# Presentation: 201175

## Chemical markers of human waste contamination in source waters: A simplified analytical approach

- TL Jones-Lepp
- United States Environmental Protection Agency
- Office of Research & Development
- NERL-Environmental Sciences Division
- Environmental Chemistry Branch
- Las Vegas,  Nevada

**Chemical markers of human waste contamination in source waters: A simplified analytical approach**

## Introduction

- Giving public water authorities a tool to monitor and measure levels of human waste contamination of waters simply and rapidly would enhance public protection.
- Chemicals shed in feces and urine might be used to detect human waste contamination of environmental waters.
- Sterols
- Bile acids
- Urobilin
- Finding human-use drugs affiliated with
-         urobilin can help define the waste as human.
- Azithromycin (antibiotic) and methamphetamine (substance of abuse) were detected

## Experimental

- Samples
- Grab samples collected, 1L or less, keep cool, above freezing but < 4oC until extraction w/in 24-hrs
- pH adjust to < 3.0 with 12N HCl
- Solid phase extraction
- OASIS HLB cartridges [Waters Corporation (Milford, MA)]
- 6-mL capacity, 0.2 g, 30-μm
- Prep cartridges 5 mL methanol followed by 5 mL DI water at a rate of  1 mL/min
- Load samples, 500 mL, into 60-mL reservoirs (60 mLs at a time); start extractions w/ pump at a rate of 4 mL/min
- Dry samples, via pump, for < 5min.
- Extract with 40 mL methanol:1%acetic acid at a rate of 1mL/min.
- Nitrogen blow-down extract to 0.5 mL – ready for LC/MS analysis.

## Experimental con’t

- μLC-electrospray-ion trap mass spectrometer (ThermoQuest Finnigan LCQTM

- _ES-ITMS_
- positive ionization mode
- Screening - scanned from 120 to 830 amu (full-scan mode)
- Heated capillary - 215°C
-  For quantifying and confirming - Two other modes, selected ion monitoring (SIM) and collision-induced dissociation (CID), were used.

- _HPLC_
- C18 RP, 5 μm particle size, 150 × 3.2 mm liquid chromatography column
- flow rate of 0.40 mL min–1, and a 40:60 split after the column, such that 40% of the flow (160 μL min–1) goes to the ES-ITMS
- Mobile phase: A: 99% water/1 mM ammonium acetate/0.1% acetic acid/1% methanol;
- B: 98% methanol/1 mM ammonium acetate/0.1% acetic acid/2% water.
- 100% mobile phase A (hold for 1 min) to 100% mobile phase B (hold for 5 min) over a 20-min gradient, with a 5-min equilibrium between runs.

## Experimental cont

**Limits-of-detection - ** Using regression analysis on the data obtained from analyzing urobilin at four different concentrations, using full scan mode, the LOD for urobilin was calculated as 32 pg (r2 = 0.999) on-column.
**CID** – Collision energy = 30%

## Sampling Sites

- Southwest – 1 site in Southern Nevada
- Great Lakes – 2 sites on Lake Michigan

- New England – 18 sites
- 9 in Maine
- 9 in Connecticut

## Southern Nevada – 1 site

## Great Lakes – 2 sites Lake Michigan beach site 1 (Silver Beach)       Lake Michigan beach site 2 (Washington Beach)

| 
Sample | Urobilin           Azithromycin 

ng/L                        ng/L | n
 |
| --- | --- | --- |
| Lake Michigan beach site 1 (Silver Beach)       
        June 29, 2004
        July 13, 2004
        July 27, 2004
        August 17, 2004
        September 8, 2004 | 
nd                            nd
nd                            nd
nd                            nd
nd                            nd
no sample | 
3
2
2
2
0 |
| Lake Michigan beach site 2 (Washington Beach)
         June 29, 2004
         July 13, 2004
         July 27, 2004          
         August 17, 2004
         September 8, 2004 | 
no sample
nd                           nd
nd                           nd
nd                           nd
nd                           nd | 
0
2
2
2
2 |

## New England – Region 1 Maine 9 sites

| 
Sample | Urobilin 
ng/L | Azithromycin 
ng/L | n |
| --- | --- | --- | --- |
| AA26900a – Sanford WWTP
AA26900b   laboratory duplicate
AA26901    400 ft downstream Sanford WWTP
AA26902a2 Yarmouth POTW
AA26902b   laboratory duplicate 2
AA26903    Royal River Landing
AA26904    Lewiston WWTP
AA26905    Androscoggin River 0.8 mi downstream 
                        Lewiston WWTP
AA26906    South Portland WWTP
AA26907    Fore River - marina
AA26909    Hampden boat landing | †
†
33
11
15
316
11
21

295
16
52 | 77
75
47
nd
nd
†
nd
†

41
†
4 | 1
1
1
1
1
1
1
1

1
1
1 |

- nd = non-detect; † positive MS/MS identification, but below LOQ; 2 Methamphetamine detected: 5 ng/L.

## S. Portland Maine WWTP & 	Fore River Marina

- S. Portland Maine WWTP & 	Fore River Marina
- AA26906 			AA26907

## Yarmouth Boat Landing     & Yarmouth POTWAA26903			AA26902

- NOTE: Yarmouth water supply tested positive for coliform Aug and Oct 2002.
-  Samples were collected Nov 2002.  Royal River Yarmouth Town Landing same area.

## Hampden Boat Landing

## New England – Region 1 Connecticut9 sites

| 
Sample | Urobilin 
ng/L | Azithromycin 
ng/L | n |
| --- | --- | --- | --- |
| AA29823 – 1.64 miles downstream Manchester WWTP
AA29824 – rural stream no housing in the immediate area
AA29825 – densely populated 60% stream flow wastewater 1.25mi upstream
AA29826 – stream flow 70% wastewater 
AA29827 --  stream flow 90% wastewater
AA29828 -- collected 0.8 km downstream from a senior housing 
            condominium complex, which has its own small WWTP 
AA29829 0.65 mi from WWTP
AA29830 field duplicate of 29829 but collected 15 min apart
AA29831 – 2mi downstream WWTP – failed septic systems 90% wastewater
AA29832 – 2mi downstream WWTP – stream flow 40 % wastewater
Control blank | †
nd
16
nd
†
nd

nd
42
17
22
nd | 13
nd
5
39
34
2

15
23
nd
nd
nd | 1
1
1
1
1
1

1
1
1
1
1 |

- None of the Connecticut samples were collected directly from WWTPs. Most were located 1 km or greater from WWTP sewage outfalls, yet both urobilin and azithromycin were detected in some samples.

- nd = non-detect; † positive MS/MS identification, but below LOQ

## Conclusions

- Detection of urobilin, along w/ a human-use drugs can equivocally show that human waste is entering a water source.
- Tertiary/secondary treatment does not seem to efficiently remove azithromycin, as evidenced by its detection at both the Sanford and Portland WWTPs, as they both have similar environmental loadings, 0.5 kg/yr and 0.6 kg/yr

***Future Research***

-    Investigate correlations between urobilin, nitrate, and coliform levels using principal component analysis

## NOTICE

- Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy. *Mention of trade names or commercial products does not constitute endorsement or recommendation for use.*

***Acknowledgments***

- Dr. Y Miyabara, Shinshu University - Education and Research Center for Inlandwater Environment, for his very thoughtful discussions regarding urobilin as a human waste marker; Dr. Jaci Batista, University of Nevada-Las Vegas for sharing Southern Nevada site information; Mr. Peter Philbrook, USEPA Region 1, for sharing his environmental extracts; and Ms. Elizabeth Sams, USEPA ORD/NHEERL, for sampling throughout the summer 2004, at Lake Michigan.