds (EDCs

)

●

Pharmaceuticals and personal care products (PPLP)

●

Steroidal hormones (estrogenic activity)

●

Flame retardants (polybrominated diphenyl ethers (PBDEs)

Representative Estrogenic Compounds

Polybrominated Diphenyl ethers

Br m

. . . . . . .

Br

. . . . . . .

n

- Flame retardants - 229 possible congeners

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.

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

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

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

Hormones and hormone mimics observed in U.S. surface waters.

Compound 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 Description 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 Detection limit (µg L -1 ) 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

Frequency of detection (%) 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

Max. (µg L -1 ) 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 Median (µg L -1 ) 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

Representative pharmaceuticals measured in the 1999–2000 UGS reconnaissance of U.S. streams. A comparison of drinking water levels with medicinal doses.

Chemical/use Medicinal dosage Caffeine/stimulant Percentage of samples with compound 71 Maximum concentration (µg L -1 ) 6 130 mg a Ibuprofen/anti-inflammatory Cimetidine/antacid 17 -ethinyl estradiol/oral contraceptive Testosterone/hormone replacement 9.5

9.5

16 2.8

1 0.58

0.831

0.214

400 mg b 800 mg day 1c 20–35 µg d Erythromycin/anti-bacterial Ciprofloxacin/anti-bacterial 21.5

2.6

1.7

0.03

150–450 mg e 1000 mg day 1f 400–800 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

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 5.43

4.48

1.2

×

10 -4 Nonylphenol monoethyoxylae Octylphenol (OP) 264 206 3.02

12.6

4.17

4.12

~10 -5

×

10 -4 17 ß-estradiol (E 2 ) 17 -ethinyl estradiol (EE 2 ) Estrone (E 1 ) 272 296 270 13 4.8

13 3.94

4.15

3.43

1.0

1.4

0.5

SUSTAINABILITY OF LAND APPLICATION

Huruy Zerzghi University of Arizona

STUDY SITE

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

DESIGN OF THE EXPERIMENT

Control (# 1) Inorganic fertilizer (# 3) Low biosolids rate (1x, # 2) High biosolids rate (3x, # 4)

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

Biosolids land application

FATE OF LAND APPLIED ENDOCRINE DISRUPTORS IN SOIL

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

Concentration (ng /g) 20 30 40 0 10 Concentration (ng/g) 20 30 40 50 BDE 47 60 0 10 50 BDE 99 60 0-30 0-30 30-60 30-60 60-90 60-90 90-120 120-150 3x 1x control 90-120 120-150 3x 1x control 0-30 30-60 0 60-90 90-120 120-150 20 Concentration (ng/g) 40 60 80 100 120 BDE 209 140 3x 1x control

Total mean concentration of PBDE congeners* in soils at the 0-30 cm soil depth compared to household indoor exposure.

Source

No biosolids 1X biosolids 3X biosolids Range for indoor dust in U.S. households*

PBDE Concentration (ng/g)

29.74

66.99

104.32

1,064 to 3,450 (mean 2,534)

From Fromme et al., 2009.

*For BDE-47, -99, -153 and -209

Soil depth 0 1’ 1400 1200 1000 800 600 400 200 0 control

Nonylphenol

1x 3x rep 1 rep 2 rep 3 rep 4 threshold

RISK ASSESSMENT FOR EXPOSURE TO PBDE

Chuck Gerba

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.

Exposure Route

Dermal Intake

Factor

Weight (Kg) Body surface area (cm 2 /day) Soil adhered to skin ( µ/cm 2 ) PBDE fraction adsorbed through the skin Outdoor exposure factor (fraction of day spent outdoors) (assumed to be 8 hours) Mean concentration of PBDE in soil

Value

65 4,615 0.096

0.03

0.33

varies

RISK ASSESSMENT FOR EXPOSURE TO PBDE

Chuck Gerba

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).

Exposure Route

Ingestion Intake

Factor

Weight (Kg) Body surface rate (g/day) Outdoor exposure factor (fraction of day spent outdoors) (assumed to be 8 hours) Mean concentration of PBDE in soil (ng/g)

Value

65 0.05

0.33

varies

Evaluation of hazard indices for BDE 47, 99, 153 and 209.

Congene r

47 99 153 209

Ingestion dose (ng/day)

0.00216

0.0075

0.00122

0.0156

Dermal dose (ng/day)

0.00057

0.00199

0.00032

0.01974

Ingestion + dermal (ng/day)

0.00273

0.00849

0.00154

0.01974

RfD µg/Kg/day

0.1

0.1

0.2

7.

Hazard index is summation of Hazard Quotient values.

Hazard Quotient*

0.0000273

0.0000849

0.0000016

0.000000282

Non Cancer Risk RISK

●

Hazard Index is several orders of magnitude < 1 suggesting very low 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

SURVIVAL OF PRIONS IN CLASS B BIOSOLIDS

Kazue Takizawa, Chuck Gerba, and Ian Pepper The University of Arizona

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

Prion Characteristics

●

A normal prion (PrP c ) is composed of mostly alpha helicies

●

An infectious prion (PrP sc) dominated by beta-sheets is

●

Resistant to inactivation by proteolytic enzymes, conventional disinfectants, and standard sterilization methods http://www.le.ac.uk/biology/research/phyto/prions.jpg

Infectious Prion Conversion

PrP c PrP sc

●

When a infectious prion PrP sc comes in contact with a normal prion PrP c , it causes PrP c to convert to PrP sc

●

PrP sc Disease occurs when concentration threshold is reached

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

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

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

POTENTIAL ROUTE OF EXPOSURE TO PRIONS VIA LAND APPLICATION

Animal Slaughterhouse Wastewater Humans Cows Wastewater Treatment Land Application Biosolids

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

Approximately 1,000 prion infected CAD5 cells 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

EVALUATION OF EXTRACTANTS OF PRION PROTEINS FROM BIOSOLID S

●

Phosphate buffer saline (PBS)

●

3% beef extract

●

Sodium dodecyl sulfate (SDS)

●

8M urea

RECOVERY OF PRIONS FROM BIOSOLIDS

Extractant PBS 3% beef extract SDS 8M urea (room temperature) *4M urea at 80

◦

C % Recovery 0.01

<0.001

4 < 0.002

17.2% with no toxicity to cells

REDUCTION OF INFECTIOUS PRIONS AT MESOPHILIC (37

◦

C) AND THERMOPHILIC (60

◦

C) TEMPERATURES

A) In PBS Mesophilic: log 10 N/N 0 Thermophilic: log 10 N/N (15 days) = ‐1.13

0 (10 days) = ‐1.80

B) Class B biosolids Mesophilic: log 10 N/N 0 Thermophilic: log 10 N/N (15 days) = ‐2.43

0 (10 days) = ‐3.41

INACTIVATION OF PRIONS IN BIOSOLIDS > PBS

●

Mechanism: protolytic enzymes that denature proteins

●

Ammonia or other substances in biosolids

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 - 99.9% reduction after 10 days at 60

◦

C

◦

C Reason: infectivity assay

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

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

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