# Presentation: 579607

## State of Analysis for Pharmaceuticals and Other Organic Contaminants in Multiple Media

**Notes:**
Toxics Program has been funding OWC research since 1998. Note that our research is broader than just pharmaceuticals (encompasses a wide variety of under investigated compounds that are associated with sources of waste (human, agricultural, commercial, industrial waste).

## The Toxics Program:

-     *toxics.usgs.gov*
- Emerging Water Quality Issues:
-     *toxics.usgs.gov/regional/emc.html*

- Dana Kolpin, Iowa City IA
*       **dwkolpin@usgs.gov*
- Larry Barber, Boulder CO
- Kymm Barnes, Iowa City IA
- Mike Focazio, Restion VA
- Ed Furlong, Denver CO
- Sheridan Haack, Lansing MI

- M.T. Meyer, Lawrence KS
*mmeyer@usgs.gov*
- Colleen Rostad, Denver CO
- Steve Zaugg, Denver CO
- E.M. Thurman, Lawrence, KS
- District expertise (e.g. Gail Cordy, Betsy Frick, Kathy Lee, Pat Phillips, Paul Stackelberg)

- Acknowledgements

**Notes:**
Our research group.

## Objectives

- Introduction
- Extractions
- Mass Spectrometry
- Immunoassay
- Selected Results

## Potential Sources – Human and Agricultural

**Notes:**
During 2004 will be collecting waste from a number of human and animal sources.

## Sources 

- What is the                                               contaminant                                                       signature of                                                         specific source types?
- What are the characteristics of the entry mechanism to the environment for different sources?

- Sources
- and Source
- Pathways

- Modified from Halling-Sorenson (1998)

## Diversity of Media

- Liquid Waste (waste-storage structures)
- Manure (dry, wet, fresh, composted)
- Ambient water (SW, GW)
- Soil & amended soils
- Sediment

## Issues and Problems

- Antibiotic Resistance (Pathogens).
- Transference of Resistance.
- Endocrine Disruption.
- Nutrient overloading of soil (what is in stored waste also goes on the field).
- Effects on Waste Treatment
- Runoff and leaching of EMC residues.

## Species Comparisons of Resistance among Enterococci

**Notes:**
Like E. coli isolates, enterococci isolates from swine exhibited a high prevalence of resistance to tetracycline 
Resistance to tylosin base reportedly confers cross resistance to erythromycin.  This may the be case among swine isolates in this study, since the WWTP isolates do not show as strong a correlation between these two resistance patterns.
No vancomycin resistance was observed among either species’ isolates

## Enterococci Resistance in Surface Waters

**Notes:**
Again, the prevalence of resistance tends to increase in surface waters downstream from swine AFOs
The background site here was impacted by human land use.
Again, the cross resistance between tylosin and erythromycin is evident downstream from the swine CAFO but not upstream

## Slide 10

**Notes:**
During the first exposure period in August of 2002, the vitellogenin concentrations were greatest in E2 or 17b-estradiol exposed fish as we expected and again these are not environmentally relevant concentrations they are 10-100 times greater than concentrations detected in WW. The GW control had very low vitellogenin concentrations below the laboratory detection limit.  The APE treatment, the wastewater and the WW passed through XAD-8 had very low concentrations of vitellogenin just slightly above the detection limit. Again indicating slight estrogenicity in the effluent during August. The APE treatment concentrations of APEs was similar to those detected in the wastewater, but were removed from the WWXAD-8 treatment with no change in vtg induction– this says that there is something else besides the APES during this first experiment that elicited the slight vtg induction.

Interestingly, we had a much different result during our second experiment.  Again the GW treatment fish had low vtg concentrations below the detection limit—good.  The Ape treatment had similar response to the first experiment. Now the difference, the WW treated fish had much greater induction during the second experiment in fact in the same order of magnitude as the E2 in the first experiment.  This really peaked our interest in how the effluent chemistry may have changed between the two experiments.  And this time the xad8 removed the extrogenic response back to concentrations that were similar with the first experiemnt. VTG concentrations went from a median of 13 to 0.01 mg/ml.

## Extractions and Sample Preparation

## Solid-Phase Extraction

- Many Types of Materials
- C2-18 on Silica backbone with varying linkages
- Polymers also with hydrophillic-lipophilic functional groups
- Anion Exchange (WC, SC, WA, SA)
- Mixed Mode
- Immunnoaffinity
- Many manufacturers

## Extraction Technologies

- Off-line Solid-Phase Extraction (liquids)
- Cartridges (syringe, sep-pak)
- Disks
- 96-well plates
- Solid-phase microextraction
- On-line Solid-Phase Extraction (liquids)
- Prospekt cartridges
- Accelerated Solvent Extraction (solids)
- Sorbents?

## Solids - Accelerated Solvent Extraction

- Pressure (atm. – 2000 psi)
- Temp. (Ambient – 200 C)
- Multiple cycles

## SPE Example

- 60 mg HLB
- Condition: 2 mL MeOH
- 	3 mL 0.5 N HCl
- 	1 mL H2O
- Load Sample

- Wash: 1 mL water
- Elute: 5 mL MeOH into test tube

- Concentrate:  N2 to 125 L
- Analyze: ESI(+)

## Typical Manual SPE Vacuum Setup

- Typical Manual SPE Vacuum Setup

- 100-1,000 ml

- 96-well plate, 2 ml

## Tandem Reverse Phase/Mixed Mode SPE

**Notes:**
Manipulation of sample pH in off-line SPE provides chance to change isomeric composition.  pH is lowered to 3 using sulfuric acid and diNa-EDTA added. Thus chlortetracycline was reported as total CTET.

## Automated Off-Line SPE (cartridge based)

## Solid-Phase Microextraction

## Sample Preparation (can configure for SPE plates)

**Sample Preparation (can configure for SPE plates)**

## Evaporation concentration

## Typical Concentration Factors:Environmental SPE

- Sample		Extract		Concentration
- Vol. (ml)	Vol. (μL)		Factor
- 100		100 			1000
- 1,000		500-1000 		1000-2000
- 1 μg/L					1-2 mg/L

## On-Line SPE – LC/MS

- 1-10 ml sample, add modifiers just prior to extraction, LC addition of IS. Extract and analyze at same time.

## Slide 24

## Prospekt cartridges available in many Flavors.

##    Methods Development

- _ __  __158 Compounds in Water_
- 45 Antibiotics
- 12 Prescription Drugs
-   8 Nonprescription Drugs
- 14 Hormones and Steroids
- 79 Household and
-          Industrial Compounds

-   _   __83 Compounds in Sediment_
-    3  Antibiotics
-  12  Prescription Drugs
-    7  Nonprescription Drugs
-  61 Household and
-            Industrial Compounds

- Special emphasis:
-  PhAC
-  HAC

*What we find in the environment depends on what we look for and how hard we look.*

## Mass Spectrometry

**Notes:**
At the Kansas OGRL lab we have on-line SPE and LC/tandem-MS.  We wanted to determine whether we could use on-SPE to determine isomeric composition of tetracyclines.

## Mass Spectrometry

- Ionization
- 	GC/MS (EI, CI)
- 	LC/MS (ESI, APCI, APPI)
- Types
- 	High Resolution Magnetic Sector
- 	Ion Trap
- 	Single Quadrapole
- 	Tandem (Triple-Stage Quadrapole)
- 	Time of Flight (TOF)

## Applications of GC/ and LC/MS

- GC/MS: Volatiles
- Priority Pollutants
- Insecticides
- Hydrocarbons
- POPs
- Derivtization for more polar compounds (e.g. surfactants, hormones)

- LC/MS: Nonvolatiles
- Hormones
- Steroids
- Pesticide Degradates
- Herbicides
- Fungicides
- Surfactants
- Pharmaceuticals
- Organometallics

## Ionization in APCI

- Vaporize the neutral pesticide with heat.
- Corona Discharge ionizes the carrier gas.   N2 + e-                 N2+ .   +    2e-
- Carrier gas reacts with water to form hydronium ion.
- Hydronium ion protonates the analyte (M+H)+.

## APCI Source

## Ionization in Electrospray

- Ionization of the solute in solution.
- Nebulize the solution and charge the droplets.
- Desolvation of the droplets by evaporation.
- Desorption of the solution ions to gas phase ions.

## Electrospray Source

## Ionization-Continuum Diagram

## Ion Trap MS/MS

**Ion Trap MS/MS**

## How Q-TOF Works

- Unique Feature is High Resolution of
- Fragment Ions

## Matrix Effects

- Suppression
- Enhancement
- Mostly occur in ESI

## Effects of Humic Material on Signal Intensity

- Relative to buffered solution

## Conclusions

- A variety of choices for sample extraction and clean-up.
- LC/MS-MS preferable for specificity.
- Sensitivity proportional to cost.
- LC/MS ESI subject to matrix effects
- On-line SPE can be used to minimize matrix effects.
- Goal is to minimize sample size and matrix effects.

## Immunoassays

## Immunoassay Types

- Enzyme-Linked Immunosorbent Assays
- 	Coated
- 		Tubes
- 		96-well plate
- 	Magnetic Particle
- Radioimmunoassay
- 	H3, C14

## Coated 96-well microtiter plate

## General Immunoassay Concept

## Tetracycline Cross-Reactivity

- Chlortetracycline*			48.8 %
- Oxytetracycline			32.7 %
- Tetracycline				41.3 %
- Isochlortetracycline		  0.3 %
- Epichlortetracycline		21.3 %

## Macrolide Cross-Reactivity

- Erytrhomycin*			72 %
- Tylosin				59 %
- Lincomycin				12 %

## Immunnoassay Kits

- Antibiotics (RIA, ELISA)
- Hormones (RIA, ELISA)
- 17β-estradiol
- Ethynylestradiol
- Estrone
- Surfactants, Industrial (ELISA)
- Alkylphenols
- Alkylphenol ethoxylates
- Linear alkylbenzene sulfonate
- Bisphenol A
- Pesticides (ELISA, RIA)
- Vitellogenin and others as effects markers
- Abraxis, Beacon, SDI, R-BioPharm, Charm Sciences

**Notes:**
After extraction samples were analyzed for tetracycline antibiotics using LC/electrospray-MS in positive-ion mode.  Mobile phase was H2O-methanol gradient with 20mM (pH 3) formic acid modifier.

## Knowledge to use IA’s Effectively

-  Detection levels vary from 1.5 ppt – 1 ppb
- Dependability in Low and Hi DOC matrices and wastewater (treated and untreated) need to be evaluated.
- Cross-reactivity must be known.
- Coupling SPE to IA can lower detection levels but must be evaluated for matrix effects.
- Can be useful screens for a wide variety of compounds.
- Cost 300-700 for 100 tests. With QA 50 to 80 samples/100 tests.
- With labor, supplies etc. can be $40-to 50/analyte.

**Notes:**
After extraction samples were analyzed for tetracycline antibiotics using LC/electrospray-MS in positive-ion mode.  Mobile phase was H2O-methanol gradient with 20mM (pH 3) formic acid modifier.

## Some General Results

- Reconnaissance Comparison
- Biosolids
- Antibiotics in swine CAFOs
- Antibiotic Residues Fish Hatcheries, CAFOs

## National Reconnaissance Studies 

- - Ground Water (2000)
- - Sources of Drinking Water (2001)
- - Streambed Sediment (2002)

- National Reconnaissance Studies

**Notes:**
Initial field efforts were to determine the occurrence of emerging contaminants to answer the fundamental questions: What compounds enter the environment? What levels do they occur at? And, in what mixtures do they occur?

## GW Recon Focus on Susceptible Settings

- Landfills

- Septic Systems

- Areas with water reuse

- CAFOs

**Notes:**
GW recon again focused on sampling sites considered susceptible to wastewater contamination.

## Summary - National Recon Studies

-                     (N=139)  (N=47)  (N=74)  (N=51)
-  Non_drugs      81%      15%       64%       50%
-  Antibiotics       48         26           26           53
-  Pharms           32           6          23         100
-  Det metabs     69         43          19            --
-  DEET                  74          35          19            --
-  Caffeine          71          11         54           24
-  Fluoxetine         1            4            1         100

**Notes:**
Compounds in yellow are those shown to have environmental effects.

## Metro-Effluent is a Complex Mixture of Compounds

**0**

**2**

**4**

**6**

**8**

**10**

**Alkylphenols**

**Triclosan**

**Plasticizers**

**Fragrances**

**DEET**

- 5-methyl-1H-benzotriazole

**Pharmaceuticals**

******g/L**

**Notes:**
One thing I want to emphasize is that the metro effluent is a complex mixture of many classes of compounds of which we analyzed a small fraction. This graph shows the concentrations of various classes including alkylphenols and other compounds such as.... 

## Municipal Biosolids - General Results

-  Relatively little variation in the number of detections or concentration for a given class of OWCs
-  Variations between products is typically an order of magnitude or less
-  Variations – demographics, time of year, production process

## Occurrence of Antibiotics Swine CAFO Lagoons: 1998-2002

## Spatial Sampling – IA (5000 feeder pigs)

**Spatial Sampling – IA (5000 feeder pigs)**

- 10-22-03

- 11-18-03

- 07-07-03

- Ctet (N, S)	   1440, <		  74, <	       67, <
- Epi-Ctet	     130,   178	  30, <	       41,     92
- Iso-Ctet	   1170, 1260      3770, 3920	   2570, 4600
- Iso-Epi-Ctet	   1120, 1160      1670, 2150       1400, 2720
- Tet		       71, <	         150,   171	      81,  210
- Anhydro-Tet         <, <		   2, <	                  2,       2
- Epi-Tet	     130,     52        170,   150	    140,   240
- _Lincomycin__	__         1,       1__	__   9,       6__	__      44,     38_
- Total-Res	    4062, 2653       5875, 6397	   4245, 7902

**Notes:**
Comparison of N and S side concentrations over time.  Data show that antibiotic composition and concentrations from N and S sides can vary quite a bit. CTET parent always higher on N side. Data also show that total concentrations can vary by a factor of 1.5 to 2 from N and S and over time.  Epi-iso and iso CTET concentrations dominant CTET forms in all samples. 

##  Antibiotics in Fish Hatcheries

-  Initial Recon
-   - Detected in 15% of samples
- 	     - SDM (12%), OTC (4%)

- Follow-up (3 hatcheries)
- - Detected in 31% of samples
-     - SDM (23%), OM (17%), OTC (8%)
- - Max conc = 36 g/L
- - Variable persistance
-     - SDM (48 d), OM (28 d), OTC (20 d)
- - Detections in untreated raceways

## IV. Transport 

- IV. Transport
-       and Fate

- Paired Watershed Study
-   - Fourmile Creek (IA)
-       low-gradient stream
-   - Boulder Creek (CO)
-       high-gradient stream

**Notes:**
In-stream transport and fate is being investigated through a paired watershed study (results still being compiled).

## IN SUMMARY

-  LC/MS-MS techniques wide variety of compounds.
- Variety of instruments. More sensitive less sample.
- Matrix effects an issue affects quantitation and detection.
- On-Line SPE with LC/MS-MS obtain 1 to 10 ppt levels on 10 ml sample can reduce matrix effects.

## IN SUMMARY

- Complex Mixtures  (HACs, PhACs).
-  Need appropriate methods for a wide variety of media.
- Need interdisciplinary studies to determine relationships, if any, between residues and on-site and off-site effects, treatment efficiency, and effectiveness of best management practices.

## IN SUMMARY

- Need sources or supplements for stable isotope labeled compounds.

## Slide 61