United States Office at Water
Environmental Protection Agency Washington, D.C. 20460
503390002
September 1985
BIOACCUMULATION
MONITORING GUIDANCE:
3. RECOMMENDED ANALYTICAL
DETECTION LIMITS
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BIOACCUMULATION
MONITORING GUIDANCE:
RECOMMENDED ANALYTICAL
DETECTION LIMITS
Prepared by:
Tetra Tech, inc.
11820 Northup Way, Suite 100
Bellevue, Washington 98005
-sj-
ou
. ,', if ;KL PROTECTION AGENCY
-' .MTGN. D.C. 20450
Prepared for:
Marine Operations Division: 301 (h) Program
Office of Marine and Estuarine Protection
U.S. Environmental Protection Agency
401 M Street SW
Washington, D.C. 20460
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EPA Contract No. 68-01-6938
TC3953-03
Final Report
BIOACCUMULATION MONITORING GUIDANCE:
3. RECOMMENDED ANALYTICAL DETECTION LIMITS
for
U.S. Environmental Protection Agency
Office of Marine and Estuarine Protection
Washington, DC 20460
September, 1985
by
Tetra Tech, Inc.
11820 Northup Way, Suite 100
Bellevue, Washington 98005
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PREFACE
This report is one element of the Bioaccumulation Monitoring Guidance
Series. The purpose of this series is to provide guidance for monitoring
of priority pollutant residues in tissues of resident marine organisms.
These guidance documents were prepared for the 301(h) sewage discharge
permit program under the U.S. EPA Office of Marine and Estuarine Protection,
Marine Operations Division. Two kinds of monitoring guidance are provided
in this series: recommendations for sampling and analysis designs, and
aids for interpretation of monitoring data.
Some basic assumptions were made in developing the guidance presented
in these documents: 1) each bioaccumulation monitoring program will be
designed to meet the requirements of the 301(h) regulations, 2) tissue
samples will be collected from appropriate locations near the sewage discharge
and from an unpolluted reference site, 3) the initial chemicals of concern
are the U.S. EPA priority pollutants and 301(h) pesticides, and 4) the
monitoring data should be suitable for a meaningful evaluation of the potential
hazards to living marine resources as well as human health. It should
be recognized that the design of a monitoring program reflects the site-
specific characteristics of the pollutant discharge and the receiving environ-
ment. Thus, site-specific considerations may lead to a modification of
the generic recommendations herein. Finally, although these guidance documents
were prepared specifically for monitoring of sewage discharges under the
301(h) program, their potential use extends to assessment and monitoring
of bioaccumulation resulting from other kinds of pollutant discharges into
marine and estuarine environments.
ii
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CONTENTS
PREFACE i i
LIST OF TABLES iv
ACKNOWLEDGMENTS v
RECOMMENDED ANALYTICAL DETECTION LIMITS 1
TRACE METALS 7
ORGANIC COMPOUNDS 12
SUMMARY OF RECOMMENDATIONS FOR DETECTION LIMITS 20
REFERENCES 22
iii
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TABLES
Number Page
1 Organic priority pollutants and 301(h) pesticides 5
2 Recommended trace metal detection limits for tissue samples 8
3 Minimum and maximum trace metal detection limits 11
reported for tissue samples
4 Minimum and maximum trace organic compound detection 14
limits reported for tissue samples
5 Recommended organic priority pollutant detection limits 16
for tissue samples
1v
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ACKNOWLEDGMENTS
This document has been reviewed by the 301(h) Task Force of the
Environmental Protection Agency, which includes representatives from the
Water Management Divisions of U.S. EPA Regions I, II, III, IV, IX, and X;
the Office of Research and Development - Environmental Research Laboratory -
Narragansett {located in Harragansett, RI and Newport, OR), and the Marine
Operations Division in the Office of Marine and Estuarine Protection, Office
of Water.
This technical guidance document was produced for the U.S. Environ-
mental Protection Agency under the 301(h) post-decision technical support
contract No. 68-01-6938, Allison J. Ouryee, Project Officer. This report
was prepared by Tetra Tech, Inc., under the direction of Dr. Thomas C. Ginn.
The primary authors were Ms. Ann C. Bailey, Mr. Robert C. Barrick, and
Mr. Harry R. Seller. Ms. Marcy B. Brooks-McAuliffe performed technical
editing and supervised report production.
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RECOMMENDED ANALYTICAL DETECTION LIMITS
The accumulation of toxic substances in marine organisms that may
lead to adverse biological effects or affect commercial or recreational
fisheries is one of the major concerns in the 301(h) program related to
evaluating the effects of sewage discharges into marine and estuarine waters.
Evaluation of differences between body burdens in organisms from relatively
uncontaminated reference areas and those from contaminated estuarine and
marine environments potentially impacted by the discharge is an important
part of bioaccumulation studies. Such comparisons will generally require
data that are reliable at low part per billion concentrations. Therefore,
low but practically attainable detection limits are a minimum requirement
to ensure the usefulness of bioaccumulation monitoring data. This report
reviews the factors that influence target pollutant detection limits and
recommends minimum detection limits for bioaccumulation studies. Although
this report is not designed to address specific analytical protocols, it
serves as a companion document to the recommended analytical protocols
in ths Bioaccumulation Monitoring Guidance series.
Achieving low detection limits for all priority pollutants during
bioaccumulation studies is difficult because a wide variety of techniques
is required to achieve optimal detection of these numerous and chemically
diverse compounds. The limited amount of tissue available for most samples
and the need to detect and identify nanogram or picogram quantities of
pollutants necessitates the use of sensitive instrumentation and complex
analytical procedures.
Environmental analytical chemists have not universally agreed upon
a convention for determining and reporting the lower detection limits of
analytical procedures. Furthermore, the basis for detection limits reported
in the literature is rarely given. Values reported as lower detection
limits are commonly based on instrumental sensitivity, levels of blank
contamination, and/or matrix interferences and have various levels of
1
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statistical significance. The American Chemical Society's Committee on
Environmental Improvement (CEI) defined the following types of detection
limits in an effort to standardize the reporting procedures of environmental
laboratories (Keith et al. 1983):
• Instrument Detection Limit (IDL) -- the smallest signal
above background noise that an instrument can detect reliably.
• Limit of Detection (LOD) -- the lowest concentration level
that can be determined to be statistically different from
the blank. The recommended value for LOD is 30, where o
is the standard deviation of the blank in replicate analyses.
• Limit of Quantitation (LOO) -- the level above which quantitative
results may be obtained with a specified degree of confidence.
The recommended value for LOQ is lOo, where a is the standard
deviation of blanks in replicate analyses.
• Method Detection Limit (MDL) -- the minimum concentration
of a substance that can be identified, measured, and reported
with 99 percent confidence that the analyte concentration
is greater than zero. The MDL is determined from seven
replicate analyses of a sample of a given matrix containing
the analyte (Glaser et al. 1981).
The CEI recommended that results below 3o should be reported as "not detected"
(NO) and that the detection limit (or LOD) be given in parentheses. In
addition, if the results are near the detection limit (3 to lOo, which
is the "region of less-certain quantitation"), the results should be reported
as detections with the limit of detection given in parentheses.
The CEI definitions are useful for establishing a conceptual framework
for detection limits, but are somewhat limited in a practical sense. The
IDL does not address possible blank contaminants or matrix interferences
and is not a good standard for complex environmental matrices, such as
tissues. The LOD and LOQ account for blank contamination, but not for
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matrix complexity and interferences. The high lOo level specified for
LOQ helps to preclude false positive findings, but may also necessitate
the rejection of valid data. The MDL is the only operationally defined
detection limit and provides a high statistical confidence level but, like
the LOQ, may be too stringent and necessitate the rejection of valid data.
The detection limits recommended in this report are not strictly based
on the CE! definitions. Instead, they are considered to be typically attainable
values based on the best professional judgment and experience of analytical
chemists who considered the instrumental sensitivity of affordable equipment,
common problems with blank contamination and matrix interferences, and
reasonable levels of laboratory analytical effort. The recommended values
are not absolute, as analytical procedures and laboratory precision can
affect attainable detection levels. The detection limits recommended herein
fall between the IDL and MDL as defined by the CEI.
Several factors determine achievable detection limits for a specific
priority pollutant, regardless of analytical procedure. The most important
factors include
• Physical sample size available - In most cases, the more
tissue available for analysis, the better the detection
levels that can be achieved. Thus, for a given method,
larger samples available for analysis will have lower detection
limits than smaller samples.
• Presence of interfering substances - For example, because
liver contains more salts than muscle, liver digestates
may require matrix matching for trace metal analyses, while
muscle digestates may not. Matrix matching may increase
the detection limit.
• Range of pollutants to be analyzed - For example, if only
one compound is of interest, a method can be optimized for
that parameter without regard to potential effects on other
parameters.
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• Level of confirmation of results - For example, gas chroma-
tography (GC) with electron capture detection (GC/ECD) is
more sensitive than GC with mass spectrometry (GC/?4S) for
pesticide analysis. However, a single GC/ECO analysis does
not provide positive identification of a compound, whereas
GC/MS provides more information for molecular confirmation.
• Level of pollutant found in the field and in analytical
blanks - For example, due to bottle preparation procedures,
analytical blanks are often contaminated with varying concen-
trations of methylene chloride. This variation in contaminant
level often precludes sensitive detection levels in tissue.
This review summarizes the detection levels generally achieved using
methods commonly employed for tissue analysis in environmental laboratories.
Because many of these levels are dependent on state-of-the-art technology,
the detection levels can be expected to decrease as methods and instruments
improve and become more commonly available.
For analytical purposes, the priority pollutant list of 126 chemicals
can be divided into five categories: trace metals (13 parameters); volatile
organic compounds (28 parameters); acid-extractable organic compounds (11
parameters); basic- and neutral-extractable organic compounds (47 parameters);
and organochlorine pesticides (25 parameters). The organic pollutants
included in each category are listed in Table 1. The remaining two priority
pollutants, asbestos and cyanide, will not be discussed because significant
bioaccumulation of these substances is not expected. Six additional pesticides
are required for the 301(h) program (Table 1).
Procedures for chemical analysis of each analytical group consist
of four sequential steps:
• Collection of organisms and preservation of tissue
• Physical preparation of tissue for analysis
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TABLE 1. ORGANIC PRIORITY POLLUTANTS AND 301(h) PESTICIDES
Acid Compounds
Base/Neutral Compounds
2,4,6-trichlorophenol
p-chloro-m-cresol
2-chlorophenol
2,4-dichlorophenol
2,4-dimethyl phenol
2-nitrophenol
4-nitropheno1
2,4-dinitrophenol
4,6-dinitro-2-methyl phenol
pentachlorophenol
phenol
Volatiles
acrolein
acrylonitrile
benzene
carbon tetrachloride
chlorobenzene
1,2-dichloroethane
1,1,1-trichloroethane
1,1-dichloroethane
1,1,2-trichloroethane
1,1,2,2-tetrachloroethane
chloroethane
2-chlorethylvinyl ether
chloroform
l.l'-dichloroethene
trans-1,2-dichloroethene
1,2-dichloropropane
cis- and trans-l,3-dichloropropene
ethylbenzene
methylene chloride
chloromethane
bromomethane
bromoform
bromod ichloromethane
c h1orod ibromomethane
tetrachloroethene
toluene
trichloroethene
vinyl chloride
acenaphthene
benzidine
1,2,4-trichlorobenzene
hexachlorobenzene
hexachloroethane
bis(2-chloroethyl)ether
2-chloronaphthalene
1,2-diehiorobenzene
1,3-dichlorobenzene
1,4-dichlorobenzene
3,3'-dichlorobenzidine
2,4-dinitrotoluene
2,6-dinitrotoluene
1,2-diphenylhydrazine
fluoranthene
4-chlorophenyl phenyl ether
4-bromophenyl phenyl ether
bis(2-chloroisopropyl)ether
bis(2-chloroethoxy)methane
hexachlorobutadiene
hexac hiorocyc1opentad i ene
isophorone
naphthalene
nitrobenzene
N-nitrosodiphenylamine
N-nitrosodimethylamine
N-nitrosodi-n-propylamine
bis(2-ethylhexyl)phtha1ate
benzyl butyl phthalate
di-n-butyl phthalate
di-n-octyl phthalate
diethyl phthalate
dimethyl phthalate
benzo(a)anthracene
benzo(a)pyrene
benzo(b)fluoranthene
benzo(k)fluoranthene
chrysene
acenaphthylene
anthracene
benzo(ghi)perylene
fluorene
phenanthrene
dibenzo(a,h)anthracene
indeno(l,2,3-cd)pyrene
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Fable 1. (Continued)
Base/Neutral Compounds (Continued) 301(h) Pesticides
pyrene Malathion
2,3,7,8-tetrachlorod ibenzo-p-dioxin Parathi on
Guthion
Pesticides Demeton
Mi rex
aldrin Methoxychlor
dieldrin
a- + ^-chlordane
4,4'-DDT
4,4'-DDE
4,4'-DDD
o-endosulfan
B-endosulfan
endosulfan sulfate
endrin
endrin aldehyde
heptachlor
heptachlor epoxide
o-HCH (hexachlorocyclohexane)
B-HCH
6-HCH
^f-HCH (lindane)
PCB-1242 (mixture)
PCB-1254 (mixture)
PCB-1221 (mixture)
PCB-1232 (mixture)
PCB-1248 (mixture)
PCB-1260 (mixture)
PCB-1016 (mixture)
toxaphene (mixture)
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• Chemical preparation of tissue for analysis
• Measurement of pollutant concentrations in the prepared
samples.
Detailed recommendations for the above procedures are beyond the scope
of this report and will be available in other reports of the Bioaccumulation
Monitoring Guidance series. In general, it is noteworthy that collection
of representative organisms is especially critical and that the samples
must be protected against contamination and degradation. Sample volume
and storage procedures are best determined after assessing specific compounds
to be measured and detection levels to be obtained, as described in the
monitoring guidance documents.
TRACE METALS
The detection of trace metals can be performed with several types
of instrumentation (e.g., neutron activation analysis, x-ray emission
spectrometry, and fluorescence spectrophotometry). However, the most widely
used types of instrumentation are
• Atomic absorption spectrophotometry (AAS)
flame
graphite furnace
cold vapor
gaseous hydride
• Inductively coupled plasma emission spectrometry (ICP).
A combination of these instrumental techniques is typically used, since
no single technique is best for all elements.
Approximate detection limits attainable with a sample size of 5 g
(wet weight) diluted to 50 ml are presented in Table 2. Sample size can
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TABLE 2. RECOMMENDED TRACE METAL
DETECTION LIMITS FOR TISSUE SAMPLES9
Element
Antimony (Sb)
Arsenic (As)
Beryllium (Be)
Cadmium (Cd)
Chromium (Cr)
Copper (Cu)
Lead (Pb)
Mercury (Hg)
Nickel (Ni)
Selenium (Se)
Silver (Ag)
Thallium (Tl )
Zinc (Zn)
Graphite
Furnace
0.02
0.02
0.003
0.01
0.02
0.01
0.03
0.02
0.02
0.01
0.02
0.2C
Detection Limitb
(ug/g wet weight)
Atomic Absorption
Gaseous
Flame Hydride
0.002
0.01
0.1
0.1
0.2
0.1
1.0
• ill ^LUIU vapor j- — — — -- — -
0.5
0.01
0.1
1.0
0.1
ICP
10
3
0.03
0.4
0.7
0.6
4
1
—
0.7
4
0.2
3 Values in boldface type are detection limits recommended for metals in
tissue samples. The most sensitive analyses for antimony, arsenic, and
selenium are attained by gaseous hydride, but this instrumentation is not
as widely available as graphite furance. When available, the use of gaseous
hydride for these elements is recommended.
b Detection limits are based on 5 g (wet weight) of muscle tissue, digested,
and diluted to 50 mL for the analysis of all elements.
c A lower detection limit of 0.02 ug/g for zinc is possible by graphite
furance, but is not required because zinc is always detected at higher
concentrations in tissues.
8
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be varied, but a minimum of 25 ml of digestate is needed for multi-element
flame AAS analysis. Sufficient dilution volumes are necessary not only
to ensure complete dissolution of the tissue but also to ensure that "dissolved
salts" have been diluted to a maximum of 2 percent of the digestate (wt/vol)
(U.S. Food and Drug Administration 1979). Thus, a maximum of 10 g of tissue
(containing 10 percent ash) could be dissolved and diluted to 50 ml for
analysis. To avoid possible matrix interferences, half of the maximum
weight (i.e., 5 g) is recommended for dissolution.
For analysis by AAS or ICP methods, tissue samples must be in solution.
A wide range of wet- (acid digestion) or dry- (ashing) oxidation methods
(U.S. EPA 1977) is available to decompose and solubilize tissue samples
(Plumb 1984). Nitric acid in combination with perchloric acid is the most
effective wet-oxidation mixture for tissue dissolution. However, hydrogen
peroxide is often used instead of perchloric acid, due to the extraordinary
care required to avoid explosions when working with perchloric acid. Although
wet-oxidation methods are less prone to loss of analytes by volatilization,
they also use more reagents and are thus more likely to result in sample
contamination than dry-ashing methods. Low-temperature or programmed-tempera-
ture ashing furnaces have been used to minimize loss of analytes during
dry-ashing. Because dry-ashing is not appropriate for all elements, elemental
recovery after dry-ashing should be monitored.
The specific analytical technique to use on digested tissue samples
depends upon the required level of sensitivity. Flame AAS is generally
the least sensitive method, but it may be adequate to analyze certain elements
(e.g., zinc) at ambient levels found in tissue samples. Graphite furnace
AAS is more sensitive than flame AAS, but is subject to more matrix and
spectral interferences. Because of its high sensitivity, graphite furnace
AAS requires particular caution with regard to laboratory contamination.
For some trace elements (e.g., cadmium, lead, silver), graphite furnace
AAS is the best analytical method because other procedures are not sensitive
enough to detect the typically low ambient tissue concentrations. In both
AAS methods, the concentration of each element is determined by a separate
analysis, making the analysis of the entire scan of priority pollutant
metals labor-intensive and relatively expensive compared to ICP. By using
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ICP for trace element analyses, several elements can be measured simul-
taneously. However, detection limits achieved with ICP are higher than
those achieved with graphite furnace AAS. Thus, ICP detection is not
recommended for any of the trace metals with the possible exception of
zinc.
Recommended detection limits for trace metals are listed in Table 2.
These detection limits are based on 5 g (wet weight) of fish tissue, digestion
with minimal elemental loss and contamination, and analysis with minimal
interference. The detection limit that may be attained for a sample depends
on the type of tissue, the digestion technique, and the choice of instrumen-
tation.
In most cases, the lowest detection limit listed in Table 2 for each
element is recommended. The most sensitive instrumental techniques listed
for beryllium, cadmium, chromium, copper, lead, nickel, silver, and thallium
is graphite furnace AAS. Graphite furnace detection of antimony is appropriate
and recommended if gaseous hydride instrumentation is unavailable. Arsenic
and selenium can be analyzed with roughly equivalent sensitivity by graphite
furnace AAS or gaseous hydride AAS. Because graphite furnace is a widely
available technique, it is recommended for analysis of arsenic and selenium.
Environmental concentrations of zinc are typically high enough for detection
by either graphite furnace AAS, flame AAS, or ICP. For mercury, cold vapor
AAS analysis is the only recommended technique.
For mercury analyses, sample dissolution with sulfuric acid and potassium
permanganate is often performed on a separate sample aliquot (Plumb 1984).
However, a separate dissolution for mercury is not necessary if precautions
are taken to prevent analyte volatilization. For the remaining elements,
wet-acid digestion using nitric acid in combination with either perchloric
acid or hydrogen peroxide is recommended. Dry-ashing is not recommended
because analytes of concern may be lost by volatilization.
For purposes of comparison with recommended detection limits (Table 2),
minimum and maximum detection limits reported in past studies of trace
metals concentrations in tissues of marine organisms are listed in Table 3.
10
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TABLE 3. MINIMUM AND MAXIMUM TRACE METAL
DETECTION LIMITS REPORTED FOR TISSUE SAMPLES
Element
Antimony
Arsenic
Beryllium
Cadmium
Chromium
Copper
Lead
Mercury
Nickel
Selenium
Silver
Thai Hum
Zinc
Detection Limit
(ug/g wet weight)
Minimum Maximum
0.01* 1.0a
Always detected^
(minimum = 0.72)
0.0033 0.253
O.OOlb 0.75b
0.005& 1.29b
Always detected*5
(minimum = 0.052)
0.030b 1.6°
0.0004b o.09b
0.019b i.o&
Always detectedb
(minimum = 0.29)
O.OOlb 0.27b
O.Oia o.5a
Always detected^1
(minimum = 1.42)
a Detection limits are based on a summary of Gahler et
al. (1982), Martin et al. (1984), and Tetra Tech{l985b).
b Detection limit ranges are summarized from Tetra Tech
(1985a, Appendix D).
11
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The detection limits in Table 3 were compiled from data in another report of
the Bioaccumulation Monitoring Guidance series (Tetra Tech 1985a, Appendix D).
The recommended detection limits tend toward the lower range of reported
detection limits.
ORGANIC COMPOUNDS
Although nationally standardized analytical protocols have been established
for organic priority pollutants in water and wastewater, no such standardized
protocols have yet been developed for tissues. Therefore, various laboratories
use different analytical procedures, which can vary significantly in their
sensitivity and minimum attainable detection limits.
Analysis of volatile organic pollutants in water is usually performed
by a vapor-stripping technique, commonly referred to as the purge and trap
technique (U.S. EPA Method 624), with subsequent GC/MS detection and quanti-
fication (U.S. EPA 1979). However, variations of this technique used for
tissue samples often produce low spike recoveries and high detection limits.
A more successful adaptation of U.S. EPA Method 624 involves a device that
vaporizes volatile organic compounds from the tissue sample under vacuum
and then condenses the volatiles in a super-cooled trap (Hiatt 1981).
The trap is then transferred to a purge and trap device, where the concentrate
is diluted to 5 ml and treated as a water sample. Using this technique,
the average recovery of volatile compounds from tissue samples spiked with
25 ng/g was found to be 74 percent (Hiatt 1981).
Analysis of semi-volatile organic compounds involves a solvent extraction
of the sample, cleanup of the characteristically complex extract, and GC
analysis and quantification. Extraction for acidic, basic, and neutral
organic pollutants in tissue often involves an initial extraction with
methylene chloride and/or methanol (Plumb 1984; Boehm 1984; MacLeod et
al. 1984). This results in an extract containing a wide range of chemicals,
including many substances that are not of concern (e.g., fats and glycerides).
For the most sensitive analysis, extracts must be cleaned up by removing
the interfering compounds. Ideally, chemically distinct fractions (i.e.,
acids, bases, and neutrals) should be separated before detection and quantifi-
12
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cation, although this is often prohibitively expensive. Efficient extract
cleanup and careful handling to minimize contamination throughout the procedure
result in ootimum detection limits. For a given kind of tissue and sample
size, variation in cleanup and extraction procedures, which differ widely
among laboratories, produces a broad range of detection levels. For example,
tissue extractions can be performed either by grinding the sample with
the solvent, refluxing the solvent through the tissue, or digesting the
tissue in a basic solution prior to solvent extraction. A comparative
study of the relative efficiency of these extraction techniques was not
reported in the literature reviewed for this report. Cleanup of the extract
can be achieved by liquid-liquid partitioning, gel permeation chromatography,
and/or normal phase liquid chromatography. The chosen methods must be
easily reproduced and must allow for a high recovery for compounds of interest.
The minimum and maximum organic compound detection limits reported
in past studies of organic compound concentrations in tissues of marine
organisms are listed in Table 4. This information was summarized from
data in another report of the Bioaccumulation Monitoring Guidance series
{Tetra Tech 1985a, Appendix D). For some chemical groups with limited
historical data for target species, detection limit ranges were determined
from a review of selected references (i.e., Gahler et al. 1982; Martin
et al. 1984; Tetra Tech 1985b). The chemical groups in Table 4 are arranged
such that compounds with similar chemistry and similar detection limits
are grouped together. The range of detection limits within each group
in Table 4 is large, indicating a wide variability among laboratories and
techniques.
The selection of organic compound minimum analytical detection limits
for 3Ql(h) bioaccumulation monitoring should be guided by tissue contaminant
levels in reference areas. This guideline will not be practical for very
clean reference areas that have undetectable contamination in the low part
per billion range. From data on the median concentrations of compounds
reported in the reference areas (Tetra Tech 1985a, Tables 3-22 and Appendix D),
concentrations for most compounds are in the low part per billion range.
Thus, optimal detection limits should be near the low end of the range
of detection limits summarized in Table 4. Another factor to consider
13
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TABLE 4. MINIMUM AND MAXIMUM TRACE ORGANIC COMPOUND
DETECTION LIMITS REPORTED FOR TISSUE SAMPLES
Priority Pollutant
Group
Detection Limit
(ug/kg wet weight)
Minimum
Maximum
Phenols
Organonitrogen compounds
Aromatic hydrocarbons (low
and high molecular weight)
Chlorinated hydrocarbons
Halogenated ethers
Phthalates
PCBs
Pesticides
Volatile compounds (halogenated
alkanes and alkenes; aromatics,
carbonyl compounds; ethers)
0.69a
1.72a
O.OSb
0.015D
0.863
0.40b
0.015b
5003
l,320b
2003
SO3
95b
200a
a Detection limits are based on a summary of Gahler et al.
(1982), Martin et al. (1984), and Tetra Tech(1985b). Detec-
tion limits summarized for Martin et al. (1984) were recom-
mended by the authors, and are not necessarily attainable
by available methods.
b Detection limit ranges are summarized from (Tetra Tech
1985at Appendix D).
14
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when selecting optimal organic poTktant detection limits for 301{h) monitoring
is that tissues need to be analyzed for many pollutants having different
chemical characteristics. Dedicated analyses developed specifically for
one group of compounds (e.g., aromatic hydrocarbons) would not be applicable
to the analysis of all compounds of concern. Some of the minimum detection
limits in Table 4 are from dedicated analyses for selected compound classes
and may not be achieved by full-scan analysis. Selection of appropriate
methods must therefore be based on a trade-off between full-scan analyses,
which are economical and feasible for a large group of users but cannot
provide optimal sensitivity for some compounds, and alternate methods that
are more sensitive for specific compound groups but can result in higher
analytical costs and large sample size requirements if multiple extractions
are required. This trade-off has been considered in the review of available
methods and associated detection limits for analyses of trace organic compounds
in tissues.
Based on a review of current extraction and detection methods for
a broad range of organic priority pollutants in tissues, detection limits
listed in Table 5 are recommended for 301(h) bioaccumulation monitoring.
Compounds that could have substantially different detection limits within
a compound class, or are difficult to recover, are footnoted in the table.
Except for volatile organic compound analyses, which are based on a separate
sample of 5 g {wet weight), the limits in Table 5 are based on the extraction
of 25 g (wet weight) of tissue. This quantity of tissue was chosen for
the detection-limit recommendations, since 25 g of tissue can be obtained
easily [reported initial wet-sample weights for tissue analyses ranged
from 3 g (MacLeod 1984) to 100 g (Boehm 1984)] and extracted efficiently.
In addition, a 25-g sample provides adequate tissue for appropriate detection
levels.
As previously discussed, extraction procedures can vary, but must
efficiently recover the broad range of compounds of interest (i.e., acids,
bases, and neutrals). Compound recovery should be carefully evaluated
for all proposed extraction procedures. A specific analytical procedure,
including sample extraction and extract cleanup, is not recommended in
this report but will be presented in another report of the Bioaccumulation
15
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TABLE 5. RECOMMENDED ORGANIC PRIORITY POLLUTANT
DETECTION LIMITS FOR TISSUE SAMPLES^
Gas Chromatography Detection Limits*3
(ug/kg-wet weight)
Priority Pollutant
Group
Mass
Spectrometry
Electron
Capture
Detections
Phenols, substituted phenols
Organonitrogen compounds
Aromatic hydrocarbons {low and
high molecular weight)
Chlorinated hydrocarbons
Halogenated ethers
Phthalates
PCBs
Pesticides
Volatile compounds (halogenated
alkanes and alkenes; aromatics,
carbonyl compounds; ethers)
10
10-20^
10-20
10
e
50
5-10J
e
0.1-19
e
0.1-5i
e
1-59
20
0.1-51
a Values in boldface type are detection limits recommended for organic
compounds in tissue samples.
b Except for the volatile compounds, detection limits are based on a 25-g
(wet weight) tissue sample extracted, concentrated to 0.5 mL after gel
permeation Chromatography cleanup, and 1-uL injected. For volatile compounds
a separate 5 g (wet weight) of tissue would be used for analysis. Bonded,
fused silica capillary GC columns, which provide better resolution than
packed columns, are assumed for analyses of semi-volatile compounds.
c Extract cleanup (e.g., removal of polar interferences by alumina column
Chromatography) is assumed.
d Substantially increased detection limits are observed for:
4-nitrophenol 100
2,4-nitrophenol 100
pentachlorophenol 80
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TABLE 5. (Continued)
e No detection limits provided since methodology does not allow adequate
recovery and/or detection.
f Benzidine and 3,3'-dichlorobenzidine may be unreported because of analytical
recovery problems.
9 Use of electron capture detection for these compounds would require dedicated
analytical protocols.
h Substantially increased detection limits are observed for:
hexachloroethane 40
hexachlorobutadiene 40
hexachlorocyclopentadiene (typically not reported because of its lability
in heated injection ports)
1 The higher range of detection limits are appropriate for pesticides such
as mirex, methoxychlor, the ODTs, and endosulfans, and for chlorinated
butadienes. Compounds such as lindane, aldrin, heptachlor, and hexachloro-
benzene can be detected at the lower limit. Toxaphene (a mixture) may
require a higher detection limit than the other organochlorine pesticides,
20 ppb.
The nonchlorinated, organophosphorous 301{h) pesticides (Malathion,
Parathion, Guthion, and Demeton) should not be analyzed with the same procedures
as the organochlorine pesticides. They require dedicated protocols (e.g.,
one- or two-step extract cleanup and GC/phosphorous specific flame photometric
or alkali flame ionization detection) for appropriate detection limits
of approximately 1-15 ppb,
J Substantially increased detection limits are observed for:
acrolein 100
acrylonitrile 100
2-chloroethylvinyl ether 100
methylene chloride 100
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Monitoring Guidance series. At a minimum, one- or two-step cleanup should
be performed following extraction to obtain adequate compound resolution.
The detection limits recommended in Table 5 are based on extract cleanup
by gel permeation chromatography and by alumina column chromatography for
ECD analyses (e.g., U.S. EPA 1984). After cleanup, the sample extract
can be concentrated to volumes usually ranging from 0.1 to 3.0 ml. The
recommended detection limits assume a final extract volume of 0.5 ml and
a minimum instrument injection volume of 1 uL.
Recommended detection limits (Table 5) are listed for either mass
spectrometry or electron capture detection. Because of the greater sensitivity
of GC/ECD relative to GC/MS for chlorinated compounds, PCBs and chlorinated
pesticides should be quantified with GC/ECD. However, analysis by GC/ECD
does not provide positive compound identification. Problems with false
readings due to interferences have been commonly reported. Thus, confirmation
of PCBs and pesticides on an alternative GC column phase (on GC/ECD}, or
preferably by GC/MS if analyte concentrations are sufficiently high, is
essential for reliable results. All other organic compound groups are
recommended for analysis by GC/MS.
A review of observed concentrations of organic compounds in marine
organisms from reference areas (Tetra Tech 1985a, Tables 3-22) indicates
that the recommended detection limits for organic compounds (Table 5) may
result in a number of "undetected" values. These levels are nonetheless
useful for purposes of comparison. By removing interferences with a one-
or two-step cleanup and using mass spectrometry confirmation (as recommended
in this report), the recommended detection limits will reliably detect
substantial elevations in organic pollutants in the vicinity of a wastewater
discharge.
As a specific monitoring program progresses, certain compounds or
compound groups may be consistently undetected near wastewater discharge
sites even with low detection limits. Such findings may justify the discon-
tinued analysis of these compounds on a site-specific basis. Focusing
on selected compound groups enables analytical methods for critical compound
groups to be optimized, and typically results in improved detection limits.
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Furthermore, If non-target organic pollutants are found to occur frequently
and at significant concentrations in tissue samples such that they are
major peaks in GC/MS reconstructed ion chromatograms, and if these compounds
can be reliably identified by comparison of their mass spectra to those
of the U.S. EPA/NIH computerized library, they should be added to the list
of 301(h) target compounds on a site-specific basis.
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SUMMARY OF RECOMMENDATIONS FOR DETECTION LIMITS
Detection limits for each sample analyzed are required to be reported
with all data sets. In general, the detection limits recommended in this
report (Tables 2 and 5) are the most sensitive that may be feasibly attained
under the requirements for full scan analyses of U.S. EPA priority pollutant
metals and organic compounds.
Detection limits for trace metals in tissue are based on a minimum
sample size of 5 g (wet weight) (Table 2). An additional 1 g (wet weight)
of tissue may be used for a separate analysis of mercury. A detection
limit of 0.003 ug/g (wet weight) is recommended for beryllium. Detection
limits of 0.01 are recommended for cadmium, copper, mercury, and silver.
Detection limits of 0.02 ug/g (wet weight) are recommended for antimony,
arsenic, chromium, nickel, selenium, and thallium. A detection limit of
0.03 ug/g (wet weight) is recommended for lead. A less sensitive detection
limit of 0.1 ug/g (wet weight) is recommended for zinc.
Detection limits for organic pollutants in tissue are based on a minimum
sample size of 25 g (wet weight), with an additional 5 g (wet weight) of
tissue recommended for a separate analysis of volatile organic compounds
(Table 5). For the majority of the volatile organic compounds, detection
limits between 5 and 10 ug/kg (wet weight) are recommended. Detection
limits of 10 ug/kg (wet weight) are recommended for aromatic hydrocarbons
and phthalates. Detection limits ranging from 10 to 20 ug/kg (wet weight)
are recomnended for chlorinated hydrocarbons and halogenated ethers. Detection
limits for the chlorinated pesticides range from 0.1 to 5 ug/kg (wet weight)
with GC/ECD. In areas where high concentrations occur, mass spectrometric
detection (with a detection limit of 50 ug/kg) will provide compound
confirmation. If GC/MS confirmation is not possible, GC/ECD analysis with
an alternative GC column should be performed. PCBs should be analyzed
by GC/ECD with a detection limit of 20 ug/kg (wet weight). PCB confirmation
20
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on an alternative GC column, or by GC/MS if concentrations permit, is strongly
recommended.
To attain the recommended detection limits, a total sample size of
35 g is recommended for a complete analysis of priority pollutant trace
metals, semivolatile, and volatile organic compounds (i.e., 5 g for trace
metals, 25 g for semivolatile organic compounds, and 5 g for volatile organic
compounds). If individual organisms selected will not provide roughly
35 g of tissue, the Region may need to evaluate modification of the monitoring
program to either reduce the scope of the analyses (e.g., eliminate volatile
organic compound analysis), raise the recommended detection limits, or
composite tissue from several organisms. To satisfy requirements for quality
assurance of the data, an additional 35 g tissue is recommended for each
replicate set of analyses conducted. Typically, replicate analyses (including
matrix spike analyses) are conducted on 5 to 10 percent of the total number
of samples.
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REFERENCES
Boehm, P.O. 1984. Workshop report on the status and trends program:
recommendations for design and implementation of the chemical measurements
segment. Battelle New England Marine Research Laboratory, Duxbury, MA.
Gahler, A.R., T.M. Cummins, J.N. Blazevich, R.H. Rieck, R.L. Arp, C.E. Gongmark,
S.V.W. Pope, and S. Filip. 1982. Chemical contaminants in edible, non-salmonid
fish and crabs from Commencement Bay, Washington. EPA-910/9-82-093. Environ.
Serv. Div. Lab., U.S. EPA, Region X, Seattle, WA. 116 pp.
Glaser, O.A., D.L. Foerst, G.O. McKee, S.A. Quave, and W.L. Budde. 1981.
Trace analyses for wastewaters. Environ. Sci. Techno!. 15:1426-1435.
Hiatt, M.H. 1981. Analysis of fish and sediment for volatile priority
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Keith, L.H., W. Crommett, J. Deegan, Jr., R.A. Libby, J.K. Taylor, and
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MacLeod, W.D., Jr., D.W. Brown, A.J. Friedman, 0. Maynes, and R.W. Pearce.
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Service, Seattle, WA. 110 pp.
Martin, M., G. Ichikawa, J. Goetzi, and M. Stephenson. 1984. Annual Summary
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of target species and review of available bioaccumulation data. Final
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investigation. Final Report. Prepared for Washington Department of Ecology
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U.S. Environmental Protection Agency. 1977 (revised October, 1980). Interim
methods for the sampling and analysis of priority pollutants in sediments
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U.S. Environmental Protection Agency. 1979 (revised March, 1983). Methods
for chemical analysis of water and wastes. EPA 600/4-79-020. Environmental
Monitoring and Support Laboratory, Cincinnati, OH.
U.S. Environmental Protection Agency. 1984 (revised January, 1985). U.S. EPA
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food. Office of Technology Assessment, Washington, DC. 229 pp.
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