United States
Environmental Protection
Agency
Environmental Monitoring-and
Support Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S4-84-063 Aug. 1984
&EPA Project Summary
EPA Method Study 20
Method 610 —PNA's
Glenn Kinzer, Ralph Riggin, Thomas Bishop, Michelle A. Birts, and Paul Strup
"V.
The U.S. Environmental Protection
Agency (US EPA) sponsored an interla-
boratory study in which 16 laboratories
participated, to provide precision and
accuracy statements for the proposed
EPA Method 610 for the 16 selected
polynuclear aromatic hydrocarbons
(PIMA's) comprising Category 9 of the
priority pollutant which may be present
in municipal and industrial aqueous
discharges. The specific PIMA's are as
follows:
Napthalene Benzo(a) anthracene
Acenaphthylene Chrysene
Acenaphthene Benzo(b)fluoranthene
Flurorene Benzo(k)fluoranthene
Phenanthrene Benzo(a)pyrene
Antharacene Dibenzo(a,h)anthracene
Fluoranthene Benzo(g.h,i)perylene
Pyrene lndeno(1,2,3-cd)pyrene
Method 610 involves extraction of the
pollutants with methylene chloride
followed by silica gel cleanup and
subsequent high performance liquid
chromatography (HPLC) analysis utiliz-
ing fluorescence and ultraviolet (UV)
detection.
The study design was based on
Youden's non-replicate design for
collaborative tests of analytical meth-
ods. Three Youden pair ampules of the
test compounds were spiked into six
types of test waters and then analyzed.
The test waters were distilled water, tap
water, a surface water, and three
different industrial wastewater efflu-
ents. The resulting data were analyzed
statistically using USEPA's computer
program entitled, Interlaboratory Meth-
od Validation Study (IMVS).
Mean recovery of the PNA's based
upon inserting analyte concentrations
into the regression equations ranged
from 43-110 percent. Overall precision
was in the range of 16-91 percent and
single-analyst precision ranged from
11 -50 percent.
A statistically significant effect due to
water type was established for six of the
16 water types. However, because
distilled water had consistently lower
recoveries than the wastewaters, and
the distilled waters were the first
samples to be analyzed, the statistical
effect was judged to be due to the
analytical learning process and therefore
of no practical importance.
This Project Summary was developed
by EPA's Environmental Monitoring
and Support Laboratory, Cincinnati,
Ohio, to announce key findings of the
research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering
information at back).
Introduction
EPA first promulgated guidelines
establishing test procedures for the
analysis of pollutants in 1973 following
the passage of the Federal Water Pollu-
tion Control Act in 1972 by Congress.
Pursuant to the amendment and publica-
tion of these guidelines EPA entered into
a Settlement Agreement—the Consent
Decree—requiring it to study and, if
necessary to regulate, 65 "priority"
pollutants and classes of pollutants of
known or suspected toxicity to the biota.
Subsequently, Congress passed the
Clean Water Act of 1977 mandating the
control of toxic pollutants discharged into
ambient waters by industry.
In order to facilitate the implementa-
tion of the Clean Water Act, EPA selected
129 specific toxic pollutants, 113 organic
and 16 inorganic, for initial study. The
organic pollutants were divided into 12
categories based on their chemical
structure. Analytical methods were .
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developed for these 12 categories by EPA
through in-house and contracted research.
Method 610 was developed in the
Battelle-Columbus Laboratories under a
contract with the Physical and Chemical
Methods Branch, Environmental Moni-
toring and Support Laboratory—Cincin-
nati. The interim Method 610 is described
in the Federal Register, Vol. 44, No. 233,
December 3, 1979. The method requires
extraction of the pollutants from the
aqueous sample with methylene chloride.
The extract is then subjected to silica gel
chromatographic cleanup. The PNA
fraction is concentrated using Kurderna-1-
Danish evaporation, exchanged to ace-
tonitrile and analyzed by HPLC with UV
absorption and fluorescence detection.
Procedure
The study was patterned after Youden's
nonreplicate design for collaborative
evaluation of precision and accuracy for
analytical methods in which samples are
analyzed in pairs, each member of a pair
having a slightly different concentration
of the constituent of interest. The analyst
was directed to do a single analysis and to
report one value for each sample, as for a
normal routine sample. Samples of three
Youden pairs used in this study contained
low, medium, and high concentrations of
the Category 9 compounds which were
spiked into each of six different water
types and then analyzed.
Prior to the formal interlaboratory
method study, participants were familiar-
ized with both the study design and the
procedure by analyzing one trial Youden
pair sample followed by attendance at a
prestudy conference. After resolving
various method interpretations and
analytical problems at the prestudy
conference, participating laboratories
were supplied with the test materials
required by the study design and instruc-
ted to begin the analyses.
The test waters were:
a. Distilled water
b. A municipal drinking water
c. A surface water, for example, a river,
vulnerable to synthetic chemical
contamination
d. Three industrial wastewaters from
industries that were potential candi-
dates to be regulated for priority
pollutants.
Analyses were conducted on distilled
water to evaluate the proficiency of the
analyst in using the method on a sample
free of interferences. Municipal drinking
and surface waters were included as
test waters since these water types are
subject to contamination. Hence, it was
considered important to obtain informa-
tion about the performance of Method
610 in such matrices as well as those
found in industrial wastewater effluents.
Statistical analyses of the data were
performed using the IMVS computer
program developed at Battelle-Columbus
Laboratories and which is a revised ver-
sion of the EPA COLST program. The pro-
gram is designed to outputthe rawdata in
tabular form and to compile summary sta-
tistics including:
• Number of data points
• True value
• Mean recovery
• Accuracy as percent relative error
• Overall standard deviation
• Overall percent relative standard
deviation
• Single-analyst standard deviation
• Single-analyst percent relative standard
deviation.
The overall standard deviations indicate
the dispersion expected among values
generated from multiple laboratories. The
single-analyst standard deviations indi-
cate the dispersion expected among
replicate determinations within a single
laboratory.
Results and Discussion
The data collected during this interla-
boratory study were analyzed statistically
to establish the relationship between pre-
cision and mean recovery, and between
accuracy and the true concentration.
These relationships are summarized by
the linear regression equations presented
in Table 1.
The results of the regression analyses
indicate apparent linear relationships
between (1) overall standard deviation
and mean recovery; (2) single-analyst
precision and mean recovery; and (3) true
concentration and mean recovery.
The percent recoveries of the PNA
compounds ranged from 43-110 percent.
The overall relative standard deviations
ranged from 16 to 91 percent and single-
analyst relative standard deviations
ranged from 11 to 48 percent.
One of the questions of interest in this
study was whether water types affected
the precision and accuracy of the method.
An analysis of variance procedure
(ANOVA) was used to test for the effect of
water type on precision and accuracy.
Based on the results of this analysis, a
statistically significant effect due to water
type was established for the following
PNA's:
acenapthalene dibenzo(a,h)anthracene
anthracene benzo(g,h,i)perylene
benzo(k)fluoranthene indenod ,2,3-cd)
pyrene
Mean recoveries of these six compounds
from distilled water were no better, and in
many cases were poorer than for the
wastewater samples. One would antici-
pate that the data would be somewhat
better for distilled water, since there is
little likelihood of interferences or matrix
affects. Since the distilled water data
were in all cases collected prior to
wastewater data, the analysts were more
experienced in utilizing the method when
they analyzed the1 wastewater samples.
This may have resulted in a learning
curve effect which improved the data for
wastewater as compared to distilled
water. Therefore, the observed statistical
effect was judged to be due to the
analytical learning process and was of no
practical importance.
For the other 10 PNA's, there were no
effects of statistical significance due to
water types among mean recoveries,
overall precisions or single-analyst
precisions. ;
Several operational problems were
reported by the laboratories while
conducting Method 610 analyses, the
most relevent of which were: ;
• HPLC column performance was
found to be somewhat variable.
Some laboratories had to examine
two or three 'commercially available
reverse phase columns, HC - ODS
Sil-X, 250 mm x 2.6 mm ID, before
one was found that would adequate-
ly separate benzo(g,h,i)perylene and
dibenzo(a,h)anthracene. The other
PNA compounds were generally well
resolved on this type of column. ;
• Fluorescence detector response for
the various Compounds was quite
different for the several types of
detectors used. Two laboratories
used filter type excitation, rather
than a grating monochromator,
which produces a much higher
relative response for anthracene
causing it to interfere with fluoran-
thene. Mercury vapor lamps were
found to give a low output at 280 nm,
resultihg in low response for; all
compounds. Use of a phosphor
coated lamp improved response
somewhat. In general, fluorescence
detectors employing deuterium lamps
and grating monochromators for
excitation gave consistent results.
Conclusions and
Recommendations
Generally, use of Method 610: by
experienced analysts should enable
industries to meet the requirements of
the National Pollutant Discharge Elimina-
tion System for discharging the subject
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pollutants into the environment.
It is recommended that the following
precautions be observed by the analyst
using Method 610 to help ensure
reliability of the resultant data.
• Some of the compounds are light
sensitive and thus exposure to light
should be kept at a minimum. All
sample extracts should be stored in
the dark prior to analysis. The elution
profile of the column should be
checked to ensure that elution of the
PNA's in the proper order is occur-
ring.
• The HPLC system performance is
important since a large number of
compounds must be separated. The
equilibration time (at 40% acetoni-
trile/60% water) between runs
should be at least 25 minutes and
should be consistent from run to run.
Solvents for HPLC must be filtered
through a submicron filter and then
degassed, either by heating or by a
helium purge, to prevent bubble
formation.
• The sensitivity of the detectors
should be checked daily.
Table 1. Regression Equations for Accuracy and Precision of Method 610 by Compound and Water Type
Water Type
Applicable Cone. Range
Distilled Water
Single-Analyst Precision
Overall Precision
Accuracy
Tap Water
Single-Analyst Precision
Overall Precision
Accuracy
Surface Water
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-94J
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-95J
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-96)
Single-Analyst Precision
Overall Precision
Accuracy
Naphthalene
(10.00 - 375.00) :
SR = 0.39X - 0. 18
S =O.41X + O,74
X =O.S7C-0.70
SR = 0.36X + 0.24
S =0.39X + O.73
X = 0.6OC - 0.62
SR = 0.24X + 1.94
S = 0.41 X+ 1.07
X = 0.6OC - 0.82
SR = 0. 19X + 1.34
S = 0.36X + O.26
X =0.62C + 0.72
SR = 0.23X - 0.48
S = 0.32X - 1.09
X = 0.58C + 1.04
SR = 0.31 X + 0.26
S = 0.41 X- 0.1 5
X =O.65C-O.76
Acenaphthylene
(10.00 - 425.00)
SR = O.36X + O.29
S = 0.42X^0.52
X =0.690-1.89
SR = 0.38X - O.O1
S = 0.44X - 0.03
X = 0.71 C- 2.58
SR = 0.27X + O.30
S = 0.30X + O.08
X =O.74C-2.07
SR = 0.19X + 1.02
S = 0.32X - 0.01
X =0.83C-1.16
SR = 0.32X - O.81
S =0.36X-0.13
X =0.75C-0.80
SR = O.17X + 0.57
S = 0.23X^1.09
X = 0.83C - 1.89
Acenaphthene
(10.00 - 260.00)
SR =0.39X^0.76
S = 0.53X + 1.32
X = 0.520 + 0.54
SR=0.29X + 0.27
S = 0.47X + 0.45
X =0.51C - 1.55
SR = 0.17X + 1.48
S = 0.48X + 0.23
X = 0.53C - 0.59
SR = 0.35X - 0.79
S = 0.50X - 0.21
X =0.59C-0.46
SR = O.24X + 0.33
S =0.47X^0.08
X = 0.570 + 0.30
SR = O.28X + O.34
S =0.43X-0.54
X = 0.620 + 0.12
Fluorene '
(10.00 - 463.00)
SR = O.44X - 1. 12
S = 0.63X - O.65
X =0.56C-0.52
SR = 0.25X + 1. 16
S =0.5OX-0.16
X = 0.59C - 1.30
SR = 0.40X - 0.93
S =0.52X-0.74
X =O.57C-O.25
SR=0.25X + 1.6O
S = O.52X - 1.26
X = 0.60C - O.03
SR = 0.21 X + 2.56
S = 0.47X - 0.44
X =0.53C + 0.73
SR = 0.35X + 0. 10
S = 0.49X - 0.39
X =0.540 + 0.36
X = Mean Recovery
C = True Value for the Concentration
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Tab/a 1. (continued)
Water Type
Applicable Cone. Range
Distilled Water
Single-Analyst Precision
Overall Precision
Accuracy
Tap Water
Single-Analyst Precision
Overall Precision
Accuracy
Surface Water
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-94)
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-95)
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-96)
Single-Analyst Precision
Overall Precision
Accuracy
Phenanthrene
(5.00 - 280.00)
SR = 0.28X + 0.05
S = 0.47X - 0.25
X =0.720-0.95
SR = 0.26X + 0. 10
S =0.35X-0.16
X = 0.71 C- 0.71
SR = 0.23X - 0.34
S = 0.37X - 0.62
X =0.700-0.26
SR = 0.1JX + 0.74
S = 0.26X - 0.22
X =0.790-0.61
SR = 0. 15X - 0.03
S = 0.28X - 0.03
X =0.730-0.48
SR = 0.35X - 0.50
S = 0.38X - 0.28
X =0.700-0.47
Anthracene
(10.00 - 400.00)
SR=0.23X+ 1.16
S = 0.41 X + 0.45
X = 0.630 - 1.26
SR = 0.22X + 0.61
S = 0.41 X + 0.10
X =0.630-2.05
SR = 0.1 9X + 0.22
S = 0.34X-0.69
X =0.640-0.45
SR = 0. 19X + 0.99
S =0.39X-0.41
X = 0.690 - 0.26
SR = 0. 19X + 0. 10
S = 0.33X - 0.31
X =0.640-0.34
SR = 0.24X - 0.29
S =0.35X-0.91
X = 0.660 + 0.08
Fluoranthene
(0.30 - 15.00)
SR = 0.22X + 0.06
S =0.32X + 0.03
X =0.680 + 0.07
SR=0.23X + 0.01
S =0.32X + 0.01
X =0.710-0.03
SR = 0.27X - 0.04
S =0.44X-0.01
X = 0.590 + 0.05
SR = 0. 12X + 0.03
S = 0.35X - 0.01
X =0.750-0.00
SR = 0.17X -0.01
S = 0.29X + 0.02
X =0.700 + 0.02
SR = 0.40X - 0.06
S = 0.38X + 0.03
X =0.750 + 0.01
\ Pyrene
(2.00 - 90.00)
' SR = 0.25X + 0.14
S = 0.42X - 0.00 :
X = 0.690 - 0. 12
SR = 0.25X + 0.02
S = 0.39X + 0.09
X = 0.680 + 0.09 ':
, SR = 0.22X -0.10
S = 0.30X -0.12
X =0.740 - 0.08 •
SR = 0.17X + 0.15
S = 0.26X - 0.02
X =0.710 + 0.02,
SR = 0.27X - 0.04 '-.
S = 0.34X-0.19
X =0.660 + 0.25
SR = 0.20X - 0.00 .
S = 0.25X + 0. 14
; X =0.770 + 0.01
X * Mean Recovery
C = True Value for the Concentration
Table 1. (Continued)
Water Type
Applicable Cone. Range
Distilled Water
Single-Analyst Precision
Overall Precision
Accuracy
Tap Water
Single-Analyst Precision
Overall Precision
Accuracy
Surface Water
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-94J
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-9S)
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-96)
Single-Analyst Precision
Overall Precision
Accuracy
Benzo(a)A nthracene
(0.50 - 16.00)
SR = 0.28X + 0.04
S = 0.34X + 0.02
X =0.730 + 0.05
SR = 0.23X + 0.13
S = 0.37X + 0.05
X =0.770 + 0.05
SR = 0.1 8X- 0.01
S =0.34X-0.05
X =0.760-0.02
SR = 0.24X + 0.03
S = 0.32X + 0.06
X =0.730 + 0.12
SR = 0.28X - 0.04
S = 0.43X - 0.04
X =0.690 + 0.03
SR = 0. 18X + 0.00
S =0.32X + 0.04
X =0.760 + 0.00
Chrysene
(2.00 - 60.00)
SR = 0.32X - 0. 18
S =0.56X-0.22
X =0.770-0.18
SR = 0.40X - 0.37
S =0.55X-0.10
X = 0.820 - 0.09
SR = 0.39X - 0.51
S = O.SOX - 0.20
X =0.770 + 0.39
SR = 0.29X - 0.06
S = 0.44X - 0.09
X =0.970-0.28
SR = 0.25X + 0.42
S = 0.48X + 0.10
X =1.220-0.58
SR = 0.24X + 0.02
S =0.45X + 0.14
X = 1.010 - 0.07
Benzo(b)Fluoranthene
(0.20 - 1 1.00)
SR = 0.21 X + 0.01
S = 0.38X - 0.00
X =0.780 + 0.01
SR = 0.24X - 0.00
S = 0.32X - 0.01
X = 0.830 + 0.00
SR = 0.26X - 0.01
S = 0.48X - 0.03
X =0.730 + 0.01
SR = 0.21 X - 0.00
S = 0.39X - 0.02
X =0.800-0.01
SR = 0.28X - 0.01
S = 0.42X - 0.02
X =0.900-0.00
SR = 0.26X - 0.01
S =0.37X-0.01
X = 0.900 + 0.00
Benzo(k)Fluoranthene
(0. 12 - 6.00)
SR = 0.44X - 0.01 ',
,S =0.69X + 0.01
X = 0.590 + 0.00
SR = 0.48X + 0.06
S = 0.91 X- 0.01
X =0.980-0.03
SR = 0. 19X + 0. 16
S =0.76X + 0.01
X =1.020+0.04
'. SR = 0. 18X - 0.01 I
S = 0.47X + 0.01
X =0.610 + 0.03
SR = 0.46X - 0.07
S =0.68X-0.01
X = 1.090 + 0.03
SR = 0.22X - 0.00
' S =0.69X-0.03
X = 0.990 - 0.05
X a Mean Recovery
C " True Value for the Concentration
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Table 1. (Continued)
Water Type
I Applicable Cone. Range
', Distilled Water
Single-Analyst Precision
Overall Precision
'Accuracy
i Tap Water
; Single-Analyst Precision
Overall Precision
Accuracy
Surface Water
Single-Analyst Precision
Overall Precision
> Accuracy
: Wastewater (0-94)
Single-Analyst Precision
Overall Precision
Accuracy
: Wastewater (C-95)
: Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (0-96)
1 Single-Analyst Precis/on
' Overall Precision
Accuracy
Benzo(a)Pyrene
(0.20 - 15.001
SR = 0.38X - 0.01
S = 0.53X - 0.01
X = 0.56C + 0.01
.SR = 0.29X - 0.01
S = 0.53X - 0.00
X = 0.54C - 0.02
SR = 0.24X -0.01
S =0.47X-0.00
X =0.650 + 0.01
SR = 0.30X - 0.01
S = 0.44X-0.01
X = 0.670 + 0.02
SR = 0.31 X + 0.01
S = 0.40X - 0.00
X =0.720-0.01
SR = 0.20X - 0.00
S = 0.41 X- 0.02
X =0.700 + 0.01
Dibenzo(a.h)A nthracene
(0.50 - 24.00)
SR = 0.24X + 0.02
S = 0.45X + 0.03
X =0.410 + 0.11
SR = 0.42X - 0.01
S = 0.44X + 0.04
X =0.680 + 0.09
SR = 0.34X + 0.04
S = 0.49X - 0.02
X =0.710-0.03
SR = 0.24X + 0.00
S = 0.35X + 0.00
X =0.710-0.05
SR = 0.25X + 0. 12
S =0.39X-0.00
X =0.770 + 0.02
SR = 0.36X - 0.07
S = 0.45X + 0.08
X =0.710 + 0.16
Benzofg, h, iJPery/ene
(1.00 - 50.00)
SR = 0.25X + 0.04
S = 0.58X + 0. 10
X = 0.44C + 0.30
SR = 0.24X - 0.06
S = 0.29X + 0.00
X =0.710-0.07
SR = 0.40X-0.16
S =0.60X-0.12
X =0.670 + 0.05
SR = 0.25X - 0.04
S = 0.36X - 0.08
X =0.720-0.05
SR = 0.27X + 0.01
S =0.48X-0.17
X =0.710 + 0.14
SR=0.34X-0.17
S =0.42X-0.04
X =0.690 + 0.20
Indenofl ,2,3-cdJPyrene
(0.75-22.00)
SR = 0.29X + 0.02
S =0.42X + 0.01
X = 0.540 + 0.06
SR = 0.33X - 0.04
S = 0.38X + 0.02
X =0.700-0.05
SR = 0.27X - 0.04
S =0.42X-0.06
X =0.600 + 0.02
SR = 0.25X - 0.06
S =0.42X-0.04
X = 0.67C + 0.01
SR = 0.39X - 0.01
S = O.SOX + 0.04
X = 0.950 - 0.05
SR = 0.37X - 0.07
S =0.44X-0.05
X =0.830-0.11
' X = Mean Recovery
\ C - True Value for the Concentration
Glenn Kinzer, Ralph Riggin, Thomas Bishop, Michelle A. Birts, and Paul Strup are
with Battelle-Columbus Laboratories, Columbus, OH 43201.
Edward L. Berg and Robert L. Graves are the EPA Project Officers (see below).
The complete report, entitled "EPA Method Study 2O, Method 610—PNA's,"
(Order No. PB 84-211 614; Cost: $14.50, subject to change) will be available
only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
'Telephone: 703-487-4650
The EPA Project Officers can be contacted at:
Environmental Monitoring and Support Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
*USGPOa, 1984-759-102-10642
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