1. Contamination of Virginia Fish by Anti-bacterials and Fragrance Compounds
Catfish on the Catwalk?
E. Mara Jacobs and Dr. Robert C. Hale
Department of Environmental & Aquatic Animal Health, Virginia Institute of Marine Science, College of William & Mary. Gloucester Point, VA 23062
E-mail: e_m_jacobs@yahoo.com
Hypotheses
Results of Sodium Sulfate Spike-Recovery Study
ABSTRACT
Personal care products, such as antibacterial agents and
fragrances, are an increasing concern in the
environment. Some show high toxicity to specific
organisms or bioaccumulate in tissues of wildlife.
Examples include the widely used bactericide triclosan
(TCS), its degradate methyltriclosan (MTCS), as well as
the fragrance compounds galaxolide (HHCB) and tonalide
(AHTN). A method was developed to identify and quantify
these in Virginia freshwater fish. Three matrices of
increasing difficulty (target compound-amended Na2SO4,
amended fish fillet composite reference material and
fillets from fish from multiple species and locations with
field-incurred contaminants) were analyzed. Samples
were dried, isotopically-labeled surrogate standards
added, and then subjected to enhanced solvent
extraction. Gel permeation chromatography (GPC) was
used to remove high molecular weight lipids from these
extracts. Polar interferences were removed by silica gel
liquid chromatography. Gas chromatography in tandem
with electron ionization mass spectrometry (GC/MS) was
used to determine analyte levels in the purified extracts.
Good recoveries of the target analytes were obtained
from the amended Na2SO4 and fish fillet composite
reference materials. Both HHCB and MTCS were detected
in fish samples with field-incurred burdens. However, no
specific trends among fish species, lipid content, or
sample location were observed. This may relate to the
extremely high lipid contents of some of the fish, which
interfered with the GC/MS analysis. Options for further
cleanup are under evaluation.
Objectives
HHCB, AHTN, TCS, MTCS be useful tracers of STP/WWTP releases
1) Develop a method of analysis for HHCB, AHTN, TCS & MTCS in fish
tissue homogenate to minimize analyte loss & optimize analyte
recovery
2) Apply method to various fish species from Virginia rivers
1) Fish sampled from VA will contain PCM & MTCS at detectable levels
2) Fish species with higher lipid contents will exhibit higher burdens of
PCMs & MTCS
Polycyclic Musk Compounds (PCMs)
•Tricyclic Galaxolide (HHCB)
•Bicyclic Tonalide (AHTN)
•Semi-volatile, hydrophobic, lipophilic, bioaccumulative, potentially toxic
Triclosan (TCS)
•Antimicrobial, preservative, disinfectant in soaps, creams, plastics,
textiles, toothpaste
•Inhibits bacterial lipid biosynthesis
- may cause antibiotic resistance
•May affect microbial degradation in STPs/WWTPs
•Identified in wastewater, sludge, sediments, surface waters, & biota
•Least bioaccumulative compound
Methyl-Triclosan (MTCS)
•Hydroxyl-group on triclosan is methylated
•More lipophilic, bioaccumulative than TCS
•Resistant to photodegradation
•No known toxicity studies to date
•Identified in surface waters, sludge, & biota (i.e., fish) primarily/exclusively
near anthropogenic sources
Results of Application to Unknown Fish
Tissues – HHCB, AHTN, & MTCS
•HHCB were determined in some individual fish samples from VA sites
•Previous study results indicated MTCS burdens in a variety of species
•However this method did not determine MTCS in all samples from
the previous study
•MTCS levels in this study all lower than previous study levels
•Both studies determined low naturally-occurring burdens
•High d3-MTCS % recoveries throughout the current study
•Previous study did not employ d3-MTCS as a surrogate
Conclusions
•Some VA freshwater fish from VA rivers contained HHCB & MTCS burdens
•Target compound concentrations did not correlate with extracted fish lipid content
•But high lipid burdens in some extracts may have impacted sample processing &
results
•Possible GC/MS chromatographic problems, i.e., GC column needs to be replaced or ion trap
cleaned frequently
•Instrument performance diminished as the project progressed, resulting in lower
recoveries, etc.
Future Research
•Use deuterated fragrance surrogate standards?
•Not available for use when the study began
•Improve clean-up & storage procedures to minimize analyte
loss & optimize analyte recovery?
•Use acid to saponify lipids in freeze-dried fish tissue,
then purify a second time using silica gel?
Acknowledgments
•Dr. C. J. Abelt, Dr. G. W. Rice
•Ellen Harvey, Matt Mainor, Mark LaGuardia, Elizabeth Bush, Drew Luellen, Gregg Mears,
Samantha Sifleet, Stacie Rice,
Da Chen, Michael Gaylor & extended lab group
•Family & friends
•Committee of Graduate Studies
•College of William & Mary
•Virginia Institute of Marine Science
•The top spectrum is for HHCB present in the fish MegaMatrix
•The bottom is the reference spectrum for a HHCB standard
•A comparison of fragments in the sample & reference spectra are used to confirm analyte identity
•The abundance of the “quantitation ions” (peak area) are used to determine levels.
• For HHCB, the quantitation ions used were 243 m/z & 213 m/z
HHCB
AHTN
d3-TCS
TCS
d3-MTCS
MTCS
p-terphenyl
Chromatograms for Target Compounds & Retention Times
•Elution from the GC column generates chromatographic peaks for each compound
• Retention time (RT) below is labeled above each peak
•RT for d3-TCS very close to RT for TCS & RT for d3-MTCS very close to RT for MTCS
•Demonstrates the analogous behavior of surrogate standards and target analytes
Fate of PCMs, TCS, & MTCS (Target Compounds)
•Sources: household use, commercial production, refuse disposal 
sewage/wastewater treatment plants (STPs/WWTPs)  released in effluent to
receiving waters, e.g. rivers
•Recalcitrant, ubiquitous
•May breakdown in the environment & in exposed organisms to more toxic
metabolites
Overview of Method Validation Procedure
• Use surrogate compounds to develop method for identifying &
quantifying target compounds (HHCB, AHTN, TCS, & MTCS)
• Detection system first necessary to evaluate all steps in the
analytical scheme
• Start at the end of the processing procedure, and work backwards
1. Establish GC/MS conditions
2. Evaluate extract purification – silica gel
3. Evaluate extraction/purification steps using simple, pure
(NaSO4) matrix.
4. Evaluate extraction/purification steps using realistic,
reproducible fish composite matrix (Mega-matrix)
GC Method MS Method
•Ion trap MS
•Electron ionization at 70 eV
•Method Limits of Quantitation/Limits of Detection ~ 1
ng/g tissue (dry weight basis)
•Used “quantitation ions” to identify target compounds
Surrogate Compounds, d3-TCS & d3-MTCS
Internal Standard, p-terphenyl
Compound Quantitation Ions
HHCB 243, 213
AHTN 243, 187
d3-TCS 291, 292, 293
TCS 288, 289, 290
d3-MTCS 305, 306, 307
MTCS 302, 303, 304
p-terphenyl 230
“We used to be
appreciated for
our natural
musk
fragrances….”
“I feel so fresh &
fragrant!
But am I safe to
eat?”
Spike Recovery Study using Fish Tissue “MegaMatrix”
•Homogenized freeze-dried fish tissues from multiple species using a blender
•All fish incorporated had been previously analyzed (using broad spectrum
analytical procedure) for MTCS
• none was detected in tissues selected for “MegaMatrix”
•Species included: Carp, suckers, catfish, quillback
•Lipid content 3.7 % +/- 0.2
•Three MegaMatrix controls were run & a Na2SO4 blank; three MegaMatrix samples
were spiked with 0.1 ug of target compounds; three MegaMatrix samples were
spiked with 1.0 ug of target compounds
HHCB AHTN
TCS
MTCS
•Criteria: Similar in structure to analytes; must not be present
in the environment
• Deuterated analogs of TCS & MTCS ideal
• These also are similar in structure to HHCB & AHTN
•Surrogate Compounds behave like target analytes (TCS,
MTCS, & even HHCB/AHTN) during method development
•Allows “real-time” monitoring of method performance
d3-TCS d3-MTCS
p-terphenyl
•Three controls were run; three samples of Na2SO4 were
spiked with 0.1 ug of target compounds; three samples
were spiked with 0.01 ug of target compounds; & one
blank
Spike Recovery Study using Sodium Sulfate
•Sodium sulfate spiked with broad range of analytes, 0.01 – 1.0 ug
•Testing a wide range of analyte concentrations contributed to the substantial range in
recoveries observed
Results of MegaMatrix Spike-Recovery Study
•Controls contained some background HHCB (24.4 ng/g, dry wt.) & AHTN (13.2 ng/g, dry wt.)
•MegaMatrix spiked with target compounds & surrogate compounds had good recoveries
•Good recoveries of d3-TCS & d3-MTCS demonstrated their usefulness as surrogate compounds
•Lower fragrance recoveries may be due to their high volatilities (i.e. evaporative losses during analysis)
Application of Method to Unknown VA Fish Samples
•Applied method to “unknown VA fish samples”
•Used archived fish samples which had been previously determined to contain MTCS
•Compared results of previous study for MTCS levels to those using this method
Dry Weights Vs. Lipid Weights
•Both dry & lipid-based concentrations were calculated for all compounds
•Dry weights were calculated based on the dry weight, e.g., 10 g MegaMatrix,
analyzed using this method
•Lipid weights were calculated based on the lipid content of
either the MegaMatrix (3.7 +/- 0.2 % lipid) or
on the individual lipid content for each “unknown” VA fish sample
•Lipid content of MegaMatrix was on average lower than that of the
“unknown” VA fish (8.4 +/- 4.3 % lipid)
&
“This fish tissue
STINKS!
It’s all in the
name of Science…”
•Both the sodium sulfate spiked-samples & spiked MegaMatrix were subjected to ASE
•ASE with methylene chloride solvent used to remove target analytes from tissue matrix
•Resulting extracts contain interfering co-extractives that must be removed by GPC and silica
Accelerated Solvent Extraction (ASE)
Packing the ASE vessels with sample (sodium sulfate, mega-matrix or selected fish)
Surrogate standards are also added at this stage Extracting the filled vessels on Dionex ASE
using high temperature & pressure methylene chloride
Lipid Determination (MegaMatrix)
•To determine the lipid content of each fish sample, an aliquot of ASE extract was removed
•Each ASE aliquot was dried under heat lamp to determine the lipid content
•If needed, ASE extracts were “split” to lower extract lipid content
prior to the GPC step to preserve cleanup efficiency
•Different species of fish exhibited variously colored extracts
& lipid contents
Gel Permeation Chromatography (GPC) to Remove Lipids from ASE Extracts
•GPC performed on sodium sulfate, MegaMatrix and individual fish samples
•Waters GPC for removal of lipids & other compounds
that might interfere with final GC/MS analyses
•Collecting GPC extracts for final purification
Silica Gel Purification Step (using Solid Phase Extraction, “SPE” columns)
•Both sodium sulfate spiked samples & spiked MegaMatrix samples were purified using SPE
•GPC extracts on silica SPE columns &
target & surrogate compounds eluted
Gas-Chromatography (GC)/Mass Spectrometry (MS) To Identify & Quantify Target Compounds
•SPE extracts ready for GC/MS
Results
Mass Spectra
•GC column used was a DB-5 (60 m, 0.32 mm i.d., 0.25 um film thickness)
•Column length increases resolution, film thickness appropriate for semi-
volatiles
•Injector splitless, temperature 325 oC
•Column temperature started at 75 oC,
•increased at a rate of 4 oC/min to 300 oC
•Total run time 57.25 minutes
•Compounds volatilize at different temperatures
• results in different
GC retention times (separation) & enter MS for detection
49.3
38.2
119.7
82.7
115.3
63.9
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
160.0
HHCB AHTN d3-TCS TCS d3-MTCSMTCS
%Recoveries
Compound
% Recoveries, MegaMatrix Spike
Study
GC Diagram
41.1
54.2
75.8
161.5
73.5
89.2
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
160.0
180.0
200.0
HHCB AHTN d3-TCS TCS d3-MTCSMTCS
%Recoveries
Compound
%Recoveries, Sodium Sulfate
Spike Study
•Added to final extract to quantitate target analytes & surrogates
•Similar in structure to analytes
•Elutes at retention time close to analyte retention times
Internal
std
Target
analyte
GC chromatogram
Silica gel purification scheme
S1 fractions eluted with 3.5 ml
hexane; sent to waste
S2 fractions eluted with 10 ml of
10/40/50
hexane/dichloromethane/acetone