&EPA
March 2023
EPA 820-R-23-003
National Pilot Study of
Pharmaceuticals and Personal Care
Products in Fish Tissue
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Notice
This report was prepared by the U.S. Environmental Protection Agency, Office of Water,
Office of Science and Technology. The results of this pilot study were published in a tech-
nical journal article (Ramirez et al., 2009) that was subjected to an external peer review
process. The EPA Project Manager for preparation of this document was Leanne Stahl who
provided overall project coordination and technical direction. Tetra Tech, Inc. provided
primary support for the development of this document under Contract Numbers EP-C-04-
030 and EP-C-09-019. Blaine Snyder was the Tetra Tech, Inc. Project Manager. Tetra Tech
subcontracted Baylor University's Center for Reservoir and Aquatic Systems Research under
Contract Numbers EP-C-04-030 and EP-C-09-019 to conduct the fish tissue analysis for
this pilot study. Additional support was provided by Computer Sciences Corporation under
Contract Number EP-W-06-046.
The U.S. Environmental Protection Agency, Office of Water, Office of Science and Technology
has approved this report for publication. Mention of trade names, commercial products, or
services does not constitute official EPA approval, endorsement, or recommendation for use.
The appropriate citation for this document is:
U.S. Environmental Protection Agency (USEPA). 2023. Pilot Study of Pharmaceuticals
and Personal Care Products in Fish Tissue. EPA 820-R-23-003. U.S. Environmental
Protection Agency, Office of Water, Washington, DC.
This document (including appendices) can be downloaded from the following
EPA Office of Water website:
https:llwww.epa.govlfish-tecl) I pilot-study-pharmaceiiticals-and-personal-care-prodiicts-
fish-tissue
Photo credits!
Blaine D. Snyder Tetra Tech. Inc., Owings Mills, MD
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National Pilot Study of
Pharmaceuticals and Personal Care
Products in Fish Tissue
U.S. Environmental Protection Agency
Office of Water
Office of Science and Technology
March 2023
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Table of Contents
Acknowledgements iii
List of Acronyms and Abbreviations v
Executive Summary vii
1.0 Introduction 1
2.0 Study Design and Approach 3
2.1 Background 3
2.2 Study Design Development 3
2.2.1 Site Selection 3
2.2.2 Target Chemicals 7
2.3 Mobilization 8
2.4 Sample Collection 9
2.5 Sample Analysis 10
2.5.1 Fish Tissue Sample Preparation 10
2.5.2 Analytical Methods 11
2.6 Data Analysis 14
2.7 Identifying and Quantifying Target Chemicals 14
3.0 Results 17
3.1 Chemicals Not Detected 18
3.1.1 Reference Site Results 18
3.1.2 Pharmaceutical Compounds 19
3.1.3 Personal Care Product Chemicals 19
3.2 Detected PPCP Chemicals 20
3.2.1 Pharmaceutical Results in Fillets 23
3.2.2 Pharmaceutical Results in Livers 23
3.2.3 Results for Personal Care Product Chemicals in Fillets 24
3.3 Lipid Results 25
4.0 Conclusions and Future Research 27
4.1 Discussion of Pilot Study Results 27
4.2 Pilot Study Conclusions 28
4.3 Future Research 29
4.3.1 National Urban River CEC Study 29
4.3.2 Great Lakes Human Health Fish Tissue Study 30
5.0 References 33
Appendix A Analytical Procedures
Appendix B Site-specific Analytical Results Tables for Pharmaceuticals in Fillet Tissue
Appendix C Site-specific Analytical Results Tables for Pharmaceuticals in Liver Tissue
Appendix D Site-specific Analytical Results Tables for Personal Care Product Chemicals
Appendix E Lipid Content in Fish Fillet and Liver Tissue Samples
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Table of Contents
Figures
Figure 1. Sampling locations for the PPCP Fish Pilot Study 5
Tables
Table 1. Wastewater Treatment and Discharge Information for Facilities in the Vicinity
of Each Sampling Location and Population Characteristics of Associated Cities 6
Table 2. Target Pharmaceutical Chemicals, Uses, and Chemical Abstracts Service (CAS)
Registry Numbers for the PPCP Fish Pilot Study 7
Table 3. Target Personal Care Product Chemicals, Uses, and Chemical Abstracts Service
(CAS) Registry Numbers for the PPCP Fish Pilot Study 8
Table 4. Fish Collected for the PPCP Fish Pilot Study 9
Table 5. Method Detection Limits for Pharmaceutical Chemicals Analyzed by
HPLC-MS/MS in Fish Fillet and Liver Composite Samples 12
Table 6. Method Detection Limits for Personal Care Products Analyzed by GC-MS/MS
in Fish Fillet Tissue Composite Samples 13
Table 7. Pharmaceutical Chemicals Not Detected in Fillet or Liver Tissue 19
Table 8. Personal Care Product Chemicals Not Detected in Fillet Tissue 20
Table 9. Analytical Results for Pharmaceutical Compounds in Fillet Composite Samples 22
Table 10. Analytical Results for Pharmaceutical Compounds in Liver Composite Samples 22
Table 11. Analytical Results for Personal Care Product Chemicals in Fillet Composite Samples. ...22
Table 12. Matrix Spike Recovery Data for Four Pharmaceutical Compounds Detected in Liver
Composite Samples that Exceeded Acceptable Limits9 24
Table 13. Lipid Percentage in Fish Fillet and Liver Composite Samples 26
ii
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Acknowledgements
The U.S. Environmental Protection Agency's (EPA) Office of Science and Technology
(OST) within the Office of Water (OW) planned, funded, organized, and implemented
this National Pilot Study of Pharmaceuticals and Personal Care Products (PPCPs) in Fish
Tissue and coordinated the efforts of participating researchers. EPA's Office of Research
and Development (ORD) provided technical guidance for study design development.
Within OST, Leanne Stahl served as the national pilot study manager and was supported
by the pilot study management team consisting of the following EPA and contractor staff:
John Wathen (EPA/OST); Blaine Snyder (Tetra Tech, Inc.); and Harry McCarty (General
Dynamics Information Technology, formerly Computer Sciences Corporation). The pilot
study management team sincerely thanks Ephraim King, Suzanne Rudzinski, Denise
Keehner, and Jim Pendergast (EPA/OST), along with John Hochheimer (Tetra Tech, Inc.) for
their leadership, oversight, and management support.
EPA/OST teamed with Tetra Tech, Inc. and Baylor University for chemical analysis of all
fish tissue samples collected for the PPCP Fish Pilot Study. John O'Donnell (Tetra Tech,
Inc.) provided oversight of analytical activities throughout the pilot study. Members of
the research team from Baylor's Department of Chemistry and Biochemistry, Department
of Environmental Science, and their Center for Reservoir and Aquatic Systems Research
included Richard Brain, Bryan Brooks, Kevin Chambliss, Laura Dobbins, Pilar Perez-
Hurtado, Mohammad Mottaleb, Alejandro Ramirez, and Sascha Usenko.
EPA would also like to express appreciation to the following professionals who assisted with
sample collection activities: Chad Barbour, Carolina Gallardo, Henry Latimer, and Jennifer
Pitt (Tetra Tech, Inc.); Gary Schiffmiller and Shann Stringer (New Mexico Environment
Department); Elizabeth Murphy and Todd Nettesheim (Great Lakes National Program
Office); and biologists from the Metropolitan Water Reclamation District of Greater
Chicago.
The principal authors of this report are Leanne Stahl (EPA/OST), Blaine Snyder, John
O'Donnell, and Ann Roseberry Lincoln (Tetra Tech, Inc.). Kevin Chambliss and Bryan
Brooks (Baylor University) provided all analytical results for this report. David Wells and
Ed Partington (EPA/OST) aided in the sampling site selection process. Tetra Tech, Inc.
formatted the statistical analysis results, integrated text and graphics developed by the
authors, and provided support for final report production.
iii
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
List of Acronyms and Abbreviations
° c
pL
BOD5
CAS
CECs
CFR
cm
d
ESI
g
GC
GC-MS/MS
GLNPO
GPC
HPLC
HPLC-MS/MS
IS
L
LC
M
MDL
mg
MGD
min
mm
MS/MS
MS/MSD
MSTFA
mTorr
n2
NA
NCCA
degrees Centigrade
microliters
5-day biological oxygen demand
Chemical Abstracts Service
contaminants of emerging concern
Code of Federal Regulations
centimeter
day
electrospray interface
gram
gas chromatography
gas chromatography-tandem mass spectrometry
Great Lakes National Program Office
gel permeation chromatography
high performance liquid chromatography
high performance liquid chromatography-tandem mass spectrometry
internal standard
liter
liquid chromatography
molar concentration
method detection limit
milligram
million gallons per day
minutes
millimeters
tandem mass spectrometry
matrix spike/matrix spike duplicate
N-methyl-N-trimethylsilyltrifluoracetamide
millitorr
nitrogen gas
not applicable
National Coastal Condition Assessment
v
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
List of Acronyms and Abbreviations
ng nanogram
NPDES National Pollutant Discharge Elimination System
NRSA National Rivers and Streams Assessment
ORD Office of Research and Development
OST Office of Science and Technology
OW Office of Water
PBDEs polybrominated diphenyl ethers
PCP personal care product
PFCs perfluorinated compounds
PFOA perfluorooctanoic acid
PFOS perfluorooctanesulfonic acid
pH hydrogen ion concentration, a measure of acidity
ppb parts per billion
PPCPs pharmaceuticals and personal care products
ppm parts per million
psi pounds per square inch
QAPP quality assurance project plan
QC quality control
R2 correlation coefficient
rpm revolutions per minute
RRF relative response factor
RSD relative standard deviation
s second
U.S. EPA United States Environmental Protection Agency
UV ultraviolet
V volt
v/v volume/volume ratio
WWTP wastewater treatment plant
vi
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Executive Summary
Pharmaceuticals and personal care products (PPCPs) are a diverse group of chemicals that
have recently received attention as potential environmental pollutants. PPCPs enter the
aquatic environment primarily as a result of their persistence through the wastewater treat-
ment process and resulting discharge to surface or ground water. Most existing information
on the environmental occurrence of PPCPs focuses on wastewater discharges and surface
waters, although an increasing body of literature indicates that certain PPCPs can accu-
mulate in fish. To date, studies of PPCPs in fish tissue generally targeted a specific chemi-
cal or chemical class at a single study location. E PA's Office of Science and Technology
(OST) responded to this data gap by designing and conducting the National Pilot Study of
Pharmaceuticals and Personal Care Products in Fish Tissue. The specific purpose of the pilot
study was to advance the
science of detecting PPCPs
in the environment by inves-
tigating the occurrence of a
broad suite of PPCPs in fish
collected from several U.S.
streams.
EPA selected fish sam-
pling sites on five effluent-
dominated streams in
population centers near
wastewater treatment plant
(WWTP) discharges based
on the assumption that
PPCPs were more likely
to occur in those areas.
These sites included the
North Shore Channel in
Chicago, Illinois; Trinity River in Dallas, Texas; Little Econlockhatchee River in Orlando,
Florida; Salt River in Phoenix, Arizona; and Taylor Run in West Chester, Pennsylvania.
EPA also obtained fish from the East Fork Gila River in the Gila River Wilderness Area of
New Mexico to represent a reference condition or an area of minimal human influences
and impacts. Field crews collected six composites of adult fish of the same resident species
from each sampling location during late summer and fall of 2006. Every composite sample
contained three or four fish that were individually wrapped as whole-body specimens and
collectively bagged as a composite. All fish were frozen on dry ice, shipped to the analytical
laboratory at Baylor University, and stored frozen at < 20° C prior to preparation of fillet
and liver tissue samples for analysis.
North Shore Channel, Chicago, Illinois
vii
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Executive Summary
r
In the laboratory, techni-
cians removed the entire
fillet (including the skin
and belly flap) from both
sides of each fish in the
composite sample, using all
available tissue to prepare
the fillet composite sample
(i.e., the batch method).
They homogenized fillet
tissue using a high-speed
enate samples in a freezer
at < 20° C prior to analy-
sis, Laboratory personnel
removed fish livers from
each fish by dissection
blender and stored homog-
Reference site, East Fork Gila River, New Mexico
and applied compositing,
homogenization, and storage techniques that mirrored those for fillet samples.
Scientists at Baylor University analyzed the fish tissue composites for 36 PPCPs, includ-
ing 24 pharmaceutical compounds and 12 personal care products. They used a method
that applies high performance liquid chromatography with tandem mass spectrometry
(HPLC-MS/MS) to analyze fillet samples for 24 pharmaceutical compounds and liver sam-
ples for 23 pharmaceuticals, following procedures described by Ramirez et al. (2007). This
analytical method provides results for a range of prescription and over-the-counter drugs,
including antibiotics, analgesics, antihistamines, along with drugs to treat high blood pres-
sure and high cholesterol, depression, seizures, and fungal infections. Following procedures
described by Mottaleb et al, (2009), laboratory staff also extracted and analyzed fillet tissue
samples for 12 personal care product (PCP) chemicals using a method that applies gas chro-
matography with tandem mass spectrometry (GC-MS/MS), This method provides results
for a range of chemicals commonly used in personal care products, including a number of
fragrances or musks, ultraviolet (UV) light filters, surfactants, antimicrobials, and insect
repellents. The laboratory measured lipid content in each of the fish tissue composites using
gravimetric (weight-based) methods.
None of the 36 target PPCPs were detected in any of the fillet and liver tissue samples from
the Gila River Wilderness reference site. A majority of the 24 pharmaceutical compounds
also did not occur in the fish tissue samples from the five effluent-dominated stream sites.
Seventeen of the pharmaceutical compounds were not detected in any of the fillet or liver
samples, including the six antibiotics (e.g., erythromycin), the three analgesics (e.g., ibu-
profen), and three of the four pharmaceuticals used to treat high blood pressure (atenolol,
metoprolol, and propranolol). Ten of the 12 personal care product chemicals did not occur
above detectable levels in the fillet tissue samples, including triclosan (widely used chemical
viii
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Executive Summary
in hand soaps), m-toluamide (an insect repellent), and three chemicals that act as ultraviolet
(UV) filters in sunscreens (benzophenone, octocrylene, and 4-methylbenzylidene camphor).
Five of the 24 target pharmaceutical compounds were found in the fillet samples from the
effluent-dominated stream sites. All of the fillet concentrations for the detected pharma-
ceutical compounds were measured in the low parts per billion (ppb). The highest fillet
concentration reported for any pharmaceutical compound was 19 ng/g of sertraline (anti-
depressant). In order of decreasing frequency, the pharmaceuticals detected in fillet samples
included diphenhydramine (18 of 30 samples), norfluoxetine and sertraline (12 of 30 samples
for each), diltiazem (8 of 30 samples), and carbamazepine (6 of 30 samples). There were
notable differences in the frequency of detections among sites. For example, none of the
pharmaceuticals were detected in fillet samples from Dallas and Orlando. The wastewater
treatment plants discharging to streams near the sampling locations in both cities employ
advanced treatment technologies that may provide more effective removal of pharmaceu-
ticals from the waste stream. Diphenhydramine occurred in every fillet sample from the
remaining three sites. These data suggest widespread discharge of this active ingredient in
over-the-counter cold medications into surface waters. In contrast, carbamazepine occurred
in all the fillet samples at only a single site (Chicago).
Seven of the target pharmaceutical compounds were detected in the liver samples, including
fluoxetine (an antidepressant) and gemfibrozil (a drug used to treat high cholesterol), in addi-
tion to the five pharmaceuticals found in the fillet samples (i.e., carbamazepine, diltiazem,
diphenhydramine, norfluoxetine, and sertraline). Pharmaceutical compounds occurred more
frequently in the liver samples than in the fillet samples. Norfluoxetine and sertraline, two of
the antidepressants, were found in liver samples from all five sampling sites. Their detection
frequencies were 26 and 23 per 30 samples, respectively. Differences in detection frequen-
cies for pharmaceuticals in the liver samples were also apparent among the sampling sites.
The fewest number of detections occurred at Orlando and Dallas, two sites where WWTPs
apply advanced treatment technologies before discharging effluents into the streams. The
concentrations of pharmaceutical compounds in fish liver samples were greater than those
measured in fillets. Differences among their mean concentrations ranged from a factor of
nearly three to more than 20. Sertraline had the highest concentrations reported for pharma-
ceuticals in liver samples with levels as high as 550 ng/g.
Personal care product (PCP) results indicated that two of the 12 target chemicals, galaxolide
and tonalide, occurred in fillet samples. Both are fragrances added to common products like
cosmetics and detergents, and both PCPs were detected in fillet samples from all five sites.
Galaxolide was measured in 29 of the 30 fillet samples, and tonalide was detected in 26 of
the 30 fillet samples. Fillet concentrations of galaxolide occurred in the low parts per million
range at a majority of the sites, and mean fillet concentrations exceeded 1000 ng/g or 1 part
per million (ppm) at three sites. Tonalide concentrations in the fillet samples were about
an order of magnitude lower than the galaxolide concentrations with mean concentrations
ranging from 55 ng/g to 240 ng/g.
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Executive Summary
This study supports conclusions from earlier studies and offers new insights, particularly
with respect to the benefits of advanced wastewater treatment technologies on the occur-
rence of PPCPs in fish tissue. Conclusions derive primarily from differences in PPCP detec-
tions and concentrations related to type of tissue, geographic location, and level of treatment
technologies applied in WWTPs before discharge of effluents into rivers or streams. They
include the following:
¦ Pharmaceutical compounds occurred in greater numbers and at higher detection
frequencies and concentrations in liver samples than in fillet samples.
¦ No significant relationships were observed between lipid content and accumulation of
pharmaceuticals in either fillet or liver tissue.
¦ Differences in wastewater treatment technologies can substantially affect the removal
efficiency of pharmaceutical compounds, which affects fish tissue concentrations.
¦ It appears that the wastewater treatment technologies applied at individual WWTPs
is a better predictor of pharmaceutical occurrence than demographics or surrogate
data for pharmaceutical use statistics; however, demographics of local populations can
influence geographic differences in detections of pharmaceutical compounds in fish
tissue.
¦ The widespread occurrence of norfluoxetine (a metabolite of the antidepressant
fluoxetine) in fish tissue samples analyzed for this study provides further evidence of
the importance of including metabolic products of target chemicals in future tissue
screening studies.
x
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
1.0 Introduction
Pharmaceuticals and personal care products (PPCPs) are a diverse group of chemicals that,
until recently, have received little attention as potential environmental pollutants. PPCPs
include all drugs (both prescription and over-the-counter medications) and non-medicinal
consumer chemicals such as fragrances (musks), sunscreens, and soaps. Recent evidence has
shown that many PPCPs enter the aquatic environment primarily as a result of their persis-
tence through the wastewater treatment process and resulting discharge to surface or ground
water (Daughton and Ternes 1999). The full extent, magnitude, and consequences of their
presence in the aquatic environment are largely unknown.
Most existing information on the environmental occurrence of PPCPs focuses on waste-
water discharges and surface waters. The limited number of studies on PPCPs in fish tissue
generally target a specific chemical (or chemical class) at a single study location. As a con-
sequence, there is a need for additional
data to provide an understanding of
PPCP accumulation in fish at a broad
scale that will support the characteriza-
tion of human health risks associated
with PPCPs in the environment. In
2006, OST initially responded to this
data gap by designing and conducting
a pilot study called the National Pilot
Study of Pharmaceuticals and Personal
Care Products in Fish Tissue with sup-
port from Baylor University and Tetra
Tech, Inc. The purpose of the PPCP Fish
Pilot Study was to advance the science
related to detecting PPCPs in the envi-
ronment by investigating the occurrence
of a broad suite of PPCPs in the tissue
of fish collected from selected U.S.
streams.
EPA's PPCP Fish Pilot Study is the
first study to assess a wide range of
PPCPs in fish from sampling loca-
tions distributed across the lower 48
states. For this study, EPA selected sites
on five effluent-dominated streams
Reference site - East Fork Gila River, Gila River
Wilderness Area, New Mexico
1
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Introduction
in population centers near wastewater treatment plant (WWTP) discharges based on the
assumption that PPCPs were more likely to occur in these areas. The study also included a
reference site located on a river in a national wilderness area. Scientists at Baylor University
analyzed the fish tissue from these sites for 36 PPCPs, including 24 pharmaceutical com-
pounds and 12 personal care products. Prior to the study, Baylor University researchers had
developed analytical methods to analyze fish tissue for this large number of PPCPs. When
EPA initiated the pilot study, their laboratory was the only one in the U.S. with this capabil-
ity. In 2009, EPA, Baylor University, and Tetra Tech collaborated on publication of the study
results in the technical journal Environmental Toxicology and Chemistry (Ramirez et al.
2009). The journal article reports summary level data for the study. This technical report
describes the planning and implementation of the pilot study in greater detail, and appendi-
ces to the report provide the complete set of site-specific data generated during this study.
2
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
2.0 Study Design and Approach
2.1 Background
The PPCP Fish Pilot Study required four years (2006-2009) for study planning, collection
and chemical analysis of the fish samples, review and statistical analysis of the tissue concen-
tration data, and publication of the results. The study team completed site selection, devel-
opment of Quality Assurance Project Plans (QAPPs) for sample collection and analysis, and
fish collection during 2006. It took about a year and a half to homogenize the fish tissue and
analyze the fish tissue samples. During this time, Baylor University chemists refined proce-
dures in their analytical method for detecting personal care products in fish tissue to address
problems with obtaining reliable results caused by lipid interference. By fall 2008, the tissue
data were reviewed and ready to report. Baylor University researchers who participated in
the study led the effort to compile and publish the results in a special issue of Environmental
Toxicology and Chemistry on PPCPs in the environment (Ramirez et al. 2009). The final
activities for this study have included production and release of this technical report and a
supporting Quality Assurance Report (USEPA 2023).
2.2 Study Design Development
Targeted sampling is an appropriate approach for initial investigation of occurrence of
PPCPs in fish. The primary objective of this pilot study was to determine which PPCPs were
accumulating in fish, so the study was designed to collect fish from surface waters where
PPCPs were most likely to occur. EPA identified a number of criteria that could increase the
likelihood of fish being exposed to PPCPs and applied these criteria in selecting sites for the
study. EPA adopted the list of target chemicals that Baylor University could detect with their
tissue methods for PPCP analysis.
2.2.1 Site Selection
EPA considered a number of factors to
identify five sampling locations around
the country where PPCPs were more
likely to occur and accumulate in fish.
The leading factor for site selection
was location on an effluent-dominated
river or stream just below a WWTP
discharge. At three of the five sites, the
flow consists of nearly 100% effluent.
On average, effluent comprises about
two-thirds of the flow at the other two
North Shore Channel, Chicago, Illinois
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Study Design and Approach
sites. EPA also assumed locations on
streams or rivers running through cities
with high population densities in con-
junction with higher median incomes
and percentages of elderly residents
could increase the potential of detect-
ing PPCPs in fish from these areas. EPA
applied two other criteria in the site
selection process. One was to include
areas where WWTP discharges receive
different levels of treatment to evaluate
the potential impact of treatment tech-
nologies on PPCP removal. Another wax
to target areas where sufficient numbers
and sizes of resident fish were avail-
able for analysis. The six criteria EPA
applied to selection of sampling loca-
tions for the pilot study can be summa-
rized as follows:
¦ Effluent-dominated river or
stream segments below WWTP
discharges;
¦ Urban or suburban areas with
high population densities;
¦ Cities with higher median incomes (used as a surrogate for pharmaceutical sales);
¦ Geographic areas with a large percentage of residents in the age category of 65 years
and older;
¦ WWT P discharges Subject to different levels of treatment; and
¦ Availability of sufficient numbers and sizes of fish.
Based on these criteria, EPA selected five sampling sites on rivers or streams in the following
cities:
¦ Chicago, Illinois (North Shore Channel)
¦ Dallas, Texas (Trinity River)
¦ Orlando, Florida (Little Econlockhatchee River)
¦ Phoenix, Arizona (Salt River)
¦ West Chester, Pennsylvania [a suburb of Philadelphia] (Taylor Run)
Trinity River, Dallas, Texas
4
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Study Design and Approach
The study design included identifying and sampling a reference site free from sources of
human contamination. The East Fork of the Gila River in the Gila Wilderness Area of
southwest New Mexico provided a suitable reference site for the study. The map in Figure 1
displays the sampling locations for the pilot study. Table 1 provides wastewater treatment
and discharge information for each facility near the sampling locations, along with popula-
tion characteristics of the cities associated with each sampling site.
Figure 1. Sampling locations for the PPCP Fish Pilot Study.
5
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Table 1. Wastewater Treatment and Discharge Information for Facilities in the Vicinity of Each Sampling Location and Population
Characteristics of Associated Cities.
LOCATION
Treatment
Receiving Water
POPULATION
Design
capacity
(MGD)a
Existing
flow
(MGD)
Effluent
(%)
65 AND
OLDER (%)
Median
income
Phoenix, Arizona
Advanced treatment I
with nutrient removal15
Salt River
1,418,041
165
153
100
8.1
$41,207
Orlando, Florida
Advanced treatment II
with nutrient removal0
Little Econlockhatchee
River
442,542
40
36
64
11.3
$35,732
Chicago, Illinois
Advanced treatment 1
with nutrient removal15
North Shore Channel
5,376,741
333
234
100d
10.3
$38,625
West Chester,
Pennsylvania
Advanced treatment 1
with nutrient removal15
Taylor Run
17,701
1.8
1.3
36-86
9.0
$37,803
Dallas, Texas
Advanced treatment II
with nutrient removal0
Trinity River
3,500,000
175
152
100d
8.1
$43,324
a Million gallons per day.
b Advanced treatment I. Wastewater discharged after receiving biological treatment, physical or chemical treatment, or both. A wastewater treatment plant with a
concentration of biochemical oxygen demand (BOD5; the amount of dissolved oxygen consumed in 5 days by biological processes breaking down organic matter) greater
than or equal to 10 mg/L but less than 20 mg/L (based on 30-d average) in its National Pollutant Discharge Elimination System (NPDES) permit is considered to be providing
advanced treatment I.
c Advanced treatment II. Wastewater discharged after receiving biological treatment, physical or chemical treatment, or both. A wastewater treatment plant with a BOD5
concentration less than 10 mg/L (based on 30-d averages) in its NPDES permit is considered to be providing advanced treatment II. Note that the addition of nutrient
removal is considered to be an improvement in effluent quality (e.g., secondary effluent with nutrient removal represents higher quality effluent than secondary effluent
without nutrient removal).
d Flow is primarily made up of effluent discharged from multiple facilities.
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Study Design and Approach
2.2.2 Target Chemicals
Two analytical methods developed by Baylor University specify the target chemicals that apply
for this study. Their tissue method for pharmaceutical analysis provides screening data for
24 compounds representing a wide range of medical uses that include antibiotics, analgesics,
antidepressants, anti-hypertension drugs, an antihistamine, and an anti-seizure drug. Their
other method screened fish tissue for 12 chemicals in personal care products, which consisted
primarily of fragrances or musks in lotions and soaps and the ultraviolet filtering chemicals in
sunscreen products. Tables 2 and 3 list the names and uses of each pharmaceutical compound
and personal care product chemical that these methods could detect in fish tissue, respectively.
Table 2. Target Pharmaceutical Chemicals, Uses, and Chemical Abstracts Service (CAS) Registry
Numbers for the PPCP Fish Pilot Study.
Pharmaceuticals using hplc-ms/ms method
Chemical
USE
CAS Number
Acetaminophen
Analgesic
103-90-2
Atenolol
Anti-hypertension
29122-68-7
Caffeine
Stimulant
58-08-2
Carbamazepine
Anti-seizure
298-46-4
Cimetidine
Anti-acid reflux
51481-61-9
Codeine
Analgesic
76-57-3
Diltiazem
Anti-hypertension
42399-41-7
1,7-Dimethylxanthine (caffeine metabolite)
Antispasmodic
611-59-6
Diphenhydramine
Antihistamine
58-73-1
Erythromycin
Antibiotic
114-07-8
Fluoxetine
Antidepressant
54910-89-3
Gemfibrozil
Antilipemic
25812-30-0
Ibuprofen
Analgesic
15687-27-1
Lincomycin
Antibiotic
154-21-2
Metoprolol
Anti-hypertension
37350-58-6
Miconazole
Antifungal
22916-47-8
Norfluoxetine (Fluoxetine metabolite)
Antidepressant
54910-89-3
Propranolol
Anti-hypertension
525-66-6
Sertraline
Antidepressant
79617-96-2
Sulfamethoxazole
Antibiotic
723-46-6
Thiabendazole
Antibiotic
148-79-8
Trimethoprim
Antibiotic
738-70-5
Tylosin
Antibiotic
1401-69-0
Warfarin
Anticoagulant
81-81-2
7
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Study Design and Approach
Table 3. Target Personal Care Product Chemicals, Uses, and Chemical Abstracts Service (CAS)
Registry Numbers for the PPCP Fish Pilot Study.
Personal Care Products using GC-ms/ms method
Chemical
USE
CAS Number
Benzophenone
UV filter
119-61-9
Celestolide (ADBI)
Fragrance/Musk
13171-00-1
Galaxolide
Fragrance/Musk
1222-05-5
4-Methylbenzylidene Camphor (4-MBC)
UV Filter
36861-47-9
Musk Ketone
Fragrance/Musk
81-14-1
Musk Xylene
Fragrance/Musk
81-15-2
p-Nonylphenol
Surfactant
104-40-5
Octocrylene
UV Filter
6197-30-4
p-Octylphenol
Surfactant
1806-26-4
m-Toluamide (DEET)
Insecticide
618-47-3
Tonalide
Fragrance/Musk
1506-02-1
Triciosan
Antimicrobial
3380-34-5
2.3 Mobilization
Prior to beginning field sampling, EPA
completed some key activities to mobi-
lize for the study. This included prepa-
ration of Quality Assurance Project
Plans for sample collection and analy-
sis. Copies of the Sample Collection
Activities QAPP (USEPA 2006a) and
the Laboratory Sample Preparation
and Analysis Activities QAPP (USEPA
2006b) are available online at
https://wiinv.epa.gov/fish-tech/pilot-
stiidy-pharmacenticals-and-personal-
care-products-fish-tissue. EPA also
formed partnerships for project coordination and sampling assistance in Chicago and for
sampling support at the reference site in New Mexico. OST coordinated with EPA's Great
Lakes National Program Office (GLNPO) to participate in a broader study of PPCPs in
Chicago's North Shore Channel and vicinity (Barber et
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Study Design and Approach
2.4 Sample Collection
Field crews collected a total of 17 to 24 adult fish
of the same resident species from each sampling
location during late summer and fall of 2006
(Table 4). This corresponds to the period when
lipid content in the fish is usually highest and water
levels in the rivers or streams are lowest. Both con-
ditions may increase the likelihood of detecting
PPCPs in fish.
The field sampling teams vised portable backpack
or boat-mounted electrofishing systems to obtain
species that are typically consumed by humans and
wildlife. At each sampling site, they retained individ-
ual fish of a single species that were similar in length
(i.e., the smallest fish in the sample was no less than
75% of the length of the largest fish) consistent with
the recommendations in U.S. EPA's Guidance for
Assessing Chemical Contaminant Data for Use
in Fish Advisories, Volume 1: Fish Sampling and
Analysis, Third Edition (USEPA 2000).
The field team recorded the weight (total body mass in grams, wet weight) and length (total
length in millimeters) of each fish before dividing the fish into six composite samples. Each
composite sample contained three or four fish individually wrapped in solvent-rinsed alu-
minum foil and secured together in a food-grade polyethylene bag. The fish samples were
frozen on dry ice, shipped in coolers to the analytical laboratory at Baylor University via
next-day air delivery, and stored in a freezer at < ~20° C prior to preparation of fillet and
liver tissue samples for analysis. A detailed description of the sampling protocols is avail-
able in the Quality Assurance Project Plan (QAPP) for Sample Collection Activities for a
Pilot Study to Investigate the Occurrence of Pharmaceuticals and Personal Care Products
(PPCPs) in Fish Tissue (USEPA 2006a), This document is available online at https-J/www.
epa.gov/fish-tech/pilot-stndy-pharmaceiiticals-and-personal-care-products-fish-tissue.
Table 4. Fish Collected for the PPCP Fish Pilot Study.
State
Sampling location
Date
Species
Number of Fish
AZ
Salt River, Phoenix
11/2006
Common carp
18
FL
Little Econlockhatchee River, Orlando
10/2006
Bow fin
17
IL
North Shore Channel, Chicago
09/2006
Largemouth bass
24
NM
East Fork Gila River (Reference
Site)
11/2006
Sonora sucker
24
PA
Taylor Run, West Chester
08/2006
White sucker
24
TX
Trinity River, Dallas
10/2006
Smallmouth buffalo
18
Electrofishing at the East Fork Gila River
reference site.
9
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Study Design and Approach
2.5 Sample Analysis
EPA contracted with an analytical laboratory at
Baylor University in Waco, Texas to prepare and
analyze fish tissue samples for the PPCP Fish Pilot
Study. At the time that EPA initiated the study,
research scientists at the university had developed
the only analytical methods available in the coun-
try to screen fish tissue for a broad suite of phar-
maceutical compounds and chemicals commonly
used in personal care products, such as fragrances
in soaps and ultraviolet filters in sunscreens. Staff
at the laboratory prepared fillet and liver tissue
samples from each fish composite sample to ana-
lyze both tissue types for pharmaceutical com-
pounds and analyzed fillet tissue only for personal
care product chemicals. They applied a method
utilizing high performance liquid chromatography
with tandem mass spectrometry (HPLC-MS/MS)
in the fillet and liver tissue analysis for pharma-
ceutical compounds. To analyze fillet samples for
personal care products, they used gas chromatog-
raphy with tandem mass spectrometry (GC-MS/MS). The laboratory also measured the lipid
content in each of the fish tissue samples using gravimetry (weight-based method),
2.5.1 Fish Tissue Sample Preparation
EPA directed the laboratory to follow tissue preparation procedures in EPA's Guidance for
Assessing Chemical Contaminant Data for Use in Fish Advisories, Volume 1: Fish Sampling
and Analysis, Third Edition (USEPA 2000) and apply the batch method to prepare compos-
ite samples of fillet tissue for analysis. The primary steps in this process include:
¦ Removing the entire fillet (including the skin and belly flap) from both sides of each
fish in the composite sample and using all the available tissue to prepare the fillet
composite sample (i.e., the batch method),
¦ Grinding frozen cubes of fillet tissue to a fine powder using a high-speed blender and
adding small amounts of dry ice during grinding to facilitate consistent blending of the
tissue,
¦ Applying quartering, mixing, and re-grinding techniques described in the guidance
document to produce a homogeneous composite mixture of fillet tissue, and
¦ Storing the homogenized fillet composite samples in a freezer at a temperature < ~20° C
until the laboratory was ready to analyze them for PPCPs.
Field packaging and labeling offish
composite samples at the Gila River
reference site.
10
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Study Design and Approach
To prepare liver composite samples, the laboratory applied tissue dissection and homogeni-
zation techniques developed for prior studies conducted by Baylor University to characterize
concentrations of PPCPs in fish tissue (Brooks et al. 2005). These techniques involved the
following steps:
¦ Removing the liver from each fish in the composite (total of three or four livers,
depending on the sampling location) and placing all of them in a clean glass container,
¦ Homogenizing the liver tissue using a motor-driven tissue homogenizer set to rotate at
30,000 revolutions per minute (rpm), and
¦ Storing the liver homogenate samples in the freezer at a temperature < 20° C until the
laboratory was ready to analyze them for pharmaceutical compounds.
2.5.2 Analytical Methods
When EPA initiated the PPCP Fish Pilot Study in 2006, the agency did not have methods
available to analyze tissue for PPCPs. However, researchers at Baylor University had devel-
oped and successfully applied two analytical methods to screen fish tissue for a wide range
of PPCPs. Based on this experience, EPA arranged for the laboratory at Baylor University
to analyze pilot study fish tissue samples since they were the only laboratory in the country
at that time with the capability to screen tissue for three dozen PPCPs. The laboratory also
measured the lipid content of each of the fish tissue samples. This section of the report pro-
vides a brief description of the methods for PPCP and lipid analysis, and Appendix A con-
tains a summary of the extraction and analytical procedures associated with each method.
2.5.2.1 Analysis of Fillet and Liver Tissue for Pharmaceutical Compounds
The laboratory at Baylor University analyzed fillet tissue samples for 24 pharmaceutical
compounds and liver tissue samples for 23 pharmaceuticals with a method that applies high
performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) using
procedures described by Ramirez et al. (2007). This method specifies a fillet tissue weight
of 1.0 gram and a liver tissue weight of 0.5 gram for analysis. It provides results for a range
of prescription and over-the-counter drugs, including antibiotics, analgesics, antihistamines,
and drugs to treat high blood pressure and high cholesterol, depression, seizures, and fungal
infections. Table 5 contains a list of the target chemicals for this pharmaceutical method,
along with the method detection limits (MDLs) for both the fillet and liver tissue. Note that
the pharmaceutical method (Ramirez et al. 2007) could reliably detect and quantify the anti-
fungal miconazole in fillet tissue, but not in liver tissue.
11
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Study Design and Approach
Table 5. Method Detection Limits for Pharmaceutical Chemicals Analyzed by
HPLC-MS/MS in Fish Fillet and Liver Composite Samples.
Chemical
MDLa (ng/g)
Fillet
Liver
Acetaminophen
4.40
34.28
Atenolol
1.48
12.86
Caffeine
3.93
25.47
Carbamazepine
0.54
1.86
Cimetidine
1.04
5.18
Codeine
6.11
31.49
Diltiazem
0.12
0.26
1,7-Dimethylxanthine (caffeine metabolite)
1.10
5.84
Diphenhydramine
0.05
0.26
Erythromycin
6.42
43.03
Fluoxetine
6.74
12.41
Gemfibrozil
6.68
24.82
Ibuprofen
45.96
172.81
Lincomycin
5.53
56.14
Metoprolol
2.50
8.90
Miconazole
10.83
NAb
Norfluoxetine (Fluoxetine metabolite)
2.90
15.31
Propranolol
1.07
3.77
Sertraline
3.56
17.29
Sulfamethoxazole
2.29
13.95
Thiabendazole
2.63
7.84
Trimethoprim
2.15
8.00
Tylosin
5.02
34.67
Warfarin
0.86
2.70
a MDL is the method detection limit.
b Miconazole was not reliably measured in liver using existing analytical method.
2.5.2.2 Analysis of Fillet Tissue for Personal Care Product Chemicals
Laboratory staff extracted and analyzed only fillet tissue samples for 12 personal care
product chemicals with a method that applies gas chromatography with tandem mass spec-
trometry (GC-MS/MS) using procedures described by Mottaleb et al. (2009). This method
requires a fillet tissue volume of 1.0 gram for analysis. It provides results for a range of
chemicals commonly used in personal care products, including a number of fragrances or
musks, ultraviolet light filters, surfactants, an antimicrobial, and an insect repellent. Table 6
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Study Design and Approach
contains a list of the specific chemicals that can be detected with this personal care product
(PCP) method, along with the detection limits for the fillet tissue.
Initially, the laboratory attempted to analyze liver tissue using the PCP method and encoun-
tered problems with interference due to the high lipid content of the liver tissue. In response,
the laboratory experimented with several modifications to the clean-up procedures for the
method to mitigate these problems. None of the PCP method modifications resolved the
interference problems sufficiently to produce reliable liver results, so no PCP data are avail-
able for liver tissue.
Table 6. Method Detection Limits for Personal Care Products Analyzed
by GC-MS/MS in Fish Fillet Tissue Composite Samples.
Chemical
Fillet MDLa (ng/g)
Benzophenone
16.4
Celestolide (ADBI)
17.7
Galaxolide
12.2
4-Methylbenzylidene-Camphor (4MBC)
120.5
Musk Ketone
321.2
Musk Xylene
397.1
p-Nonylphenol
9.7
Octocrylene
36
p-Octylphenol
8.2
m-Toluamide (DEET)
5.1
Tonalide
13.4
Triclosan
37.8
a MDL is the method detection limit.
2.5.2.3 Analysis of Fish Tissue for Lipids
To identify any correlations between PPCP and lipid concentrations, the laboratory mea-
sured the lipid content of each fillet and liver composite sample prepared from the six fish
composite samples collected at every sampling location. The method for lipid analysis
involves extracting lipids from two grams of tissue using a mixture of solvents, evaporating
the solvents from the tissue mixture, determining the lipid content gravimetrically (i.e., mea-
suring the lipid content based on weight) after drying the residue to a constant weight, and
calculating the percent lipid by dividing the weight of the lipid residue by the initial weight
of the tissue aliquot (approximately two grams). Appendix A provides a detailed description
of the lipid method.
EPA requested that the laboratory also use lipid analysis as a quality control procedure
to assess the homogeneity of the fillet and liver tissue samples. This procedure included
triplicate lipid testing of all six fillet tissue samples and one of the six liver tissue samples
13
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Study Design and Approach
prepared from the six fish composite samples collected at every sampling location. For every
fillet tissue sample, the laboratory analyzed three 2-gram tissue aliquots for lipids using the
method summarized above. Since liver tissue was scarce compared to the fillet tissue, the
laboratory tested three 2-gram tissue aliquots for lipid content from only one in every set
of six liver tissue samples. If the relative standard deviation for the triplicate lipid measure-
ments was less than 15%, then the tissue sample met the criterion for homogeneity.
2.6 Data Analysis
The analytical laboratory reported quantitative results for each pharmaceutical compound
and personal care product chemical in fish fillets, and for each pharmaceutical compound
in fish liver tissue. The MDL was the reporting limit for the PPCP data, so only values
greater than or equal to the MDL were included when determining frequency of detection,
mean detected concentration, or maximum detected concentration. Frequency of detection
is defined as equal to the number of composites in which a chemical was detected at a con-
centration greater than or equal to the MDL compared to the total number of composites.
Similarly, the mean and maximum detected concentrations presented in this report refer to
the mean or maximum of the detected concentrations greater than or equal to the MDL.
The final results do not include data for three chemicals. Miconazole, an antifungal phar-
maceutical, is not reported for fish liver tissue samples because the laboratory determined
that it could not be reliably measured using the specified analytical method. Benzophenone
and octocrylene, UV filters found in personal care products, were identified in blank control
samples at concentrations comparable to those reported in the analytical samples. Therefore,
the analytical results for these two chemicals were considered inconclusive by the laboratory
and are excluded from the final results.
2.7 Identifying and Quantifying Target Chemicals
The HPLC-MS/MS and GC-MS/MS methods used several characteristics to identify the
target analytes. The first characteristic was retention time, or the time at which chemicals
elute from the liquid or gas chromatograph. Retention time is a key indicator of the presence
of a compound of interest in chromatographic analysis. Retention time profiles were deter-
mined by analysis of mixtures of high concentration standards during the development of
chromatographic conditions. In addition to retention time, mass spectral data were used to
confirm the identification of chemicals of interest and yield optimized quantitation of target
chemicals of interest in the presence of co-eluting non-target interferences (i.e., chemicals
not included in the study that produce peaks on the chromatogram that overlap with peaks
of chemicals included in the study). While the retention time of a specific peak in the chro-
matogram may suggest the presence of a chemical of interest, it is the presence of specific
precursor and quantitation ions in the tandem mass spectrometers that allows certainty in
the identification.
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Study Design and Approach
After a chromatographic peak is identified as a chemical of interest, its concentration in the
sample is calculated by the instrument data system. The tandem MS/MS detectors used in
the HPLC-MS/MS and GC-MS/MS methods typically produce two or more ions that are
characteristic of the chemical. The responses (i.e., peak areas) for these characteristic ions
are used to calculate the concentrations in the sample. The pharmaceutical method used
matrix-matched calibration standards (i.e., fish tissue matrix to the extent possible), and
both instrumental methods used internal standard calibration and quantitation equations.
Appendix A provides details on internal standard calibrations and general quantitation
reporting.
15
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
16
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
3.0 Results
The PPCP Fish Pilot Study is the first research effort to screen fish tissue samples from sev-
eral sites across the United States for a broad suite of PPCPs. Since the focus of the study
was to investigate the occurrence of PPCPs in fish, EPA selected sampling locations for this
national pilot study based on factors that could increase the likelihood of encountering
PPCPs in the environment. Refer to Section 2.1,1 for a list of these factors. With the targeted
study design, it is appropriate to apply routine calculations for statistical analysis of the fish
tissue concentration data (e.g., mean concentrations and corresponding standard deviations).
Sections 3.1 and 3.2 describe the occur-
rence of PPCPs in fish tissue. Tables 7
and 8 in Section 3.1 list the pharma-
ceuticals and personal care products
not detected in any of the fish tissue
samples, respectively. Tables 9, 10, and
11 in Section 3.2 identify the method
detection limit associated with each
pharmaceutical compound detected in
fillet and liver samples (Tables 9 and
10) and with each personal care prod-
uct detected in fillet samples (Table 11). Bowfin 0r|and0) Rorjda
Additional information about method
detection limits is provided below.
These three tables also report the fre-
quency of detection (ratio based on a
total of six composite samples from
each site) and the mean and maximum
tissue concentrations for each of the five
sampling locations (Chicago, Dallas,
Orlando, Phoenix, and West Chester,
PA). Ramirez et al. (2009) provides
summary-level data and a discussion of
the pilot study results. Consistent with
the reporting approach in Ramirez et al.
i Sonora sucker, East Fork Gila River
(2009), the mean fish tissue concentra-
tions reported in the three tables were
derived from detected concentrations only (i.e., only values from detected concentrations
were vised to calculate the mean concentrations without assigning any values to non-detects
and factoring them into the mean concentration calculations). Table 12 presents quality
control data for pharmaceutical compounds detected in liver composite samples. These data
17
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Results
can be applied in interpretation of the liver results at four of the sampling locations. In addi-
tion, three appendices provide site-specific PPCP data for the six fillet and liver composites
analyzed from each sampling site: Appendix B contains the pharmaceutical data for fillet
samples; Appendix C contains the pharmaceutical data for liver samples; and Appendix D
contains the personal care products data for fillet samples.
Section 3.3 presents the lipid results for the pilot study fish tissue samples. Table 13 in
Section 3.3 provides a summary of the mean lipid content (% lipid) in the fillet and liver
samples from each sampling location and the standard deviations that correspond to the
mean lipid values. Appendix E contains a series of tables that provide the site-specific lipid
data for the fillet and liver samples. All of the lipid data in Section 3.3 and Appendix E,
along with the site-specific PPCP data in Appendices B, C, and D, are unique to this report.
None of the site-specific data were published in Ramirez et al. (2009).
All of the data tables report the fish tissue results as wet-weight concentrations. These
concentrations are expressed as the mass of the chemical per unit of fish tissue mass. The
reporting unit for both the pharmaceutical method (HPLC-MS/MS) and the personal care
product method (GC-MS/MS) is nanograms per gram (ng/g) or parts per billion (ppb).
The Code of Federal Regulations (CFR) provides a definition and description of the method
detection limit (MDL) in 40 CFR, part 136, Appendix B. The MDL varies for different
chemicals, matrices (e.g., water or tissue), and analytical methods. Tables 5 and 6 (Section
2.5.2) list method detection limits for each target pharmaceutical compound and personal
care product chemical, respectively. The MDL is designed to provide a 99% level of confi-
dence that when a chemical is reported as being present at the MDL level, it is really present.
The opposite is not true, however. If a chemical is reported as not being present at the MDL
level, there is a 50% possibility that the chemical is really present (i.e., the result is a false
negative).
3.1 Chemicals Not Detected
3.1.1 Reference Site Results
One challenge that EPA faced in selecting sampling sites for the pilot study was identifying
a reference site free from human influence with respect to the 36 chemicals of interest for
the pilot study (Tables 2 and 3 in Section 2.1.2). Fishery biologists from the New Mexico
Environment Department offered their technical expertise about southwest fisheries to assist
EPA in selecting the reference site. They recommended a site on the East Fork of the Gila
River that flows through the Gila Wilderness Area in southwest New Mexico. EPA followed
their recommendation and it turned out to be an appropriate one. Results from the analysis
of fillet and liver tissue from the six reference site fish composite samples showed that none
of the 36 target PPCPs were detected in either type of fish tissue samples.
18
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Results
3.1.2 Pharmaceutical Compounds
Results for the 24 target pharmaceutical compounds in fillet tissue samples and 23 target
pharmaceutical compounds in liver tissue samples revealed that the majority of these com-
pounds did not occur in the fish tissue. Seventeen of the pharmaceutical compounds were
not detected in any of the fillet or liver samples from fish composite samples collected at the
five sites located on effluent-dominated rivers or streams. None of the six antibiotics
(e.g., erythromycin) or the three analgesics (e.g., ibuprofen) occurred at detectable levels in
the fish tissue samples. Three of the four pharmaceuticals used to treat high blood pressure
(atenolol, metoprolol, and propranolol) were also not found in the fish tissue. Table 7 con-
tains a complete list of the 17 pharmaceuticals not detected in any of the fish tissue samples.
Table 7. Pharmaceutical Chemicals Not Detected in Fillet or Liver Tissue.
Chemicals Not Detected
USE
Acetaminophen
Analgesic
Atenolol
Anti-hypertension
Caffeine
Stimulant
Cimetidine
Anti-acid reflux
Codeine
Analgesic
1,7-Dimethylxanthine
Antispasmodic
Erythromycin
Antibiotic
Ibuprofen
Analgesic
Lincomycin
Antibiotic
Metoprolol
Anti-hypertension
Miconazole
Antifungal
Propranolol
Anti-hypertension
Sulfamethoxazole
Antibiotic
Thiabendazole
Antibiotic
Trimethoprim
Antibiotic
Tylosin
Antibiotic
Warfarin
Anticoagulant
3.1.3 Personal Care Product Chemicals
Results for the 12 target chemicals commonly found in personal care products showed that
10 of these compounds did not occur in the fish tissue. The chemicals not detected in fillet
samples included triclosan and w-toluamide, which are used widely in hand soaps and insect
repellents, respectively. This group also included three chemicals that act as ultraviolet filters
in sunscreens (benzophenone, octocrylene, and 4-methylbenzylidene camphor or 4-MBC).
Table 8 provides a list of the 10 personal care product chemicals not detected in any of the
19
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Results
fillet samples. Liver tissue samples were not analyzed for any of the chemicals in personal
care products as explained in Section 2.5.2.2.
Table 8. Personal Care Product Chemicals Not Detected in Fillet Tissue.
Chemicals Not Detected
USE
Benzophenone
UV filter
Celestolide (ADBI)
Fragrance/Musk
4-Methylbenzylidene Camphor (4-MBC)
UV filter
Musk Ketone
Fragrance/Musk
Musk Xylene
Fragrance/Musk
p-Nonylphenol
Surfactant
Octocrylene
UV filter
p-Octylphenol
Surfactant
m-Toluamide (DEET)
Insecticide
Triclosan
Antimicrobial
3.2 Detected PPCP Chemicals
This report summarizes the fillet and liver concentration data for PPCPs detected in the fish
tissue composite samples using the same approach as Ramirez et al. (2009). The fillet con-
centration data in Tables 9 (pharmaceuticals) and 11 (personal care products) and the liver
concentration data in Table 10 (pharmaceuticals) are based on six individual tissue com-
posite sample results from each of the five sampling sites (i.e., 30 fillet composite samples
and 30 liver composite samples). All values are expressed in nanograms per gram (ng/g) or
parts per billion (ppb) mass of compound per mass of wet-weight fish tissue. In addition to
the summary-level data, this report provides results for individual tissue samples at each site
in Appendix B through Appendix D. Data from these appendices were used to generate the
result summaries reported in Tables 9-11.
Tables 9 and 10 present the pharmaceutical results for the fillet and liver samples analyzed
from each site. These data include frequency of occurrence, mean tissue concentration, and
maximum tissue concentration for each detected pharmaceutical compound at every sam-
pling location. The frequency of occurrence is a ratio identifying the number of fillet or liver
samples with quantifiable detections for a specific chemical out of the six samples analyzed
from each location. In Table 9, for example, the frequency reported under Chicago for
diphenhydramine is 6/6, which indicates that this antihistamine was detected in all six fillet
samples analyzed from Chicago. Another example is the frequency of 2/6 reported under
Chicago for norfluoxetine (a metabolite or breakdown product of the commonly prescribed
antidepressant fluoxetine), indicating that this chemical was detected in only one-third of
the Chicago fillet samples. Note that frequencies greater than zero are highlighted in bold
type. As described in Section 3.0, the mean tissue concentrations were calculated using
only the values from detected concentrations. The two fillet concentrations measured above
20
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Results
the detection limit for norfluoxetine at Chicago were 3.19 ng/g and 3.21 ng/g (Table B-l,
Appendix B). In Table 9, the mean concentration for these two detected values is 3.2 ng/g (or
ppb). In this particular case, the maximum concentration reported to two significant figures
is also 3.2 ng/g (or ppb). Tables B-l (Chicago), B-2 (Dallas), B-3 (Orlando), B-4 (Phoenix),
and B-5 (West Chester, PA) contain site-specific concentration data for the pharmaceutical
compounds detected in individual fillet samples. These data were used to derive the sum-
mary results displayed in Table 9. Tables C-l through C-5 in Appendix C provide the site-
specific pharmaceutical data for individual liver samples that were used to derive the data
presented in Table 10.
Table 11 provides summary-level concentration data for personal care product chemicals in
fillet samples. There are only fillet tissue results for this group of chemicals, and they are
reported in the same format described above for the pharmaceutical compounds. Tables
D-l through D-5 in Appendix D contain the site-specific data for personal care products
detected in the individual fillet samples that were used to derive the data summarized in
Table 11.
21
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Table 9. Analytical Results for Pharmaceutical Compounds in Fillet Composite Samples.
Chicago
Dallas
Orlando
Phoenix
West Chester
Chemical
MDL (ng/g)
Freq
Mean
Max
Freq
Mean
Max
Freq
Mean
Max
Freq
Mean
Max
Freq
Mean
Max
Carbamazepine
0.54
6/6
2.3
3.1
0/6
*
*
0/6
*
*
0/6
*
*
0/6
*
*
Diltiazem
0.12
5/6
0.13
0.16
0/6
*
*
0/6
*
*
0/6
*
*
3/6
0.15
0.20
Diphenhydramine
0.05
6/6
1.4
1.7
0/6
*
*
0/6
*
*
6/6
1.2
1.4
6/6
1.7
2.5
Fluoxetine
6.7
0/6
*
*
0/6
*
*
0/6
*
*
0/6
*
*
0/6
*
*
Gemfibrozil
6.7
0/6
*
*
0/6
*
*
0/6
*
*
0/6
*
*
0/6
*
*
Norfluoxetine
2.9
2/6
3.2
3.2
0/6
*
*
0/6
*
*
4/6
4.0
4.8
6/6
3.9
5.0
Sertraline
3.6
0/6
*
*
0/6
*
*
0/6
*
*
6/6
5.0
6.5
6/6
11
19
Table 10. Analytical Results for Pharmaceutical Compounds in Liver Composite Samples.
Chicago
Dallas
Orlando
Phoenix
West Chester
Chemical
MDL (ng/g)
Freq
Mean
Max
Freq
Mean
Max
Freq
Mean
Max
Freq
Mean
Max
Freq
Mean
Max
Carbamazepine
1.9
6/6
6.0
8.0
0/6
*
*
0/6
*
*
0/6
*
*
0/6
*
*
Diltiazem
0.26
6/6
0.71
0.90
0/6
*
*
0/6
*
*
4/6
0.32
0.44
6/6
0.69
0.76
Diphenhydramine
0.26
6/6
7.0
9.6
5/6
0.52
0.93
0/6
*
*
6/6
6.7
11
6/6
10
11
Fluoxetine
12
3/6
19
23
2/6
13
14
0/6
*
*
0/6
*
*
6/6
70
80
Gemfibrozil
25
0/6
*
*
0/6
*
*
0/6
*
*
6/6
70
90
2/6
27
27
Norfluoxetine
15
6/6
73
130
4/6
37
48
5/6
57
78
5/6
33
44
6/6
38
48
Sertraline
17
6/6
84
150
4/6
27
28
1/6
NA
21
6/6
71
110
6/6
380
550
Table 11. Analytical Results for Personal Care Product Chemicals in Fillet Composite Samples.
Chicago
Dallas
Orlando
PHOENIX
West Chester
Chemical
MDL (ng/g)
Freq
Mean
Max
Freq
Mean
Max
Freq
Mean
Max
Freq
Mean
Max
Freq
Mean
Max
Galaxolide
12
6/6
1,300
1,800
6/6
840
1,800
5/6
110
290
6/6
1,800 2,100
6/6
1,800
2,000
Tonalide
13
6/6
150
230
6/6
72
150
1/6
NA
21
6/6
240
290
6/6
55
72
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Results
3.2.1 Pharmaceutical Results in Fillets
Results for pharmaceutical compounds show that five of the 24 target chemicals in this
group were found in the fillet samples (Table 9). In order of decreasing detection frequency,
they include:
¦ diphenhydramine (18 of 30 samples),
¦ norfluoxetine and sertraline (12 of 30 samples for each antidepressant),
¦ diltiazem (8 of 30 samples), and
¦ carbamazepine (6 of 30 samples).
There are notable differences in the frequency of detections among sites. For example, none
of the pharmaceuticals were detected in fillet samples from Dallas and Orlando. The waste-
water treatment plants (WWTPs) discharging to streams near the sampling locations in both
cities employ advanced treatment technologies that may provide more effective removal of
pharmaceuticals from the waste stream. Diphenhydramine occurred in every fillet sample
from the remaining three sites (Chicago, Phoenix, and West Chester near Philadelphia).
These data suggest widespread discharge of this active ingredient in over-the-counter cold
medications into surface waters. In contrast, carbamazepine occurred in all the fillet samples
at a single site (Chicago). On a site-specific basis, the largest number of pharmaceutical
detections occurred in the fillet samples from West Chester (21 of 30 possible detections),
followed by Chicago (19) and Phoenix (16).
All of the fillet concentrations for the detected pharmaceutical compounds were measured in
the low parts per billion (ppb) range. None of the concentrations exceeded 20 ng/g (or ppb),
and the majority of these concentrations were below 5 ng/g. The highest fillet concentration
reported for any pharmaceutical compound was 19 ng/g. This was the level of sertraline
(antidepressant) measured in a fillet sample from West Chester, PA. Sertraline was only
detected in the fillet samples from West Chester and Phoenix (all six samples from each site).
The mean concentration of sertraline in the West Chester samples (11 ng/g) was greater than
twice the mean sertraline concentration in the Phoenix samples (5 ng/g).
3.2.2 Pharmaceutical Results in Livers
Seven of the 23 pharmaceutical compounds were detected in the liver samples. These com-
pounds include fluoxetine (an antidepressant) and gemfibrozil (a drug used to treat high
cholesterol), in addition to the five pharmaceuticals found in the fillet samples (i.e., car-
bamazepine, diltiazem, diphenhydramine, norfluoxetine, and sertraline). Pharmaceutical
compounds were detected more frequently in the liver samples than in the fillet samples.
Norfluoxetine and sertraline, two of the antidepressants, occurred in liver samples from all
five sampling sites. Their detection frequencies were 26 and 23 per 30 samples, respectively.
Diphenhydramine was detected in the liver samples with the same frequency as sertraline
(23 detections in the 30 samples). Differences in detection frequencies for pharmaceuticals
in the liver samples are apparent among the sampling sites. Consistent with the fillet results,
carbamazepine occurred in only the liver samples from Chicago. The fewest number of
23
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Results
detections occurred at Orlando and Dallas, two sites where WWTPs apply advanced treat-
ment technologies before discharging effluents into the streams. Site-specific data show that
six of the seven pharmaceuticals found in liver samples were detected in the fish liver sam-
ples from Chicago and West Chester. Gemfibrozil was not detected in Chicago liver samples
and carbamazapine was not detected in West Chester liver samples.
Compared to the fillet concentrations, the concentrations of pharmaceutical compounds
measured in fish liver samples were greater. Differences among their mean concentrations
ranged from a factor of nearly three to more than 20. All of the diltiazem concentrations
in the liver samples were less than 1 ng/g (1 ppb). Sertraline had the highest concentrations
reported for pharmaceuticals in liver samples, which included maximum concentrations
of 100 ng/g at Phoenix, 150 ng/g at Chicago, and 550 ng/g at West Chester. The levels of
sertraline in the liver samples from West Chester and Phoenix may be anomalous based on
recovery data for quality control samples analyzed from these locations (i.e., matrix spike
samples) that exceeded the defined acceptability limits.
Matrix spike samples for liver tissue from West Chester exceeded the acceptable limit for
sertraline recovery by a factor of about three (acceptable limit for percent recovery of 150%
and percent recovery of 473%), while the recovery of sertraline from Phoenix liver tissue was
172% compared to the acceptable limit of 150%. Table 12 provides the matrix spike recov-
ery data for the four pharmaceutical compounds detected in liver tissue with recoveries that
exceeded the acceptable limit (fluoxetine, gemfibrozil, norfluoxetine, and sertraline). Note
that the recovery data for liver samples from Chicago and the reference site did fall within
the acceptable limits, so the liver results for Chicago and reference site samples are not
affected by the matrix spike recovery issue. However, the matrix spike recovery data may be
a confounding factor for the liver results reported for these four pharmaceuticals at Dallas,
Orlando, Phoenix, and West Chester.
Table 12. Matrix Spike Recovery Data for Four Pharmaceutical Compounds Detected in Liver
Composite Samples that Exceeded Acceptable Limits3.
Chemical
Matrix Spike Recovery Percentage
Chicago
Dallas
Orlando
Phoenix
WEST
Chester
Reference
(New Mexico)
Fluoxetine
144
335
349
271
362
105
Norfluoxetine
92
398
350
197
247
115
Sertraline
96
584
407
172
473
120
Gemfibrozil
172
166
527
246
218
106
a The acceptable range was from 60% to 150%.
3.2.3 Results for Personal Care Product Chemicals in Fillets
Concentration data for personal care product (PCP) chemicals in fish tissue are available
only for fillet samples. In applying their PCP method to liver samples, Baylor University
encountered problems with interferences due to the high lipid content of the liver tissue
24
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Results
(Section 2.5.2.2). PCP results show that two of the 12 target chemicals occurred in the
fillet samples. These chemicals include galaxolide and tonalide, which are both fragrances
added to common products like cosmetics and detergents. Both chemicals were detected
in the fillet samples at all five sites. Galaxolide was detected more frequently in the fillet
samples than any of the pharmaceutical compounds were detected in either the fillet or
liver samples. It occurred in 29 of the 30 fillet samples. Tonalide was detected in 26 of the
30 fillet samples, a detection frequency that matched the occurrence of nor fluoxetine in liver
samples. Site-specific data reveal that galaxolide and tonalide occurred in every fillet sample
from Chicago, Dallas, Phoenix, and West Chester. Orlando fillet samples had the lowest fre-
quencies of detection for the PCP chemicals with five detections for galaxolide and one for
tonalide.
Fillet concentrations of galaxolide occurred in the low parts per million range at a major-
ity of the sites. The mean concentrations of galaxolide exceeded 1,000 ng/g or 1 part per
million (ppm) in fillet samples from Chicago, Phoenix, and West Chester (1,300 ng/g for
Chicago samples and 1,800 ng/g for Phoenix and West Chester samples). The maximum
concentrations of galaxolide ranged from 1,800 ng/g to 2,100 ng/g (1.8 ppm to 2.1 ppm)
in fillet samples from Chicago, Dallas, Phoenix, and West Chester. Orlando fillet samples
contained the lowest concentrations of galaxolide with a mean concentration of 100 ng/g
(0.1 ppm) and a maximum concentration of 290 ng/g (0.29 ppm). Tonalide concentrations
in the fillet samples were about an order of magnitude lower than the galaxolide concentra-
tions. The mean tonalide concentrations in the fillet samples from the five sites ranged from
55 ng/g to 240 ng/g, and the maximum tonalide concentration of 290 ng/g was measured in
a fillet sample from Phoenix.
3.3 Lipid Results
Fish sampling efforts resulted in the collection of different species at each site since field
teams found that the resident species differed from site to site. Table 13 lists the fish spe-
cies collected at each sampling location, along with the mean percentage of lipids measured
in the fillet and liver tissue samples for the individual species. The lipid content of both the
fillet and liver samples varied widely among the six species analyzed for this study. The
mean lipid measurements ranged from 1.0% to 4.9% in the fillet samples and from 2.2%
to 11.6% in the liver samples. Sonora sucker fillet samples from the reference site in New
Mexico had the highest mean percentage of lipids in the fillet tissue, while the common
carp samples from Phoenix contained the highest mean percentage of lipids in liver tissue.
Appendix E contains tables that provide the site-specific lipid data used to summarize the
data in Table 13.
Lipid concentrations in fish are often closely correlated to the accumulation of chemicals
in their tissue. Groups of non-polar organic chemicals, such as pesticides, polychlorinated
biphenyls (PCBs), and dioxins and furans, tend to accumulate in lipid-rich tissues, particu-
larly in the livers. For this study, Ramirez et al. (2009) examined the relationship between
lipid content and accumulation of PPCPs in fish tissue by plotting percent lipid versus
25
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Results
chemical concentration for all six composite samples from each sampling site. The plots indi-
cated that there was no relationship between these two variables for any of the pharmaceu-
tical compounds detected in the fish tissue. In contrast, the two PCP chemicals detected in
fillet samples, galaxolide and tonalide, showed significant positive correlations between lipid
content and chemical concentrations in the fillet samples at Orlando and Dallas for galax-
olide and at Chicago and Dallas for tonalide.
Table 13. Lipid Percentage in Fish Fillet and Liver Composite Samples.
location
Species
% Lipid in Fillets
% Lipid in Liver
Mean
SD
Mean
SD
Chicago
Largemouth bass
2.3
0.6
2.2
0.4
Dallas
Smallmouth buffalo
2.2
1.1
8.1
2.7
Orlando
Bowfin
1.0
0.7
2.9
1.6
Phoenix
Common carp
3.9
0.8
11.6
2.1
West Chester
White sucker
1.9
0.4
4.7
0.9
Reference (NM)
Sonora sucker
4.9
1.6
4.9
2.5
26
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
4.0 Conclusions and Future Research
To advance the science related to detecting PPCPs in the environment, EPA teamed with
research scientists at Tetra Tech, Inc. and Baylor University to conduct the first national pilot
study for investigating the occurrence of PPCPs in fish tissue. Since the majority of human
pharmaceuticals and personal care product chemicals enter surface waters from point-source
release of WWTP effluents (Daughton and Ternes 1999), EPA obtained fish for the study
from locations on effluent-dominated streams near WWTP discharges at five sites across
the country. Chemists at Baylor University applied methods they developed to analyze fillet
and liver samples for up to 24 pharmaceuticals (Ramirez et al. 2007) and fillet samples
for 12 chemicals commonly used in personal care products (Mottaleb et al. 2009). Of the
24 pharmaceuticals and 12 PCPs analyzed for this study, 17 of the pharmaceuticals (71%)
and 10 of the PCPs (83%) were not detected in the fish tissue samples even though EPA
targeted sampling locations that potentially represented worst-case scenarios for studying
occurrence of PPCPs. However, some of the PPCPs that occurred in the fish tissue samples
were widely distributed among the study sites, including the following compounds: the anti-
histamine, diphenhydramine; the antidepressant, sertraline; and the musk fragrances, galax-
olide and tonalide.
This final section of the report provides information that relates pilot study results to results
from earlier studies of PPCPs in fish tissue, summarizes significant findings from the pilot
study, and describes work that EPA is pursuing to expand investigations of the occurrence
of contaminants of emerging concern (CECs) in fish tissue. Ramirez et al. (2009) contains a
detailed discussion of the pilot study results, and this report summarizes highlights of that
discussion in Section 4.1. Section 4.2 presents the pilot study conclusions and Section 4.3
describes further research that EPA is conducting on CECs in fish tissue.
4.1 Discussion of Pilot Study Results
An important topic of discussion in Ramirez et al. (2009) is the greater occurrence of PPCPs
in effluent-dominated rivers and streams compared to other aquatic environments based on
results from this study and from previous research. WWTPs releasing effluents into rivers
and streams continually introduce PPCPs not removed by plant treatment processes into
these aquatic systems. Fish living in the vicinity of these point-source discharges over their
entire life cycle encounter exposures to constantly replenished concentrations of PPCPs that
can be characterized as pseudopersistent exposures. Given this continuous exposure sce-
nario, PPCPs can accumulate in fish tissue as demonstrated by the high concentrations of
galaxolide measured in the fillet samples analyzed for the pilot study.
The core discussion in Ramirez et al. (2009) compares results of the pilot study to results
from previous studies of PPCPs in fish tissue. This comparison emphasizes earlier work
27
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Conclusions and Future Research
conducted by Baylor University on fish tissue samples collected from Pecan Creek (an efflu-
ent-dominated stream in the Denton area of north central Texas) and analyzed for PPCPs
(Brooks et al. 2005). Pharmaceutical results from these studies show detections for the same
pharmaceutical compounds as the pilot study (i.e., carbamazepine, diltiazem, diphenhydr-
amine, fluoxetine, norfluoxetine, and sertraline) except for gemfibrozil. In addition, the
concentration ranges measured for the pharmaceuticals in the fish tissue samples are simi-
lar between the Pecan Creek and national pilot studies. Chemists at Baylor University also
detected and quantified the same PCPs in fillet samples from Pecan Creek that occurred in
the pilot study fillet samples, i.e., galaxolide and tonalide, although at slightly lower con-
centrations. Overall, there is good agreement between the Brooks et al. (2005) research and
the pilot study results for the number and types of PPCPs detected in fish tissue and for
the range of concentrations measured in the tissue. The discussion of pilot study results in
Ramirez et al. (2009) includes data cited from Canadian, Danish, German, and Swiss stud-
ies of PPCPs in fish tissue. These data comparisons demonstrate similarities in the specific
PPCPs detected and in the concentrations of those compounds.
Fillet concentrations of musk compounds detected in fish tissue from this study, specifically
galaxolide, are up to an order of magnitude higher than whole-body concentrations detected
in recent studies (Osemwengi and Gerstenberger 2004). Given the potential for both ecologi-
cal and human health effects of galaxolide (Luckenbach and Epel 2005), its detection in fish
tissue at the ppm-level may be of concern.
4.2 Pilot Study Conclusions
Data from EPA's National Pilot Study of PPCPs in Fish Tissue support conclusions from
earlier studies and offer new insights, particularly with respect to the benefits of advanced
wastewater treatment technologies on the occurrence of PPCPs in fish tissue. Conclusions for
this study derive primarily from differences in PPCP detections and concentrations related to
type of tissue, geographic location, and level of treatment technologies applied in WWTPs
before discharge of effluents into rivers or streams. They include the following:
¦ At all five of the sampling locations, pharmaceutical compounds occurred in greater
numbers and at higher detection frequencies and concentrations in liver samples than
in fillet samples. No significant relationships were observed between lipid content and
accumulation of pharmaceuticals in either fillet or liver tissue. Ramirez et al. (2009)
noted that these differences are consistent with the liver being the primary site of
metabolism of xenobiotics (compounds that are foreign to an organism, such as drugs
and environmental pollutants) in fish.
¦ Differences in wastewater treatment technologies can substantially affect the removal
efficiency of pharmaceutical compounds from wastewater discharges. By design, the
pilot study included sampling locations near discharges from WWTPs that apply
various levels and types of wastewater treatment processes. Fewer detections at lower
frequencies and concentrations occurred at sites with more advanced wastewater
treatment (Dallas, TX and Orlando, FL) than those with lower levels of wastewater
28
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Conclusions and Future Research
treatment (Chicago, IL and West Chester, PA). The advanced wastewater treatment at
the Dallas WWTP involves ozonation, while the process line at the Orlando WWTP
diverts nearly half of the daily load of treated wastewater through a constructed
wetland to significantly reduce the amount of wastewater directly discharged into
the adjacent river. In contrast, the wastewater entering the Chicago and West Chester
WWTPs receives a less advanced level of treatment (secondary treatment) before being
discharged from the plants.
¦ Results from this study do not provide evidence that factors such as percentage of
population age 65 and older or median income can serve as reliable indicators of areas
where human pharmaceuticals are more likely to occur and accumulate in fish tissue.
Based on EPA's evaluation of data from a limited number of sites, it appears that the
wastewater treatment technologies applied in local WWTPs is a better predictor of
pharmaceutical occurrence. However, demographics of local populations can influence
geographic differences in detections of pharmaceutical compounds in fish tissue. For
example, carbamazepine (an anti-seizure drug) was only detected in fillet and liver
samples from Chicago.
¦ The widespread occurrence of norfluoxetine in fish tissue samples analyzed for this
study provides further evidence of the importance of including metabolic products of
target chemicals in future studies. Norfluoxetine is a metabolite of the antidepressant
fluoxetine that has been identified in previous studies (Brooks et al. 2005). Koplin et
al. (2002) documented the importance of obtaining data on metabolites for analysis
of PPCPs in water, and results from EPA's PPCP Fish Pilot Study demonstrate the
importance of targeting metabolites in fish tissue. It will be critical for future research
efforts to identify other metabolites that are biologically active and persistent in fish.
However, future analysis of these metabolic products may be limited by the availability
of pure standards and the performance of the analytical method.
4.3 Future Research
Obtaining environmental data on CECs continues to be an area of interest for EPA. Since
completing the PPCP Fish Pilot Study, EPA has initiated a national study of CECs in urban
rivers and a regional assessment of CECs in the Great Lakes. The designs for both studies
involve analysis of perfluorinated compounds (PFCs, including PFOA and PFOS) in fish fillet
samples. Sections 4.3.1 and 4.3.2 provide summaries of the design and status for the urban
river and Great Lakes studies, respectively.
4.3.1 National Urban River CEC Study
Results from recent studies (e.g., Barcelo and Petrovic 2007, and Lau et al. 2007) prompted
EPA to explore options to expand investigation of CECs in the environment to a national
scale. In 2008, an opportunity to conduct a nationally representative study of CECs in
surface water and fish tissue samples became available under EPA's National Rivers and
Streams Assessment (NRSA). The NRSA is a probability-based study that involved collec-
tion of physical, chemical, and biological indicator data at approximately 1800 randomly
29
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Conclusions and Future Research
selected sites (across all stream orders) in the lower 48 states. Data from the NRSA will
allow a statistically valid characterization of the condition of rivers and streams throughout
the country.
The statistical design of the NRSA included a representative subset of 163 urban river sam-
pling locations. EPA planned and is currently implementing a special study of CECs at these
urban river sites called the National Urban River CEC Study. This is EPA's first broad assess-
ment of CECs on a national level using a statistically based sampling design. The urban river
study includes the following components:
¦ Sampling at 163 randomly selected urban river sites (5th order or larger) throughout the
lower 48 states;
¦ Collecting one surface water grab sample and one fish composite sample (five similarly-
sized adult fish of a single species that is commonly consumed by humans) from each
site;
¦ Analyzing the surface water samples for 54 pharmaceutical compounds; and
¦ Analyzing fish fillet composite samples for 13 PFCs, and 6 musks (including galaxolide
and tonalide).
Two EPA laboratories and one commercial laboratory are analyzing the urban river fish and
water samples for CECs. EPA's National Exposure Research Laboratories in Cincinnati, OH
and Las Vegas, NV analyzed surface water samples for pharmaceuticals and fish fillet sam-
ples for musks, respectively. AXYS Analytical in Sydney, British Columbia analyzed fillet
samples for PFCs. The CEC assessments are in various stages of data review and analyses.
EPA anticipates reporting CEC results in 2013.
4.3.2 Great Lakes Human Health Fish Tissue Study
EPA identified an opportunity to assess CECs in fish on a regional scale during planning for
its 2010 National Coastal Condition Assessment (NCCA). EPA's field effort for the NCCA
during 2010 consisted of collecting physical, chemical, and biological data from about
680 randomly selected marine sites along the coasts of the United States and from a repre-
sentative set of 225 nearshore sites throughout the Great Lakes. EPA will use results from
the NCCA to characterize the condition of the Nation's coastal and Great Lakes waters.
EPA's Office of Water, Great Lakes National Program Office, and Office of Research and
Development combined resources and expertise to conduct the first statistically based assess-
ment of Great Lakes fish contamination relevant to human health under the NCCA. The
team initiated the Great Lakes Human Health Fish Tissue Study to collect and analyze fish
samples for a number of contaminants, including mercury, polychlorinated biphenyls (PCBs),
polybrominated diphenyl ethers (PBDEs), and PFCs. This assessment also includes analysis
of fillet tissue for fatty acids to obtain species-specific data on compounds that may offer
health benefits. Field crews collected fish samples from a representative subset of 157 Great
Lakes sites (about 30 sites per lake) in the nearshore regions (up to 30 meters deep or
30
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Conclusions and Future Research
5 kilometers from shore). The Baltimore, MD Division of Microbac Laboratories prepared
the fish samples for analysis (i.e., filleting and grinding the tissue). The following labora-
tories are analyzing Great Lakes fillet samples: Brooks Rand Labs in Seattle, WA for mer-
cury; AXYS Analytical in Sydney, British Columbia for 209 PCB congeners; ALS Canada
in Burlington, Ontario for 52 PBDEs; and TestAmerica Laboratories in West Sacramento,
CA for 13 PFCs. Southwest Research Institute in San Antonio, TX is analyzing the tissue
samples for 5 fatty acids. Results for the target chemicals will be available in 2013.
EPA will evaluate results from the National Urban River CEC Study and the Great Lakes
Human Health Fish Tissue Study to determine future directions for assessment of CECs in
fish tissue. EPA is archiving tissue from both studies to provide more cost-effective alterna-
tives for assessing accumulation of new CECs in fish tissue. Forming creative partnerships
with other public or private entities will also be critical for continuing agency efforts to
monitor levels of CECs in fish.
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
32
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
5.0 References
Barber, L.B., G.K. Brown, T.G. Nettesheim, E.W. Murphy, S.E. Bartelland, and H.L.,
Schoenfuss. 2011. Effects of biologically-active chemical mixtures on fish in a
wastewater-impacted urban stream. Science of the Total Environment 409 (22):
4720-4728.
Barcelo, D. and and M. Petrovic. 2007. Pharmaceuticals and personal care products (PPCPs)
in the environment. Analytical and Bioanalytical Chemistry 387(4): 1141-1142.
Brooks, B.W., C.K. Chambliss, J.K. Stanley, A.J. Ramirez, K.E. Banks, R.D. Johnson, and
R.J. Lewis. 2005. Determination of select antidepressants in fish from an effluent-
dominated stream. Environmental Toxicology and Chemistry 24:464-469.
Daughton, C.G. and T.A. Ternes. 1999. Pharmaceuticals and personal care products
in the environment: Agents of subtle change? Environmental Health Perspectives
107(6):907-938.
Koplm, D.W., E.T. Furlong, M.T. Meyer, E.M. Thurman, S.D. Zaugg, L.B. Barber, and
H.T. Buxton. 2002. Pharmaceuticals, hormones, and other organic wastewater
contaminants in U.S. streams, 1999-2000: A national reconnaissance. Environmental
Science and Technology 36:1202-1211.
Lau, C., K. Anitole, C. Hodes, D. Lai, A. Pfahles-Hutchens, and J. Seed. 2007.
Perfluoroalkyl acids: A review of Monitoring and Toxicological findings. Toxicological
Sciences 99(2): 366-394.
Luckenbach, T. and D. Epel. 2005. Nitromusk and polycyclic musk compounds as long-term
inhibitors of cellular xenobiotic defense systems mediated by multidrug transporters.
Environmental Health Perspectives 113(l):17-24.
Mottaleb, M.A., S. Usenko, J.G. O'Donnell, A.J. Ramirez, B.W. Brooks, and C.K.
Chambliss. 2009. Gas chromatography-mass spectrometry screening methods for
select UV-filters, synthetic musks, alkylphenols, an antimicrobial agent, and an insect
repellent in fish. Journal of Chromatography A 1216:815-823.
Osemwengi, L.I., and S.L. Gerstenberger. 2004. Levels of synthetic musk compounds in
municipal wastewater for potential estimation of biota exposure in receiving waters.
Journal of Environmental Monitoring 6:533-539.
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
References
Ramirez A.J, M.A. Mottaleb, B.W. Brooks, and C.K. Chambliss. 2007. Analysis of
pharmaceuticals in fish using liquid chromatography-tandem mass spectrometry.
Analytical Chemistry 79:3155-3163.
Ramirez A.J., R.A. Brain, S. Usenko, M.A. Mottaleb, J.G. O'Donnell, L.L. Stahl, J.B.
Wathen, B.D. Snyder, J.L. Pitt, R Perez-Hurtado, L.L. Dobbins, B.W. Brooks, and C.K.
Chambliss. 2009. Occurrence of pharmaceuticals and personal care products in fish:
Results of a national pilot study in the United States. Environmental Toxicology and
Chemistry 28(12):2587-2597.
U.S. Environmental Protection Agency (USEPA). 2000. Guidance for Assessing Chemical
Contaminant Data for Use in Fish Advisories, Volume 1: Fish Sampling and Analysis,
Third Edition. U.S. Environmental Protection Agency, Office of Water, Washington,
D.C. EPA-823-B-00-007.
U.S. Environmental Protection Agency (USEPA). 2006a. Quality Assurance Project
Plan for Sample Collection Activities for a Pilot Study to Investigate the
Occurrence of Pharmaceuticals and Personal Care Products (PPCPs) in Fish Tissue.
U.S. Environmental Protection Agency, Office of Water, Washington, D.C.
EPA 820-F-23-003.
U.S. Environmental Protection Agency (USEPA). 2006b. Quality Assurance Project
Plan for Laboratory Sample Preparation and Analysis Activities in the National
Pilot Study of Pharmaceuticals and Personal Care Products (PPCPs) in Fish Tissue.
U.S. Environmental Protection Agency, Office of Water, Washington, D.C.
EPA 820-F-23-004.
U.S. Environmental Protection Agency (USEPA). 2023. Quality Assurance Report for the
National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue.
U.S. Environmental Protection Agency, Office of Water, Washington, D.C.
EPA 820-F-23-002.
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix A
Analytical Procedures
-------
National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
-------
National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix A: Analytical Procedures
Analysis of Pharmaceuticals by HPLC-MS/MS
Following homogenization, fillet and liver tissue samples were subsequently extracted and
analyzed for 24 and 23 pharmaceutical compounds, respectively, by high performance liquid
chromatography with tandem mass spectrometry (HPLC-MS/MS) using methods described
in Ramirez et al. (2007). The method utilizes matrix-matched calibration standards (aliquots
of control matrix from outside of the study area that are expected to be reasonably free of
target compounds) spiked at a minimum of five concentrations, and extracted and analyzed
along with study samples. By extracting standards, matrix effects and bias were minimized
in the final analytical results.
Sample Extraction for Pharmaceuticals Analysis
Preparation of homogenates for analysis involved the following steps:
1) weighing out 1.0 gram (g) of fillet tissue and 0.5 g of liver tissue for each standard or
sample composite individually to the nearest 0.01 g and placing each tissue aliquot
into a 20-mL borosilicate glass screw-cap vial,
2) spiking with the appropriate surrogates (acetaminophen-d4, diphenhydramine-d3,
carbamazepine-d10, and ibuprofen-13C3) and standard mixtures (full target list
spikes for calibration standards, control and matrix spike [MS/MSD] samples), as
appropriate to complete the required batch quality control (method blank, low and
high level control samples, and a pair of spiked MS/MSD samples from each site) in
each analytical batch of 20 or fewer samples,
3) combining sample homogenates with 8 milliliters (mL) of 1:1 mixture of 0.1 M acetic
acid buffer (pH 4) and methanol extraction solvent, and tightly replacing the cap,
4) sonicating the homogenate mixture in an ultrasonic bath for 15 minutes at 25° C,
5) shaking the mixture vigorously for 20 seconds to further ensure mixing and
extraction,
6) quantitatively transferring each extract to a separate 50 mL polypropylene copolymer
round-bottomed centrifuge tube with several rinses of the extraction solvent,
7) centrifuging the extracts at 16,000 rpm for 40 minutes at 4° C to achieve a full
separation of residual solid and liquid phases,
8) transferring the supernatant into a clean 18 mL disposable borosilicate glass culture
tube with rinses using disposable Pasteur pipettes,
9) evaporating the sample extracts to dryness under a stream of dry nitrogen at 45° C,
10) reconstituting the extracts in 1 mL of mobile phase 0.1% formic acid in reagent water,
11) adding internal standards (7-aminoflunitrazapam-d7, fluoxetine-d6, and meclofenamic
acid),
12) sonicating for 1 min at 25° C,
13) filtering extracts using a Teflon membrane syringe filter into an amber HPLC injection
vial, and
14) sealing the vial with a fluoropolymer lined cap.
A-l
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix A: Analytical Procedures
Instrumental Analysis of Pharmaceuticals by HPLC-MS/MS
Pharmaceutical compounds were determined using analytical methods described in Ramirez
et al. (2007). Calibration standard extracts (7 concentrations for fillet and 8 concentrations
for liver tissue calibration) and sample extracts were analyzed on a Varian HPLC equipped
with a binary pump and auto sampler connected to a 12.5 mm by 2.1 mm (inside diameter)
C18 guard column, which preceded a 15 cm by 2.1 mm (inside diameter) C18 chromato-
graphic column. Chromatographic separations were completed under a binary gradient con-
sisting of 0.1% (v/v) formic acid in water and 100% methanol. Additional chromatographic
parameters included:
¦ 10 microliter (pL) injection volume,
¦ 30°C column temperature, and
¦ 350 pL/min mobile phase flow rate.
Eluted analytes were monitored by MS/MS using a Varian triple-quadrupole mass analyzer
equipped with an electrospray interface (ESI).
Target analytes were introduced individually into the mass spectrometer in both positive and
negative ionization modes to determine the best ionization mode for analysis and to identify
the most intense precursor ions for each target analyte. Once these variables were isolated,
the energy at the collision cell was adjusted, while the third quadrupole was scanned to iden-
tify and optimize the intensity of a product ion for each compound. Precursor and product
ions were identified for each target analyte. Additional instrumental parameters held con-
stant for all analytes were as follows:
¦ nebulizing gas — nitrogen (N2) at 60 pounds per square inch (psi),
¦ drying gas — N2 at 19 psi,
¦ temperature —300 °C,
¦ needle voltage — 5000 V ESI+, 4500 V ESI-,
¦ declustering potential — 40 V, and
¦ collision gas — argon at 2.0 mTorr.
Sample results were calculated using the instrument software against a linear internal
standard calibration curve for each tissue type. No additional multi-point calibration was
required after the initial calibration curve analysis (i.e., analysis of a single calibration
curve for fillets and a single calibration curve for livers). Analytical performance was veri-
fied through analysis of continuing calibration standards or daily standard calibration
verification.
A-2
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix A: Analytical Procedures
Analysis of Personal Care Products by GC-MS/MS
Following sample homogenization, fillet tissue samples were subsequently extracted and
analyzed for 12 personal care product (PCP) chemicals by gas chromatography with tandem
mass spectrometry (GC-MS/MS) using methods described by Mottaleb et al. (2009). Unlike
the pharmaceutical method, matrix-matched calibration standards were not utilized in the
PCP analytical method since matrix interferences were not as prevalent following the rigor-
ous clean-ups used in the sample extraction procedures. Standards for the PCP analysis were
prepared in solvent and subjected to derivatization to enhance measurement response, but
were not subjected to the full preparation procedures used for study samples.
Sample Extraction for Personal Care Products Analysis
Preparation of homogenates for analysis involved the following steps:
1) weighing out 1.0 g of fillet tissue for each sample composite and 1.0 g of control
matrix for each QC sample individually to the nearest 0.01 g and placing each tissue
aliquot into a 20-mL borosilicate glass screw-cap vial,
2) spiking all samples with the appropriate surrogates (benzophenone-d10 and 13C6
p-nonylphenol) and spiking QC samples (control and MS/MSD samples) with standard
mixtures containing all target analytes, as appropriate, to complete the required batch
quality control (method blank, low and high level control samples, and a pair of MS/
MSD samples from each site) in each analytical batch of 20 samples or fewer,
3) adding 10 mL of acetone to each spiked homogenate aliquot,
4) sonicating samples for 15 min at 25° C,
5) shaking vigorously on removal for 20 seconds to further ensure mixing and
extraction,
6) transferring samples into 50-mL polypropylene copolymer round-bottomed centrifuge
tubes using 1 mL acetone as a rinse,
7) centrifuging at 16,000 rpm for 40 min at 4° C,
8) transferring the supernatant into 18-mL disposable glass test tubes,
9) evaporating the solvent to dryness under a stream of nitrogen at 30° C,
10) reconstituting the samples in 200 pL of 65:35 (v/v) hexane:acetone in preparation for
silica gel clean-up,
11) loading sample extracts onto a preconditioned (8 mL of 65:35 hexane:acetone by
volume) silica gel column (1 g), and eluting with 30 mL of hexane:acetone,
12) evaporating the resultant extract to near-dryness and reconstituting in 700 pL of
methylene chloride to allow a solvent exchange in preparation for gel permeation
chromatography (GPC) clean-up,
A-3
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix A: Analytical Procedures
13) injecting one half (350 pL) of the extract into the GPC to separate the target analytes
from co-extracted interferences by 5 mL/min elution through a cross-linked styrene
divinylbenzene copolymer guard (30 mmx4.6 mm) and analytical (150 mmxl9 mm)
columns connected in series,
14) discharging co-extracted interferences to waste and collecting the fraction eluting
between 11.4 and approximately 19.4 minutes,
15) concentrating the methylene chloride extract to near dryness and reconstituting it to
approximately 200 pL in hexane:acetone,
16) adding 100 pL of MSTFA derivatizing agent, capping the GC vial, and heating the
mixture in an oven at 60° C for 45 min,
17) concentrating the derivatized extract one final time to near dryness at room
temperature under a stream of nitrogen, then reconstituting it in 180 pL of 77-hexane,
and
18) spiking the extracts prior to analysis with 20 pL of the internal standards
(phenanthrene-d10 and mirex) as described by Mottaleb et al. (2009).
Instrumental Analysis of Personal Care Products by GC-MS/MS
Sample extracts were analyzed on a Varian GC system equipped with an autosampler and
ion trap mass spectrometer. Analytes were separated using a 30 mm by 0.25 mm, 0.25 pm
VF-5 MS capillary column. A temperature program, starting at 100° C and ramping up
to 290° C, allowed separations in approximately 25 minutes with additional bake-out and
equilibration resulting in approximately 50 minutes between injections. Helium was used
as the carrier gas at a constant flow rate of 1 mL/min (linear velocity 37.2 cm/s). Injections
of 1.0 pL were made using splitless mode and an injection port temperature of 275° C. The
transfer line between the GC and the mass spectrometer was kept at 280° C.
Sample results were calculated using the available instrument software against a linear inter-
nal standard calibration curve. A total of two calibration curves were required to complete
the analysis of samples from all 6 sites (i.e., once derivatized, standards exhibited a degraded
response, and a second set of calibration curve and continuing calibration verification stan-
dards were required to complete the sample analyses). Initial daily calibration verification
standards and frequent calibration verifications were distributed throughout the analytical
sequences to minimize reanalysis required due to the cumulative effects of co-extractables
(in excess of those removed during preparatory chromatography), which degraded chromato-
graphic performance.
A-4
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix A: Analytical Procedures
Analysis of Lipids
Three replicate tissue aliquots (approximately 2 g) from each fillet composite were analyzed
for lipids using the procedure described in Mottaleb et al. (2009). This procedure was
modified slightly for liver specimens. Due to limited sample mass, triplicate measurements
were made for only one liver composite from each sampling site except Phoenix, for which a
duplicate was analyzed (Appendix E). All other lipid determinations for liver samples were
based on a single measurement.
Lipid analysis involved the following steps:
1) combining 2 g of tissue with 15 mL of a 1:1 mixture of dichloromethane:hexane in a
borosilicate vial,
2) homogenizing each mixture for 3 minutes using a motor-driven tissue homogenizer,
3) placing the vials in an incubator for 24 hours at 35°C and periodically agitating by
gentle end-over-end rotation,
4) adding 2 g of solid anhydrous sodium sulfate for each 1 g of sample (g Na2S04 =
2 x g tissue) following extraction,
5) filtering the mixture through Grade 415 filter paper,
6) washing the solid residue with an additional 15 mL of 1:1 dichloromethane:hexane,
7) collecting the combined filtrate for each sample in a pre-weighed test tube,
8) evaporating the solvent with dry nitrogen for 8 hours at 45° C using an evaporator
concentration workstation, and
9) drying each residue after evaporation to a constant weight in a vacuum oven at 40° C.
Lipid content was determined gravimetrically by weighing the three replicates from each
sample. Percent lipid determinations were then calculated as shown in the following
example, where t.t = test tube:
Sample weight (g)
t.t WEIGHT (g)
t.t+LIPID WEIGHT (g)
LIPID w(g)
Lipid %
2.1630
9.6768
9.7060
0.0292
1.35
Lipid weight = ((t.t + lipid) weight) - t.t weight
= (9.7060-9.6768)g
= 0.0292g
Lipid % = Lipid weight x 100
Sample weight
= 0.0292g x 100
2.1630g
= 1.35%
A-5
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix A: Analytical Procedures
Quantifying Target Chemicals
The pharmaceutical method used matrix-matched (to the extent possible) calibration stan-
dards, and both instrumental methods used internal standard calibration and quantitation
equations. Internal standard calibration required the determination of relative response fac-
tors (RRF) defined by the following equation:
Ris = the area response of the quant ion m/z for the internal standard,
Rx = the area response of the quant ion m/z for the target chemical,
Ax = the amount (concentration in ng/g) of the target compound, and
Ajs = the amount (concentration in ng/g) of the internal standard.
The quantitation ion (quant ion) is the primary ion used in the calculation of calibration
curve coefficients (or linearity) and sample analysis. Generally speaking, quantitation ions
are the most responsive, as they are compared to other spectral data to complete the qualita-
tive confirmation both by the instrument data system software and by the analytical chemist
conducting the measurements. At the onset of analysis, multiple quant ions may be selected
in order to afford the analyst an opportunity to select the one least impacted by the sample
matrix, if necessary.
Calibration data were evaluated through assessment of the relative standard deviation (RSD)
of the RRFs in the calibration curve using the following equations:
where s is the standard deviation, % is the mean RRF over the standard curve, and the stan-
dard deviation of the curve is calculated as follows:
R A
RRF — —— x——
Ris 4
Where:
RSD
X
where X is the measured value of the replicate, % is the mean of the measured values, and n
is the number of replicates.
A-6
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix A: Analytical Procedures
Both analytical methods included an optional relative standard deviation criterion of less
than 30% to demonstrate linearity across the calibration range. If the calibration curve
produced an evaluation of < 30% RSD, the average RRF could be used to calculate sample
results in subsequent analyses. Alternatively, a curve can be plotted and used to calculate
sample results, provided the correlation coefficient (R2) is greater than 0.995. The latter
quantitation technique was used for sample analysis, as it is a more commonly used feature
in the instrument's data handling capabilities and it gave the analyst the ability to assess
data in real time.
Linear regression calibration curve output reports provided slope values for each target
chemical, and sample results were calculated as in the following example for a control
sample copied from one of the study reports. A requirement for all laboratory deliverables
was to include an example calculation verifying the data system results for a control sample
(the only samples where analytes are certain to be found).
General Quantitation Report
Data File: c:\varianws\data\tetratech\2-21-2008\REF-CCV_F.SMS Acquisition Date: 2/21/200811:35
Comment: M3
SamplelD: REF-CCV_F Analyst: AM
Calibration File: C:\VarianWS\data\Tetra Tech\2-05-2008\rerun cal levels (L1-L7)\L7_.SMS
Cal. Sample Dates: First: 2/5/2008 17:49 Last: 2/5/2008 22:41
Compound
R. T.
SCAN#
Q lON(s)
Area
CONC
UNITS
Match
RF
7)* Phenanthane-d10
10.40
1094
160.3
390415
40.00
ng/g
995
1.000
ng/g
r\r\r\
TTzTTj
1) m-Toluamide
7.14
481
145.0
13489
157.40
ng/g
999
0.009
3)Benzophenone
7.90
623
153.0
66833
299.92
ng/g
999
0.023
4) Celestolide
8.83
798
172.9
16490
219.69
ng/g
999
0.008
9^2
r\r~t
ng/g
r\r\r\
TTzTTj
u) I UNLJ ^SU[fO]
8) Octylphenol
10.72
1153
178.9
21197
72.48
ng/g
100
0.030
9) Galaxolide
11.50
1303
213.1
49746
460.13
ng/g
975
0.011
10) Musk Xylene
11.65
1332
265.0
24349
4529.73
ng/g
991
0.001
11) Tonalide
11.69
1341
187.0
27683
509.39
ng/g
990
0.006
13) Nonylphenol
13.06
1615
178.9
37592
127.20
ng/g
100
0.030
O
ng/g
942
15) 4MBC
16.41
2245
169.1
77836
2397.15
ng/g
945
0.003
16) Musk Ketone
16.47
2258
287.1
23502
8694.87
ng/g
925
0.000
17) Triclosan
17.30
2427
200.0
56898
212.40
ng/g
983
0.027
^./UU
e
\J.\J\J
119/9
20) Octocrylene
25.02
3926
248.2
153167
320.26
ng/g
788
0.049
* Indicates Internal Standard.
A-7
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix A: Analytical Procedures
Example calculation:
Concentration of target chemical = (area of Target * IS concentration) /
(area of the IS * slope of target)
m-toluamide concentration = (13489 * 40) / (390415 * 0.0088) = 539560 /
3435.65 = 157 ng/g
The quantitation reports were consulted by the analyst to access data which were near or
above established target detection limits, and that these data were used to further investigate
spectral data for product ions and relative responses between the two to complete qualita-
tive identification of results. While the "Match" column was a useful tool in identifying
poor spectral matches, analyst judgment remained integral to analysis and interpretation
of results. Initially, all sample data that provided spectral confirmation were tabulated with
appropriate qualification indicating the results were estimates reported below the calibra-
tion range, or that the results were below the calculated method detection limit. Results were
later more closely scrutinized and the decision was made to exclude only results below the
method detection limit and report them as
-------
National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix B
Site-specific Analytical Results Tables for
Pharmaceuticals in Fillet Tissue
-------
National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
-------
Table B-1. Analytical Results for Pharmaceuticals in Fish Fillet Samples from Chicago, Illinois.
Chemical
MDL
ng/g (ppb)
Chicago 1
Chicago 2
Chicago 3
Chicago 4
Chicago 5
Chicago 6
Acetaminophen
4.40
*
*
*
*
*
*
Atenolol
1.48
*
*
*
*
*
*
Caffeine
3.93
*
*
*
*
*
*
Carbamazepine
0.54
1.79
1.95
1.95
2.58
2.62
3.13
Cimetidine
1.04
*
*
*
*
*
*
Codeine
6.11
*
*
*
*
*
*
Diltiazem
0.12
0.14
0.12
*
0.12
0.12
0.16
1,7-Dimethylxanthine
1.10
*
*
*
*
*
*
Diphenhydramine
0.05
1.12
1.15
1.24
1.68
1.33
1.74
Erythromycin
6.42
*
*
*
*
*
*
Fluoxetine
6.74
*
*
*
*
*
*
Gemfibrozil
6.68
*
*
*
*
*
*
Ibuprofen
45.96
*
*
*
*
*
*
Lincomycin
5.53
*
*
*
*
*
*
Metoprolol
2.50
*
*
*
*
*
*
Miconazole
10.83
*
*
*
*
*
*
Norfluoxetine
2.90
*
*
*
3.19
*
3.21
Propranolol
1.07
*
*
*
*
*
*
Sertraline
3.56
*
*
*
*
*
*
Sulfamethoxazole
2.29
*
*
*
*
*
*
Thiabendazole
2.63
*
*
*
*
*
*
Trimethoprim
2.15
*
*
*
*
*
*
Tylosin
5.02
*
*
*
*
*
*
Warfarin
0.86
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
Table B-2. Analytical Results for Pharmaceuticals in Fish Fillet Samples from Dallas, Texas.
to
Chemical
MDL
ng/g (ppb)
Dallas 1
Dallas 2
Dallas 3
Dallas 4
Dallas 5
Dallas 6
Acetaminophen
4.40
*
*
*
*
*
*
Atenolol
1.48
*
*
*
*
*
*
Caffeine
3.93
*
*
*
*
*
*
Carbamazepine
0.54
*
*
*
*
*
*
Cimetidine
1.04
*
*
*
*
*
*
Codeine
6.11
*
*
*
*
*
*
Diltiazem
0.12
*
*
*
*
*
*
1,7-Dimethylxanthine
1.10
*
*
*
*
*
*
Diphenhydramine
0.05
*
*
*
*
*
*
Erythromycin
6.42
*
*
*
*
*
*
Fluoxetine
6.74
*
*
*
*
*
*
Gemfibrozil
6.68
*
*
*
*
*
*
Ibuprofen
45.96
*
*
*
*
*
*
Lincomycin
5.53
*
*
*
*
*
*
Metoprolol
2.50
*
*
*
*
*
*
Miconazole
10.83
*
*
*
*
*
*
Norfluoxetine
2.90
*
*
*
*
*
*
Propranolol
1.07
*
*
*
*
*
*
Sertraline
3.56
*
*
*
*
*
*
Sulfamethoxazole
2.29
*
*
*
*
*
*
Thiabendazole
2.63
*
*
*
*
*
*
Trimethoprim
2.15
*
*
*
*
*
*
Tylosin
5.02
*
*
*
*
*
*
Warfarin
0.86
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
Table B-3. Analytical Results for Pharmaceuticals in Fish Fillet Samples from Orlando, Florida.
Chemical
MDL
ng/g (ppb)
Orlando 1
Orlando 2
Orlando 3
Orlando 4
Orlando 5
Orlando 6
Acetaminophen
4.40
*
*
*
*
*
*
Atenolol
1.48
*
*
*
*
*
*
Caffeine
3.93
*
*
*
*
*
*
Carbamazepine
0.54
*
*
*
*
*
*
Cimetidine
1.04
*
*
*
*
*
*
Codeine
6.11
*
*
*
*
*
*
Diltiazem
0.12
*
*
*
*
*
*
1,7-Dimethylxanthine
1.10
*
*
*
*
*
*
Diphenhydramine
0.05
*
*
*
*
*
*
Erythromycin
6.42
*
*
*
*
*
*
Fluoxetine
6.74
*
*
*
*
*
*
Gemfibrozil
6.68
*
*
*
*
*
*
Ibuprofen
45.96
*
*
*
*
*
*
Lincomycin
5.53
*
*
*
*
*
*
Metoprolol
2.50
*
*
*
*
*
*
Miconazole
10.83
*
*
*
*
*
*
Norfluoxetine
2.90
*
*
*
*
*
*
Propranolol
1.07
*
*
*
*
*
*
Sertraline
3.56
*
*
*
*
*
*
Sulfamethoxazole
2.29
*
*
*
*
*
*
Thiabendazole
2.63
*
*
*
*
*
*
Trimethoprim
2.15
*
*
*
*
*
*
Tylosin
5.02
*
*
*
*
*
*
Warfarin
0.86
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
Table B-4. Analytical Results for Pharmaceuticals in Fish Fillet Samples from Phoenix, Arizona.
Chemical
MDL
ng/g (ppb)
Phoenix 1
Phoenix 2
Phoenix 3
Phoenix 4
Phoenix 5
Phoenix 6
Acetaminophen
4.40
*
*
*
*
*
*
Atenolol
1.48
*
*
*
*
*
*
Caffeine
3.93
*
*
*
*
*
*
Carbamazepine
0.54
*
*
*
*
*
*
Cimetidine
1.04
*
*
*
*
*
*
Codeine
6.11
*
*
*
*
*
*
Diltiazem
0.12
*
*
*
*
*
*
1,7-Dimethylxanthine
1.10
*
*
*
*
*
*
Diphenhydramine
0.05
1.04
1.20
1.41
1.21
1.12
1.24
Erythromycin
6.42
*
*
*
*
*
*
Fluoxetine
6.74
*
*
*
*
*
*
Gemfibrozil
6.68
*
*
*
*
*
*
Ibuprofen
45.96
*
*
*
*
*
*
Lincomycin
5.53
*
*
*
*
*
*
Metoprolol
2.50
*
*
*
*
*
*
Miconazole
10.83
*
*
*
*
*
*
Norfluoxetine
2.90
*
3.42
4.83
4.19
*
3.53
Propranolol
1.07
*
*
*
*
*
*
Sertraline
3.56
4.05
4.55
4.97
5.36
4.70
6.47
Sulfamethoxazole
2.29
*
*
*
*
*
*
Thiabendazole
2.63
*
*
*
*
*
*
Trimethoprim
2.15
*
*
*
*
*
*
Tylosin
5.02
*
*
*
*
*
*
Warfarin
0.86
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
Table B-5. Analytical Results for Pharmaceuticals in Fish Fillet Samples from West Chester, Pennsylvania.
Chemical
MDL
ng/g (ppb)
W. Chester 1
W. Chester 2
W. Chester 3
W. Chester 4
W. Chester 5
W. Chester 6
Acetaminophen
4.40
*
*
*
*
*
*
Atenolol
1.48
*
*
*
*
*
*
Caffeine
3.93
*
*
*
*
*
*
Carbamazepine
0.54
*
*
*
*
*
*
Cimetidine
1.04
*
*
*
*
*
*
Codeine
6.11
*
*
*
*
*
*
Diltiazem
0.12
*
*
*
0.14
0.12
0.20
1,7-Dimethylxanthine
1.10
*
*
*
*
*
*
Diphenhydramine
0.05
1.23
1.49
1.82
1.74
1.67
2.48
Erythromycin
6.42
*
*
*
*
*
*
Fluoxetine
6.74
*
*
*
*
*
*
Gemfibrozil
6.68
*
*
*
*
*
*
Ibuprofen
45.96
*
*
*
*
*
*
Lincomycin
5.53
*
*
*
*
*
*
Metoprolol
2.50
*
*
*
*
*
*
Miconazole
10.83
*
*
*
*
*
*
Norfluoxetine
2.90
3.15
2.95
4.19
4.39
3.74
4.99
Propranolol
1.07
*
*
*
*
*
*
Sertraline
3.56
7.01
6.51
13.85
12.64
9.98
18.63
Sulfamethoxazole
2.29
*
*
*
*
*
*
Thiabendazole
2.63
*
*
*
*
*
*
Trimethoprim
2.15
*
*
*
*
*
*
Tylosin
5.02
*
*
*
*
*
*
Warfarin
0.86
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
Table B-6. Analytical Results for Pharmaceuticals in Fish Fillet Samples from Gila Wilderness Area, New Mexico.
Chemical
MDL
ng/g (ppb)
Reference 1
Reference 2
Reference 3
Reference 4
Reference 5
Reference 6
Acetaminophen
4.40
*
*
*
*
*
*
Atenolol
1.48
*
*
*
*
*
*
Caffeine
3.93
*
*
*
*
*
*
Carbamazepine
0.54
*
*
*
*
*
*
Cimetidine
1.04
*
*
*
*
*
*
Codeine
6.11
*
*
*
*
*
*
Diltiazem
0.12
*
*
*
*
*
*
1,7-Dimethylxanthine
1.10
*
*
*
*
*
*
Diphenhydramine
0.05
*
*
*
*
*
*
Erythromycin
6.42
*
*
*
*
*
*
Fluoxetine
6.74
*
*
*
*
*
*
Gemfibrozil
6.68
*
*
*
*
*
*
Ibuprofen
45.96
*
*
*
*
*
*
Lincomycin
5.53
*
*
*
*
*
*
Metoprolol
2.50
*
*
*
*
*
*
Miconazole
10.83
*
*
*
*
*
*
Norfluoxetine
2.90
*
*
*
*
*
*
Propranolol
1.07
*
*
*
*
*
*
Sertraline
3.56
*
*
*
*
*
*
Sulfamethoxazole
2.29
*
*
*
*
*
*
Thiabendazole
2.63
*
*
*
*
*
*
Trimethoprim
2.15
*
*
*
*
*
*
Tylosin
5.02
*
*
*
*
*
*
Warfarin
0.86
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix C
Site-specific Analytical Results Tables for
Pharmaceuticals in Liver Tissue
-------
National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
-------
Table C-1. Analytical Results for Pharmaceuticals in Fish Liver Tissue Samples from Chicago, Illinois.
Chemical
MDL
ng/g (ppb)
Chicago 1
Chicago 2
Chicago 3
Chicago 4
Chicago 5
Chicago 6
Acetaminophen
34.28
*
*
*
*
*
*
Atenolol
12.86
*
*
*
*
*
*
Caffeine
25.47
*
*
*
*
*
*
Carbamazepine
1.86
4.27
5.15
6.64
6.77
5.32
7.52
Cimetidine
5.18
*
*
*
*
*
*
Codeine
31.49
*
*
*
*
*
*
Diltiazem
0.26
0.50
0.54
0.78
0.88
0.64
0.90
1,7-Dimethylxanthine
5.84
*
*
*
*
*
*
Diphenhydramine
0.26
5.20
4.54
7.46
9.13
5.91
9.59
Erythromycin
43.03
*
*
*
*
*
*
Fluoxetine
12.41
*
*
18.42
14.44
*
22.76
Gemfibrozil
24.82
*
*
*
*
*
*
Ibuprofen
172.81
*
*
*
*
*
*
Lincomycin
56.14
*
*
*
*
*
*
Metoprolol
8.90
*
*
*
*
*
*
Norfluoxetine
15.31
41.06
20.96
127.71
81.33
38.26
129.65
Propranolol
3.77
*
*
*
*
*
*
Sertraline
17.29
41.19
42.08
96.40
148.70
34.15
140.93
Sulfamethoxazole
13.95
*
*
*
*
*
*
Thiabendazole
7.84
*
*
*
*
*
*
Trimethoprim
8.00
*
*
*
*
*
*
Tylosin
34.67
*
*
*
*
*
*
Warfarin
2.70
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
Table C-2. Analytical Results for Pharmaceuticals in Fish Liver Tissue Samples from Dallas, Texas.
Chemical
MDL
ng/g (ppb)
Dallas 1
Dallas 2
Dallas 3
Dallas 4
Dallas 5
Dallas 6
Acetaminophen
34.28
*
*
*
*
*
*
Atenolol
12.86
*
*
*
*
*
*
Caffeine
25.47
*
*
*
*
*
*
Carbamazepine
1.86
*
*
*
*
*
*
Cimetidine
5.18
*
*
*
*
*
*
Codeine
31.49
*
*
*
*
*
*
Diltiazem
0.26
*
*
*
*
*
*
1,7-Dimethylxanthine
5.84
*
*
*
*
*
*
Diphenhydramine
0.26
0.93
*
0.26
0.80
0.31
0.28
Erythromycin
43.03
*
*
*
*
*
*
Fluoxetine
12.41
12.44
*
*
13.75
*
*
Gemfibrozil
24.82
*
*
*
*
*
*
Ibuprofen
172.81
*
*
*
*
*
*
Lincomycin
56.14
*
*
*
*
*
*
Metoprolol
8.90
*
*
*
*
*
*
Norfluoxetine
15.31
46.26
*
26.90
48.17
27.44
*
Propranolol
3.77
*
*
*
*
*
*
Sertraline
17.29
28.21
*
27.54
23.19
28.07
*
Sulfamethoxazole
13.95
*
*
*
*
*
*
Thiabendazole
7.84
*
*
*
*
*
*
Trimethoprim
8.00
*
*
*
*
*
*
Tylosin
34.67
*
*
*
*
*
*
Warfarin
2.70
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
Table C-3. Analytical Results for Pharmaceuticals in Fish Liver Tissue Samples from Orlando, Florida.
Chemical
MDL
ng/g (ppb)
Orlando 1
Orlando 2
Orlando 3
Orlando 4
Orlando 5
Orlando 6
Acetaminophen
34.28
*
*
*
*
*
*
Atenolol
12.86
*
*
*
*
*
*
Caffeine
25.47
*
*
*
*
*
*
Carbamazepine
1.86
*
*
*
*
*
*
Cimetidine
5.18
*
*
*
*
*
*
Codeine
31.49
*
*
*
*
*
*
Diltiazem
0.26
*
*
*
*
*
*
1,7-Dimethylxanthine
5.84
*
*
*
*
*
*
Diphenhydramine
0.26
*
*
*
*
*
*
Erythromycin
43.03
*
*
*
*
*
*
Fluoxetine
12.41
*
*
*
*
*
*
Gemfibrozil
24.82
*
*
*
*
*
*
Ibuprofen
172.81
*
*
*
*
*
*
Lincomycin
56.14
*
*
*
*
*
*
Metoprolol
8.90
*
*
*
*
*
*
Norfluoxetine
15.31
48.27
44.09
48.99
78.39
62.95
*
Propranolol
3.77
*
*
*
*
*
*
Sertraline
17.29
*
*
*
21.18
*
*
Sulfamethoxazole
13.95
*
*
*
*
*
*
Thiabendazole
7.84
*
*
*
*
*
*
Trimethoprim
8.00
*
*
*
*
*
*
Tylosin
34.67
*
*
*
*
*
*
Warfarin
2.70
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
Table C-4. Analytical Results for Pharmaceuticals in Fish Liver Tissue Samples from Phoenix, Arizona.
Chemical
MDL
ng/g (ppb)
Phoenix 1
Phoenix 2
Phoenix 3
Phoenix 4
Phoenix 5
Phoenix 6
Acetaminophen
34.28
*
*
*
*
*
*
Atenolol
12.86
*
*
*
*
*
*
Caffeine
25.47
*
*
*
*
*
*
Carbamazepine
1.86
*
*
*
*
*
*
Cimetidine
5.18
*
*
*
*
*
*
Codeine
31.49
*
*
*
*
*
*
Diltiazem
0.26
*
*
0.29
0.28
0.28
0.44
1,7-Dimethylxanthine
5.84
*
*
*
*
*
*
Diphenhydramine
0.26
6.32
4.38
5.79
5.48
7.28
11.09
Erythromycin
43.03
*
*
*
*
*
*
Fluoxetine
12.41
*
*
*
*
*
*
Gemfibrozil
24.82
74.43
77.60
60.10
49.14
66.95
90.39
Ibuprofen
172.81
*
*
*
*
*
*
Lincomycin
56.14
*
*
*
*
*
*
Metoprolol
8.90
*
*
*
*
*
*
Norfluoxetine
15.31
*
25.02
41.25
27.96
28.70
43.65
Propranolol
3.77
*
*
*
*
*
*
Sertraline
17.29
57.28
64.80
67.79
62.19
68.86
105.24
Sulfamethoxazole
13.95
*
*
*
*
*
*
Thiabendazole
7.84
*
*
*
*
*
*
Trimethoprim
8.00
*
*
*
*
*
*
Tylosin
34.67
*
*
*
*
*
*
Warfarin
2.70
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
Table C-5. Analytical Results for Pharmaceuticals in Fish Liver Tissue Samples from West Chester, Pennsylvania.
Chemical
MDL
ng/g (ppb)
W. Chester 1
W. Chester 2
W. Chester 3
W. Chester 4
W. Chester 5
W. Chester 6
Acetaminophen
34.28
*
*
*
*
*
*
Atenolol
12.86
*
*
*
*
*
*
Caffeine
25.47
*
*
*
*
*
*
Carbamazepine
1.86
*
*
*
*
*
*
Cimetidine
5.18
*
*
*
*
*
*
Codeine
31.49
*
*
*
*
*
*
Diltiazem
0.26
0.59
0.60
0.71
0.75
0.72
0.76
1,7-Dimethylxanthine
5.84
*
*
*
*
*
*
Diphenhydramine
0.26
7.86
9.40
10.67
11.44
10.73
11.17
Erythromycin
43.03
*
*
*
*
*
*
Fluoxetine
12.41
63.24
66.13
66.28
65.11
79.70
78.26
Gemfibrozil
24.82
*
*
27.34
*
26.88
*
Ibuprofen
172.81
*
*
*
*
*
*
Lincomycin
56.14
*
*
*
*
*
*
Metoprolol
8.90
*
*
*
*
*
*
Norfluoxetine
15.31
37.56
47.73
33.29
38.02
34.40
37.79
Propranolol
3.77
*
*
*
*
*
*
Sertraline
17.29
358.62
431.96
432.17
545.34
326.03
189.66
Sulfamethoxazole
13.95
*
*
*
*
*
*
Thiabendazole
7.84
*
*
*
*
*
*
Trimethoprim
8.00
*
*
*
*
*
*
Tylosin
34.67
*
*
*
*
*
*
Warfarin
2.70
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
Table C-6. Analytical Results for Pharmaceuticals in Fish Liver Tissue Samples from the Gila Wilderness Area, New Mexico.
Chemical
MDL
ng/g (ppb)
Reference 1
Reference 2
Reference 3
Reference 4
Reference 5
Reference 6
Acetaminophen
34.28
*
*
*
*
*
*
Atenolol
12.86
*
*
*
*
*
*
Caffeine
25.47
*
*
*
*
*
*
Carbamazepine
1.86
*
*
*
*
*
*
Cimetidine
5.18
*
*
*
*
*
*
Codeine
31.49
*
*
*
*
*
*
Diltiazem
0.26
*
*
*
*
*
*
1,7-Dimethylxanthine
5.84
*
*
*
*
*
*
Diphenhydramine
0.26
*
*
*
*
*
*
Erythromycin
43.03
*
*
*
*
*
*
Fluoxetine
12.41
*
*
*
*
*
*
Gemfibrozil
24.82
*
*
*
*
*
*
Ibuprofen
172.81
*
*
*
*
*
*
Lincomycin
56.14
*
*
*
*
*
*
Metoprolol
8.90
*
*
*
*
*
*
Norfluoxetine
15.31
*
*
*
*
*
*
Propranolol
3.77
*
*
*
*
*
*
Sertraline
17.29
*
*
*
*
*
*
Sulfamethoxazole
13.95
*
*
*
*
*
*
Thiabendazole
7.84
*
*
*
*
*
*
Trimethoprim
8.00
*
*
*
*
*
*
Tylosin
34.67
*
*
*
*
*
*
Warfarin
2.70
*
*
*
*
*
*
* Less than the Method Detection Limit (
-------
National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix D
Site-specific Analytical Results Tables for
Personal Care Product Chemicals
-------
National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
-------
Table D-1. Analytical Results for Personal Care Products in Fish Fillet Samples from Chicago, Illinois.
Chemical'
MDL
ng/g (ppb)
Chicago 1
Chicago 2
Chicago 3
Chicago 4
Chicago 5
Chicago 6
Celestolide
17.7
*
*
*
*
*
*
Galaxolide
12.2
657.50
624.60
1,211.95
1,582.40
1,806.64
1,760.75
4-Methylbenzylidene
Camphor (4-MBC)
120.5
*
*
*
*
*
*
Musk Ketone
321.2
*
*
*
*
*
*
Musk Xylene
397.1
*
*
*
*
*
*
p-Nonylphenol
9.7
*
*
*
*
*
*
p-Octylphenol
8.2
*
*
*
*
*
*
m-Toluamide
5.1
*
*
*
*
*
*
Tonalide
13.4
80.99
79.09
138.02
176.57
230.21
222.67
Triclosan
37.8
*
*
*
*
*
*
f Benzophenone and octocrylene were identified in blank control samples at concentrations comparable to those reported in the analytical samples. The analytical results
for these two chemicals were considered inconclusive by the laboratory and are excluded from the final results.
* Less than the Method Detection Limit (
-------
Table D-2. Analytical Results for Personal Care Products in Fish Fillet Samples from Dallas, Texas.
Chemical'
MDL
ng/g (ppb)
Dallas 1
Dallas 2
Dallas 3
Dallas 4
Dallas 5
Dallas 6
Celestolide
17.7
*
*
*
*
*
*
Galaxolide
12.2
516.10
606.42
202.91
1,146.77
1,842.27
731.02
4-Methylbenzylidene
Camphor (4-MBC)
120.5
*
*
*
*
*
*
Musk Ketone
321.2
*
*
*
*
*
*
Musk Xylene
397.1
*
*
*
*
*
*
p-Nonylphenol
9.7
*
*
*
*
*
*
p-Octylphenol
8.2
*
*
*
*
*
*
m-Toluamide
5.1
*
*
*
*
*
*
Tonalide
13.4
45.12
54.86
19.99
102.74
149.68
57.67
Triclosan
37.8
*
*
*
*
*
*
f Benzophenone and octocrylene were identified in blank control samples at concentrations comparable to those reported in the analytical samples. The analytical results
for these two chemicals were considered inconclusive by the laboratory and are excluded from the final results.
* Less than the Method Detection Limit (
-------
Table D-3. Analytical Results for Personal Care Products In Fish Fillet Samples from Orlando, Florida.
Chemical'
MDL
ng/g (ppb)
Orlando 1
Orlando 2
Orlando 3
Orlando 4
Orlando 5
Orlando 6
Celestolide
17.7
*
*
*
*
*
*
Galaxolide
12.2
288.53
115.17
62.44
39.66
33.42
*
4-Methylbenzylidene
Camphor (4-MBC)
120.5
*
*
*
*
*
*
Musk Ketone
321.2
*
*
*
*
*
*
Musk Xylene
397.1
*
*
*
*
*
*
p-Nonylphenol
9.7
*
*
*
*
*
*
p-Octylphenol
8.2
*
*
*
*
*
*
m-Toluamide
5.1
*
*
*
*
*
*
Tonalide
13.4
21.44
*
*
*
*
*
Triclosan
37.8
*
*
*
*
*
*
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Benzophenone and octocrylene were identified in blank control samples at concentrations comparable to those reported in the analytical samples. The analytical results
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Less than the Method Detection Limit (
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Table D-4. Analytical Results for Personal Care Products in Fish Fillet Samples from Phoenix, Arizona.
Chemical'
MDL
ng/g (ppb)
Phoenix 1
PHOENIX 2
Phoenix 3
Phoenix 4
Phoenix 5
Phoenix 6
Celestolide
17.7
*
*
*
*
*
*
Galaxolide
12.2
2,038.93
1,960.68
1,961.63
2,099.75
1,414.39
1,049.12
4-Methylbenzylidene
Camphor (4-MBC)
120.5
*
*
*
*
*
*
Musk Ketone
321.2
*
*
*
*
*
*
Musk Xylene
397.1
*
*
*
*
*
*
p-Nonylphenol
9.7
*
*
*
*
*
*
p-Octylphenol
8.2
*
*
*
*
*
*
m-Toluamide
5.1
*
*
*
*
*
*
Tonalide
13.4
259.54
283.56
290.57
272.81
191.69
162.12
Triclosan
37.8
*
*
*
*
*
*
f Benzophenone and octocrylene were identified in blank control samples at concentrations comparable to those reported in the analytical samples. The analytical results
for these two chemicals were considered inconclusive by the laboratory and are excluded from the final results.
* Less than the Method Detection Limit (
-------
Table D-5. Analytical Results for Personal Care Products In Fish Fillet Samples from West Chester, Pennsylvania.
Chemical'
MDL
ng/g (ppb)
W. Chester 1
W. Chester 2
W. Chester 3
W. Chester 4
W. Chester 5
W. Chester 6
Celestolide
17.7
*
*
*
*
*
*
Galaxolide
12.2
2,017.65
1,812.86
2,006.32
1,864.33
1,960.51
1,236.54
4-Methylbenzylidene
Camphor (4-MBC)
120.5
*
*
*
*
*
*
Musk Ketone
321.2
*
*
*
*
*
*
Musk Xylene
397.1
*
*
*
*
*
*
p-Nonylphenol
9.7
*
*
*
*
*
*
p-Octylphenol
8.2
*
*
*
*
*
*
m-Toluamide
5.1
*
*
*
*
*
*
Tonalide
13.4
72.26
53.16
66.93
62.48
40.82
35.57
Triclosan
37.8
*
*
*
*
*
*
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for these two chemicals were considered inconclusive by the laboratory and are excluded from the final results.
Less than the Method Detection Limit (
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Table D-6. Analytical Results for Personal Care Products In Fish Fillet Samples from the Gila Wilderness Area, New Mexico.
Chemical'
MDL
ng/g (ppb)
Reference 1
Reference 2
Reference 3
Reference 4
Reference 5
Reference 6
Celestolide
17.7
*
*
*
*
*
*
Galaxolide
12.2
*
*
*
*
*
*
4-Methylbenzylidene
Camphor (4-MBC)
120.5
*
*
*
*
*
*
Musk Ketone
321.2
*
*
*
*
*
*
Musk Xylene
397.1
*
*
*
*
*
*
p-Nonylphenol
9.7
*
*
*
*
*
*
p-Octylphenol
8.2
*
*
*
*
*
*
m-Toluamide
5.1
*
*
*
*
*
*
Tonalide
13.4
*
*
*
*
*
*
Triclosan
37.8
*
*
*
*
*
*
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Benzophenone and octocrylene were identified in blank control samples at concentrations comparable to those reported in the analytical samples. The analytical results
for these two chemicals were considered inconclusive by the laboratory and are excluded from the final results.
Less than the Method Detection Limit (
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National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
Appendix E
Lipid Content in
Fish Fillet and Liver Tissue Samples
-------
National Pilot Study of Pharmaceuticals and Personal Care Products in Fish Tissue
-------
Table E-1. Analytical Results for Lipid Content in Fish Fillet Samples from Chicago, Illinois.
Chicago 1
Chicago 2
Chicago 3
Chicago 4
Chicago 5
Chicago 6
Replicate 1
Lipid weight (g)
0.0292
0.0440
0.0419
0.0533
0.0647
0.0532
Sample weight (g)
2.1630
2.0040
2.0369
2.0717
2.0213
2.0439
Lipid %
1.3500
2.1956
2.0570
2.5728
3.2009
2.6029
Replicate 2
Lipid weight (g)
0.0273
0.0451
0.0429
0.0513
0.0674
0.0594
Sample weight (g)
2.0724
2.0842
2.0211
2.0075
2.0327
2.2188
Lipid %
1.3173
2.1639
2.1226
2.5554
3.3158
2.6771
Replicate 3
Lipid weight (g)
0.0304
0.0353
0.0425
0.0510
0.0693
0.0539
Sample weight (g)
2.1974
1.5580
2.1166
2.0566
2.1468
2.0682
Lipid %
1.3835
2.2657
2.0079
2.4798
3.2281
2.6061
Summary
Average Lipid %
1.3502
2.2084
2.0625
2.5360
3.2483
2.6287
Standard Deviation
0.0331
0.0521
0.0575
0.0494
0.0600
0.0420
-------
Table E-2. Analytical Results for Lipid Content in Fish Fillet Samples from Dallas, Texas.
Dallas 1
Dallas 2
Dallas 3
Dallas 4
Dallas 5
Dallas 6
Replicate 1
Lipid weight (g)
0.0357
0.0429
0.0181
0.0691
0.0875
0.0325
Sample weight (g)
2.1415
2.0787
2.0795
2.2466
2.1908
2.0099
Lipid %
1.6671
2.0638
0.8704
3.0758
3.9940
1.6170
Replicate 2
Lipid weight (g)
0.0346
0.0418
0.0176
0.0593
0.0867
0.0387
Sample weight (g)
2.1395
2.0062
2.0221
2.0066
2.0905
2.1314
Lipid %
1.6172
2.0835
0.8704
2.9552
4.1473
1.8157
Replicate 3
Lipid weight (g)
0.0334
0.0458
0.0162
0.0598
0.0790
0.0344
Sample weight (g)
2.0600
2.2592
2.0075
2.0463
2.0136
2.0865
Lipid %
1.6214
2.0273
0.8070
2.9223
3.9233
1.6487
Summary
Average Lipid %
1.6352
2.0582
0.8493
2.9845
4.0215
1.6938
Standard Deviation
0.0277
0.0286
0.0366
0.0808
0.1145
0.1068
-------
Table E-3. Analytical Results for Lipid Content in Fish Fillet Samples from Orlando, Florida.
Orlando 1
Orlando 2
Orlando 3
Orlando 4
Orlando 5
Orlando 6
Replicate 1
Lipid weight (g)
0.0488
0.0337
0.0182
0.0100
0.0100
0.0122
Sample weight (g)
2.0660
2.2852
2.1320
2.1662
2.1196
2.0219
Lipid %
2.3621
1.4747
0.8537
0.4616
0.4718
0.6034
Replicate 2
Lipid weight (g)
0.0459
0.0334
0.0178
0.0097
0.0104
0.0111
Sample weight (g)
2.0712
2.1211
2.1038
2.0359
2.0596
2.1061
Lipid %
2.2161
1.5747
0.8461
0.4764
0.5050
0.5270
Replicate 3
Lipid weight (g)
0.0455
0.0325
0.0180
0.0097
0.0098
0.0127
Sample weight (g)
2.0680
2.1734
2.1157
2.0998
2.0300
2.0854
Lipid %
2.2002
1.4954
0.8508
0.4619
0.4828
0.6090
Summary
Average Lipid %
2.2595
1.5149
0.8502
0.4667
0.4865
0.5798
Standard Deviation
0.0892
0.0528
0.0038
0.0085
0.0169
0.0458
-------
Table E-4. Analytical Results for Lipid Content in Fish Fillet Samples from Phoenix, Arizona.
Phoenix 1
Phoenix 2
Phoenix 3
Phoenix 4
Phoenix 5
Phoenix 6
Replicate 1
Lipid weight (g)
0.0721
0.0847
0.0969
0.1095
0.0841
0.0542
Sample weight (g)
2.1314
2.0962
2.1160
2.3087
2.0537
2.0260
Lipid %
3.3828
4.0406
4.5794
4.7429
4.0950
2.6752
Replicate 2
Lipid weight (g)
0.0675
0.0799
0.0919
0.1037
0.0926
0.0591
Sample weight (g)
2.1020
2.0458
2.0174
2.1917
2.2806
2.2439
Lipid %
3.2112
3.9056
4.5554
4.7315
4.0603
2.6338
Replicate 3
Lipid weight (g)
0.0677
0.0870
0.0951
0.0908
0.0859
0.0540
Sample weight (g)
2.0898
2.2060
2.0705
2.0013
2.0072
1.9991
Lipid %
3.2395
3.9438
4.5931
4.5371
4.2796
2.7012
Summary
Average Lipid %
3.2778
3.9633
4.5760
4.6705
4.1450
2.6701
Standard Deviation
0.0920
0.0696
0.0191
0.1157
0.1179
0.0340
-------
Table E-5. Analytical Results for Lipid Content in Fish Fillet Samples from West Chester, Pennsylvania.
W. Chester 1
W. Chester 2
W. Chester 3
W. Chester 4
W. Chester 5
W. Chester 6
Replicate 1
Lipid weight (g)
0.0312
0.0490
0.0442
0.0414
0.0510
0.0274
Sample weight (g)
2.1707
2.2533
2.1216
2.2320
2.2832
2.1425
Lipid %
1.4373
2.1746
2.0833
1.8548
2.2337
1.2789
Replicate 2
Lipid weight (g)
0.0329
0.0433
0.0446
0.0378
0.0467
0.0256
Sample weight (g)
2.1878
2.0429
2.0096
2.0244
2.1188
2.0200
Lipid %
1.5038
2.1195
2.2193
1.8672
2.2041
1.2673
Replicate 3
Lipid weight (g)
0.0326
0.0438
0.0447
0.0394
0.0475
0.0269
Sample weight (g)
2.0320
2.0120
2.0712
2.1265
2.0214
2.1129
Lipid %
1.6043
2.1769
2.1582
1.8528
2.3499
1.2731
Summary
Average Lipid %
1.5151
2.1570
2.1536
1.8583
2.2625
1.2731
Standard Deviation
0.0841
0.0325
0.0681
0.0078
0.0770
0.0058
-------
Table E-6. Analytical Results for Lipid Content in Fish Fillet Samples from the Gila Wilderness Area, New Mexico.
Reference 1
Reference 2
Reference 3
Reference 4
Reference 5
Reference 6
Replicate 1
Lipid weight (g)
0.0749
0.0918
0.1794
0.0852
0.1027
0.1055
Sample weight (g)
2.0917
2.1683
2.1260
2.0070
2.2311
2.0474
Lipid %
3.5808
4.2337
8.4384
4.2451
4.6031
5.1529
Replicate 2
Lipid weight (g)
0.0699
0.0841
0.1748
0.0915
0.0968
0.0995
Sample weight (g)
2.0614
2.1780
2.2158
2.0980
2.0299
2.0297
Lipid %
3.3909
3.8613
7.8888
4.3613
4.7687
4.9022
Replicate 3
Lipid weight (g)
0.0733
0.0838
0.1609
0.0897
0.0919
0.1069
Sample weight (g)
2.1426
2.0323
2.0893
2.0100
2.0239
2.1366
Lipid %
3.4211
4.1234
7.7011
4.4627
4.5407
5.0033
Summary
Average Lipid %
3.4643
4.0728
8.0094
4.3564
4.6375
5.0195
Standard Deviation
0.1021
0.1913
0.3831
0.1089
0.1178
0.1261
-------
Table E-7. Analytical Results for Lipid Content in Fish Liver Samples from Chicago, Illinois.
Chicago 1
Chicago 2
Chicago 3
Chicago 4
Chicago 5
Chicago 6
Replicate 1
Lipid weight (g)
0.0465
0.0327
0.0421
0.0474
0.0588
0.0236
Sample weight (g)
2.0089
1.9991
2.0839
2.0627
2.1197
1.0467
Lipid %
2.3147
1.6357
2.0203
2.2980
2.7740
2.2547
Replicate 2
Lipid weight (g)
0.0214
Sample weight (g)
1.0236
Lipid %
2.0907
Replicate 3
Lipid weight (g)
0.0244
Sample weight (g)
1.0198
Lipid %
2.3926
Summary
Average Lipid %
2.3147
1.6357
2.0203
2.2980
2.7740
2.2460
Standard Deviation
0.1512
-------
Table E-8. Analytical Results for Lipid Content in Fish Liver Samples from Dallas, Texas.
Dallas 1
Dallas 2
Dallas 3
Dallas 4
Dallas 5
Dallas 6
Replicate 1
Lipid weight (g)
0.1181
0.1809
0.1963
0.2067
0.2071
0.0759
Sample weight (g)
2.0128
2.0269
2.0876
2.0189
2.0648
2.0893
Lipid %
5.8674
8.9250
9.4031
10.2382
10.0300
3.6328
Replicate 2
Lipid weight (g)
0.2175
Sample weight (g)
2.0940
Lipid %
10.3868
Replicate 3
Lipid weight (g)
0.2149
Sample weight (g)
2.0677
Lipid %
10.3932
Summary
Average Lipid %
5.8674
8.9250
9.4031
10.2382
10.2700
3.6328
Standard Deviation
0.2079
-------
Table E-9. Analytical Results for Lipid Content in Fish Liver Samples from Orlando, Florida.
Orlando 1
Orlando 2
Orlando 3
Orlando 4
Orlando 5
Orlando 6
Replicate 1
Lipid weight (g)
0.0750
0.1157
0.0612
0.0227
0.0334
0.0497
Sample weight (g)
2.0795
2.0509
2.0524
2.0153
2.0127
2.0190
Lipid %
3.6066
5.6414
2.9819
1.1264
1.6595
2.4616
Replicate 2
Lipid weight (g)
0.0755
Sample weight (g)
2.0125
Lipid %
3.7516
Replicate 3
Lipid weight (g)
0.0720
Sample weight (g)
2.0244
Lipid %
3.5566
Summary
Average Lipid %
3.6383
5.6414
2.9819
1.1264
1.6595
2.4616
Standard Deviation
0.1012
-------
Table E-10. Analytical Results for Lipid Content in Fish Liver Samples from Phoenix, Arizona.
W
Hi
o
Phoenix 1
Phoenix 2
Phoenix 3
Phoenix 4
Phoenix 5
Phoenix 6
Replicate 1
Lipid weight (g)
0.2146
0.3042
0.2140
0.2703
0.1965
0.2069
Sample weight (g)
2.0390
2.0138
2.0276
2.0882
2.0918
2.0071
Lipid %
10.5248
15.1058
10.5543
12.9442
9.3938
10.3084
Replicate 2
Lipid weight (g)
0.2225
Sample weight (g)
2.0336
Lipid %
10.9412
Replicate 3
Lipid weight (g)
0.2189
Sample weight (g)
2.0493
Lipid %
10.6817
Replicate 4
Lipid weight (g)
0.2225
Sample weight (g)
2.0211
Lipid %
11.0089
Replicate 5
Lipid weight (g)
0.2262
Sample weight (g)
2.0109
Lipid %
11.2487
Replicate 6
Lipid weight (g)
0.2114
Sample weight (g)
2.0006
Lipid %
10.5668
Summary
Average Lipid %
10.5248
15.1058
10.5543
12.9442
9.3938
10.7926
Standard Deviation
0.3390
-------
Table E-11. Analytical Results for Lipid Content in Fish Liver Samples from West Chester, Pennsylvania.
W. Chester 1
W. Chester 2
W. Chester 3
W. Chester 4
W. Chester 5
W. Chester 6
Replicate 1
Lipid weight (g)
0.0946
0.1003
0.0665
0.0372
0.0573
0.0753
Sample weight (g)
2.0732
1.9288
1.1284
1.0104
1.0637
2.0077
Lipid %
4.5630
5.2001
5.8933
3.6817
5.3869
3.7506
Replicate 2
Lipid weight (g)
0.0531
Sample weight (g)
1.0054
Lipid %
5.2815
Replicate 3
Lipid weight (g)
0.0652
Sample weight (g)
1.0114
Lipid %
6.4465
Summary
Average Lipid %
4.5630
5.2001
5.8738
3.6817
5.3869
3.7506
Standard Deviation
0.5828
-------
Table E-12. Analytical Results for Lipid Content in Fish Liver Samples from the Gila Wilderness Area, New Mexico.
Reference 1
Reference 2
Reference 3
Reference 4
Reference 5
Reference 6
Replicate 1
Lipid weight (g)
0.0838
0.0928
0.0745
0.0613
0.2048
0.0910
Sample weight (g)
2.0757
2.0269
2.0059
2.0621
2.0853
2.1054
Lipid %
4.0372
4.5784
3.7140
2.9727
9.8211
4.3222
Replicate 2
Lipid weight (g)
0.0654
Sample weight (g)
2.0350
Lipid %
3.2138
Replicate 3
Lipid weight (g)
0.0692
Sample weight (g)
2.0138
Lipid %
3.4363
Summary
Average Lipid %
4.0372
4.5784
3.4547
2.9727
9.8211
4.3222
Standard Deviation
0.2506
------- |