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151 Occurrence of Antibiotics_ Pharmaceuticals and Sterols at Select

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151 Occurrence of Antibiotics_ Pharmaceuticals and Sterols at Select Powered By Docstoc
					      Occurrence of Antibiotics, Pharmaceuticals and Sterols at Select Surface and
                               Wastewater Sites in Iowa

                 Terry G. Cain†, Dana W. Kolpin‡, John D. Vargo†, and Michael D. Wichman†
†
  University of Iowa (State) Hygienic Laboratory, #H101 OH, 102 Oakdale Campus, Iowa City, Iowa 52242-
  5002
‡
  United States Geological Survey, 400 South Clinton Street, Box 1230, Iowa City, Iowa 52244


Abstract

Interest has been expressed in tracing possible sources of contamination (e.g. from human or other waste) by
using compounds such as pharmaceuticals, caffeine, and sterols in waste as potential source markers. This pilot
study was designed to identify and measure a limited set of compounds in aqueous environmental samples to
determine possible relations between these compounds to animal and human activity. Field samples were
collected quarterly for one year from three stream basins – a control stream; a stream potentially affected by
concentrated swine production, and a stream potentially affected by concentrated cattle production. In addition,
samples were collected from two wastewater treatment plant discharges in an urban area. Samples were
analyzed for 34 organic wastewater compounds. A preliminary examination of the data suggests that caffeine,
acetaminophen, ibuprofen, trimethoprim, sulfamethoxazole, bis(2-ethylhexyl)phthalate, and diethyl phthalate,
have the strongest relation to human activity.


Introduction

          Widespread concern has been expressed in Europe and the United States in recent years regarding the
entry of human and animal pharmaceuticals into the environment.1 Chronic effects of exposures to these
compounds has been documented,2,3 but much is yet to be understood regarding their ultimate environmental
effects. Some interest has been expressed in tracing sources of contaminants — for example, from human waste
or concentrated animal feeding operations — by using compounds such as pharmaceuticals, caffeine, patterns of
sterols in waste, or animal feed components such as soybean isoflavones as potential source markers.3-13
          Currently (2004) there are no USEPA approved methods for measuring pharmaceuticals in
environmental samples and research indicates that most investigators have developed methods utilizing such
instrumentation as liquid chromatography/mass spectrometry (LC/MS), liquid chromatography/tandem mass
spectrometry (LC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) to measure these compounds
primarily for their specific studies.14-21 For example, the U.S. Geological Survey Toxic Substances Hydrology
Program implemented a reconnaissance of stream waters throughout the United States in 1999, and USGS
researchers are developing and/or refining the analytical methods to measure emerging prescription, non-
prescription, and other suspected endocrine disrupting compounds.22, 23
          This paper briefly describes GC/MS and LC/MS/MS methods developed to determine human and
animal pharmaceuticals in environmental samples for the compounds listed in Table 1. Three Iowa watersheds
and two wastewater treatment plant discharges were selected for sampling. Water and sediment samples were
collected quarterly from each of these sites over 1-year to evaluate the effect of human and animal activities on
stream water quality. This research provides data on the occurrence of select pharmaceuticals, sterols, and other
organic compounds in the environment, and an examination of their use as potential indicators of human and/or
livestock activity.




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                                            Table 1. Study Analytes

                                                                                       Other Organic
  Non-prescription Drugs           Antibiotics                Sterols             Wastewater Compounds
 Acetominophen                Lincomycin               Cholesterol            1,2,4-Trichlorobenzene
 Caffeine                     Sulfadimethoxine         Coprostan-3-ol         1,4-Dichlorobenzene
 Cotinine                     Sulfamethazine           Dihydrocholesterol     4-Chloro-3-methylphenol
 Ibuprofen                    Sulfamethoxazole         Ergosterol             4-Methylphenol
 Nicotine                     Sulfathiazole            Sitosterol             Acenaphthene
                              Trimethoprim             Stigmastanol           Anthracene
                              Tylosin                  Stigmasterol           Benzo(a)pyrene
                                                                              bis(2-Ethylhexyl)phthalate
                                                                              Diethyl phthalate
                                                                              Fluoranthene
                                                                              Naphthalene
                                                                              N-Nitroso-di-n-propylamine
                                                                              Phenanthrene
                                                                              Phenol
                                                                              Pyrene


Site Selection, Sampling and Analysis
Site Selection

          Three stream basins were selected for sampling to represent a stream affected by intense swine
production, a stream affected by intense cattle production, and a stream relatively unaffected by human activity.
Because of Iowa’s intensive agriculture and numerous “family” hog farms, a control site was difficult to
identify, but Sny Magill Creek, a relatively pristine stream located in northeast Iowa was utilized as the control
site for baseline comparison. Municipal wastewater treatment plant outfalls chosen for this study are in the Iowa
City area. Wastewater received and treated at the Iowa City north plant (NWWTP) includes university, hospital
and residential waste. The Iowa City south plant (SWWTP) receives and treats wastewater from residential and
industrial sources.

Sampling

          Aqueous samples were collected unfiltered, in solvent-rinsed, 1-liter glass bottles with Teflon-lined
lids. Sediment samples were collected in solvent-rinsed, one-pint glass bottles with Teflon-lined lids. All
samples were stored in the dark at 4 °C, extracted within 14 days of collection, and analyzed within 40 days of
extraction. Four aqueous samples (3-clear glass and 1-amber glass liter containers) were collected at each site
for the antibiotics, non-prescription pharmaceuticals, other wastewater contaminants and sterols. One sediment
sample was collected from each site for sterols. Additional samples were collected for quality control (QC)
purposes, either as duplicate and spike samples or matrix spike and matrix spike duplicate samples (MS/MSD)
at a different site each quarter. There was enough sediment sample in the pint container and the amber quart
container to perform QC analysis for the antibiotics and sterols in sediment. Two additional 1-liter samples
were collected for each of the other three analytical series; sterols, caffeine, and wastewater contaminants.

Analysis

      The analytical methods for the determination of non-prescription pharmaceuticals, antibiotics, sterols and
other organic compounds were refined and implemented at the University Hygienic Laboratory (UHL). The
sterol samples were extracted using USEPA SW-846 Method 351024 for water samples or Method 355025 for



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sediment samples. Aqueous samples for caffeine, nicotine, cotinine and other potential organic contaminants
were extracted according to USEPA SW-846 Method 3510.24 The determinative method for caffeine, nicotine,
cotinine, sterols and other organic contaminants was a variation of USEPA Method 8270,26 analysis of
extractable compounds by GC/MS. To improve sensitivity, selected ion monitoring was used for the all the
GC/MS determinations, and the trimethyl silyl derivative was formed for the sterols.
      The analytical method for the determination of the antibiotics as well as the non-prescription analgesics,
acetaminophen and ibuprofen, listed in Table 1, was developed at the UHL.27 Aqueous samples were extracted
using solid phase extraction (SPE) with subsequent analysis by reversed phase HPLC using a tandem
quadrupole LC/MS/MS system equipped with an electrospray (ESI) interface. Most analytes could be
monitored in both positive and negative ion monitoring modes.

Results and Discussion

Non-prescription Drugs

         Maximum concentrations measured for the non-prescription drugs are summarized in Table 2.
Caffeine was detected at all study sites at concentrations ranging from less than reporting limits of 40 ng/L at
the control site to 3,600 ng/L at the north wastewater treatment plant. Nicotine and cotinine, the human
metabolite of nicotine, were detected primarily at the two wastewater treatment plant outfalls. Detected
concentrations of cotinine were greatest at the north wastewater treatment plant outfall at 350 ng/L. Ibuprofen
was detected at all study sites except for the control site. The highest detected concentrations for ibuprofen were
1,100 ng/L at the north plant and 280 ng/L at the south plant. Acetaminophen was found at all sites except the
south wastewater treatment outfall. The detected concentration of acetominophen was greatest at the north
wastewater site at 420 ng/L. These results suggest that non-prescription drugs are relatively ubiquitous in the
sites sampled, but the highest concentrations were found at the sites with the highest component of human waste
(WWTP outfalls).

                           Table 2 – Maximum Concentrations of Non-prescription Study Drugs

                                                                       Sampling Site
              Study Analytes              Control          Cattle          Swine     N WWTP              S WWTP
                                            ng/L            ng/L            ng/L       ng/L                 ng/L
    Caffeine                                    35J              68             93      3,600                  200
    Cotinine                                    14J              20      <      40        350                   34J
    Nicotine                             <      40               11J     <      40        130                   24J
    Acetominophen                              1.1                6            8.2        420            <       1
    Ibuprofen                            <        2              14            2.1      1,100                  280
    Note – J flag represents an estimated concentration, compound positively detected at concentration less than detection
    limit.

Antibiotics

         Maximum concentrations measured for the antibiotics included are summarized in Table 3. Antibiotics
were detected in at least one sample at all study sites. Trimethoprim (65%) and sulfamethoxazole (60%), were
the antibiotics detected with the greatest frequency. Concentrations of these two antibiotics were much higher at
the two WWTP outfalls than at other study sites (Table 3). Sulfathiazole, an animal antibiotic, was detected in
30% of the samples with the highest measured concentrations in the cattle and swine sites. The detection of
three antibiotics in the control basin documents the difficulty in finding a true control basin that is completely
unaffected by human or animal waste in Iowa. Lincomycin and tylosin were not detected in any of the samples
collected in this study (Table 3).




                                                                                                                      153
                                Table 3 – Maximum Concentrations of Study Antibiotics

                                                                    Sampling Site
            Study Analytes             Control           Cattle         Swine       N WWTP      S WWTP
                                         ng/L             ng/L           ng/L         ng/L         ng/L
    Lincomycin                        <        1     <         1      <       1     <      1    <       1
    Sulfadimethoxine                  <        1             2.1      <       1            1          2.8
    Sulfamethazine                           15              1.2      <       1            2    <       1
    Sulfamethoxazole                  <        1             6.4             11        3,100          810
    Sulfathiazole                           1.5              7.3            3.5     <      1            2
    Trimethoprim                            8.2              36              16        1,300        1,100
    Tylosin                           <        1     <         1      <       1     <      1    <       1

Sterols

         Maximum concentrations measured for the sterols included in this study are summarized in Tables 4
and 5. Although sterol compounds are heavily associated with sediment and are not very soluble in water, select
sterols have been detected in unfiltered aqueous samples from streams.21 Consequently, both aqueous and
sediment samples were collected and analyzed for sterols at all study sites. As expected, concentrations
measured in the sediment samples (Table 5) were generally greater than concentrations measured in the aqueous
samples (Table 4). Additional study of these data are necessary to determine if any patterns are present in sterol
concentrations that can be utilized to characterize and differentiate among the study sites.

                           Table 4 – Maximum Concentrations of Sterols in Aqueous Samples

                                                                    Sampling Site
            Study Analytes             Control         Cattle           Swine     N WWTP         S WWTP
                                        µg/L            µg/L             µg/L       µg/L            µg/L
    Cholesterol                             1.9             4.2             1.8         81              10
    Coprostan-3-ol                    <    0.05            0.13            0.17         90             7.5
    Dihydrocholesterol                     0.12            0.23            0.21         11             2.3
    Ergosterol                        <    0.25      <     0.25       <    0.25        3.2               3
    Sitosterol                              2.2               6             1.4         32             6.9
    Stigmastanol                      <    0.05            0.26       <    0.05        4.3             1.1
    Stigmasterol                           0.32             3.2            0.56         15             6.4

                          Table 5 – Maximum Concentrations of Sterols in Sediment Samples

                                                                    Sampling Site
            Study Analytes             Control           Cattle        Swine      N WWTP        S WWTP
                                        µg/kg             µg/kg         µg/kg       µg/kg         µg/kg
    Cholesterol                          2,400               480          1,400      4,300           680
    Coprostan-3-ol                          110                34            61        210           350
    Dihydrocholesterol                      620              130            260        270           210
    Ergosterol                               78              230            870   <     10      <     10
    Sitosterol                           6,300             2,400          4,700      1,200         2,300
    Stigmastanol                         1,400               320            590        160           630
    Stigmasterol                         1,500               490          1,400        240           660




                                                                                                             154
Other Organic Compounds

          Maximum concentrations measured for other organic compounds are summarized in Table 6. Only
seven of the fifteen other organic compounds listed in Table 1 were detected in this study. All seven were
detected at the north wastewater outfall. Bis(2-ethylhexyl)phthalate, a common plasticizer was detected the
most frequently and at the greatest concentration at the north wastewater treatment outfall (Table 6). Phenol was
detected at least once at all study sites. Few detections of these other organic compounds were measured at the
control, cattle and swine sites.

                                Table 6 – Maximum Concentrations of Other Organic Compounds

                                                                               Sampling Site
             Study Analytes                   Control            Cattle            Swine     N WWTP                   S WWTP
                                                ng/L              ng/L              ng/L       ng/L                      ng/L
     1,4-Dichlorobenzene                     <      50         <       50        <      50         60                        90
     4-Methylphenol                          <      50         <       75        <      50        370                 <      50
     bis(2-Ethylhexyl)phthalate                  1,200         < 1,000           < 1,000       1,0000                     1,200
     Diethyl phthalate                       <     250         <     250         <     250        260                 <     250
     Naphthalene                             <      50         <       50        <      50        140                 <      50
     Phenol                                         71                 79               67        220                        67
     Pyrene                                  <      50         <       50        <      50        110                 <      50

     Note - 1,2,4-Trichlorobenzene, 4-Chloro-3-methylphenol, Acenaphthene, Anthracene, Benzo(a)pyrene, Fluoranthene,
     N-Nitroso-di-n-propylamine and Phenanthrene were not detected any samples collected in this study.
     Note – J flag represents an estimated concentration, compound positively detected at concentration less than detection
     limit.

Acknowledgment
The authors wish to acknowledge the assistance provided by Dr. George M. Breuer for his initial guidance for
this project. The authors also wish to acknowledge the staff at the UHL for collection, processing and analyses
of all samples associated with this study. This research was funded by the National Institute for Environmental
Health Sciences through the University of Iowa Environmental Health Sciences Research Center, NIEHS/NIH
P30 ES05605.



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Biographical Sketches
Terry G. Cain: Mr. Cain is a chemist and the supervisor of the GC Analysis section of the University of Iowa
Hygienic Laboratory (UHL) having worked for the UHL in various capacities since 1984. Mr. Cain’s analytical
group works closely with industrial clients, university departments, government agencies, and private clients
offering technical assistance, collaborative research, and testing for a number of programs for regulatory
compliance. Mr. Cain is a trained mass spectral interpretation specialist and served as the Quality Assurance
Coordinator for the UHL. Cain may be contacted by telephone at 319-335-4345 or via e-mail at terence-
cain@uiowa.edu

Dana W. Kolpin: Dana Kolpin is a research hydrologist and has worked for the USGS since 1984. His
research interests include the occurrence of pesticides, pharmaceuticals, and other emerging contaminants in the
environment. Kolpin may be contacted by telephone at 319-358-3614 or via e-mail at dwkolpin@usgs.gov




                                                                                                                                  156
John D. Vargo: Dr. Vargo is a Program Manager at the Iowa Hygienic Laboratory providing oversight to the
HPLC, LCMS, Sample Preparation, and Research and Development sections. He has an extensive background
in environmental methods development, focusing on the use of solid phase extraction with subsequent analysis
by LC/MS/MS techniques. Dr. Vargo has numerous publications and has made many presentations on the use
of LC/MS for the analysis of environmental samples for trace chemical residues. Vargo may be contacted at by
telephone at 319-335-4478 or via e-mail at john-vargo@uiowa.edu

Michael D. Wichman: Dr. Wichman is a Senior Program Manager for Environmental Health Programs at the
Iowa Hygienic Laboratory. He has appointments as an Adjunct Assistant Professor for the Department of
Occupational and Environmental Health in the College of Public Health and as an Investigator in the
Environmental Assessment and Control Research Core and Exposure Assessment Facility for the
Environmental Health Sciences Research Center at the University of Iowa. His current research interests
include simplifying sample preparation techniques, environmental monitoring for pesticide and industrial
chemical degradates, as well as biomonitoring for human metabolites resulting from environmental exposure.
Wichman may be contacted by telephone at 319-335-4479 or via e-mail at michael-wichman@uiowa.edu




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