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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