RESIDUES AND TRACE ELEMENTS
Ion Chromatographic Method for Dissolved Hexavalent
Chromium in Drinking Water, Groundwater, and Industrial
Wastewater Effluents: Collaborative Study
EDGELL ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 77, NO. 4, 1994
KENNETH W. EDGELL
Bionetics Corp., 16 Triangle Park Dr, Cincinnati, OH 45246
JAMES E. LONGBOTTOM
U.S. Environmental Protection Agency, 26 W. Martin Luther King Dr, Cincinnati, OH 45268
ROBERT J. JOYCE
Dionex Corp., 501 Mercury Dr, Sunnyvale, CA 94086
Collaborators: E. Arar; T. Arnold; J. Capito; R. Dovi; D. Dragotta; P. Grohse; P. Jackson; R. Joyce; B. Mitchell; J. Northington;
W. Pratt; R. Roehl; C. Scutt; J. Sherwell; C. Shoop; T. Surveski; D. Tucker; V. White; F. Williams; W. Wolf; J. Stanley
A collaborative study was conducted by the U.S.
Environmental Protection Agency (U.S. EPA) and
the American Society for Testing Materials (ASTM)
on an ion chromatographic method for the determi-
nation of hexavalent chromium (U.S. EPA method
218.6, and the ASTM equivalent method). This
study was designed to determine the mean recov-
ery and precision of analyses for hexavalent chro-
mium in reagent water, drinking water, groundwa-
ter, and industrial wastewaters. The study design
was based on Youden’s nonreplicate plan for col-
laborative studies of analytical methods. The test
waters were spiked with hexavalent chromium at
8 concentration levels, prepared as 4 Youden pairs.
A fifth Youden concentration pair was also includ-
ed to determine method performance close to the
method detection limit. Twenty-one laboratories
were instructed to filter their test waters through a
0.45 µm filter and to adjust the pH of the filtrate to
9–9.5 with an ammonium sulfate/ammonium hy-
droxide buffer solution before spiking with the
hexavalent chromium concentrates. A known vol-
ume, 50–250 µL, was injected into an ion chroma-
tograph which separated the Cr(VI), as CrO42−−, on
an anion exchange column. After separation, the
Cr(VI) was derivatized with diphenylcarbazide and
the colored complex was detected at 530 nm. The
submitted data were corrected for background con-
centrations and analyzed by applying ASTM D-2777-
Submitted for publication July 23, 1992.
The recommendation was approved by the Committee on Environment-
al Quality, and was adopted by the Official Methods Board of the Associa-
tion. See “Changes in Official Methods of Analysis” (1994) J. AOAC Int.
77, Jan/Feb issue, and “Official Methods Board Actions” (1993) The Ref-
eree, 17, July issue.
86 statistical procedures and a U.S. EPA computer
program. U.S. EPA method 218.6 and the equivalent
ASTM method were judged acceptable for the
measurement of hexavalent chromium concentra-
tions at 1–1000 µg Cr(VI)/L. The study found that
the use of a single linear calibration extending over
3 orders of magnitude yielded biased results at the
very lowest concentration levels. Shorter range cali-
bration curves yielded more accurate results.
Analysis of variance (ANOVA) tests indicated that
method performance was significantly different be-
tween the reagent water matrix and the other ma-
trixes used. The recovery of Cr(VI) from the other
matrixes was lower at all concentration levels with
slightly less precision when compared with the re-
agent water data set. For reagent water, the mean
recovery and the overall and single-analyst relative
standard deviations were 105%, 7.8% and 3.9%, re-
spectively. For the other matrixes, the same values
were 96.7%, 11.9% and 6.3%, respectively. The
method was adopted first action by AOAC INTER-
NATIONAL.
exavalent chromium salts are used extensively in the
H metal plating and leather industries and in the manufac-
ture of paints, dyes, explosives, and ceramics. Chro-
mate compounds are also added to cooling water for corrosion
control. As a result, chromium salts are found in the waste-
waters of many industries, potentially contaminating drinking
water supplies. The hexavalent chromium concentration of the
United States drinking waters varies between 3 and 40 µg/L
with a mean of 3.2 µg/L (1). Since hexavalent chromium is
toxic to humans, animals, and aquatic life (“Health Effects As-
sessment for Hexavalent Chromium” EPA/540/1-86/019,
1986), and is a suspected carcinogen, the U.S. Environmental
Protection Agency (U.S. EPA) is very interested in monitoring
the levels of hexavalent chromium in drinking water supplies.
Sensitive and highly selective colorimetric methods for the
determination of hexavalent chromium are well established (1,
2); when these methods are used however, matrix interferences
may produce inaccurate results. To this end, U.S. EPA has de-
veloped an ion chromatography method 218.6 (Arar, E.J.,
Long, S.E., & Pfaff, J.D. (1990) Determination of Dissolved
Hexavalent Chromium in Drinking Water, Groundwater, and
Industrial Wastewater Effluents by Ion Chromatography, Envi-
ronmental Monitoring Systems Laboratory, Office of Research
and Development, U.S. EPA, Washington, DC), which selec-
tively removes hexavalent chromium from these matrix inter-
ferences before derivatization and colorimetric determination.
The U.S. EPA Environmental Monitoring Systems Labora-
tory at Cincinnati, Ohio (EMSL-Cincinnati), develops or se-
lects analytical methods and provides quality assurance (QA)
support for Agency programs that involve water and wastewa-
ter regulations. In EMSL-Cincinnati, the responsibility for pro-
viding QA support is assigned to the Quality Assurance Re-
search Division (QARD). One of the Division’s activities is to
conduct collaborative studies, either independently or jointly
with other government or not-for-profit organizations, to evalu-
ate analytical methods selected for the Agency’s operating pro-
grams. U.S. EPA and the American Society for Testing and Ma-
terials (ASTM) have agreed to conduct joint collaborative
studies. ASTM, through the consensus standard process, pro-
vides for review of the methods, study designs, and selection of
appropriate test materials. U.S. EPA conducts the studies and
evaluates the data by using statistical procedures acceptable to
both organizations.
The joint study for U.S. EPA method 218.6 was conducted
under the direction of QARD through its contract with The
Bionetics Corp. U.S. EPA prepared and distributed sample con-
centrates, user instructions, and report forms, solicited partici-
pants through an announcement in the Federal Register (55 FR
14349, April 17, 1990), and evaluated the returned data.
Additional volunteer laboratories were located by ASTM
Committee D-19 through ion chromatography task group an-
nouncements, contacts with ion chromatography instrument
manufacturers, and announcements at analytical conferences.
Twenty-three laboratories participated in the study and 21 sub-
mitted data.
The study had the following objectives: characterization of
multi-laboratory performance of U.S. EPA method 218.6 in
terms of recovery, overall precision, and single-analyst preci-
sion; comparison of the recovery and precision for hexavalent
chromium in reagent water and various waters-of-choice; de-
termination of the interlaboratory method detection limit
(IMDL) for Cr(VI) in reagent water and comparison of IMDL
with the method detection limit (MDL) in U.S. EPA method
218.6; and development of appropriate performance-based ac-
ceptance limits for use in quality control.
Collaborative Study
The study design was based on Youden’s nonreplicate de-
sign for collaborative evaluation of overall precision, single-
analyst precision, and mean recovery for analytical methods (3,
4). Two samples differing in analyte concentration by approxi-
mately 2 standard deviations were analyzed as a Youden pair to
provide data for estimating single-analyst precision. In this
study, 4 such concentration pairs were used to encompass the
range 6–960 µg/L. The study design called for each participant
to spike hexavalent chromium into reagent water and a water-
of-choice. In addition to the spiked matrixes, each participant
was asked to analyze both a single unspiked sample (blank) of
the reagent water and the selected matrix water. The evaluation
of the results obtained for the spiked reagent water demon-
strated the proficiency of the method in recovering Cr(VI) from
a sample free of interferences. Analyses of the spiked matrix
waters tested the suitability of the method on representative
water types and provided a basis for comparison with the re-
sults from reagent water.
Quality control (QC) requirements consistent with the
method were incorporated into this study to monitor the “in
control” status of the various stages of the analytical process.
Each participant was sent a concentrate for preparing a QC
sample at 40 µg/L. The participants were instructed to analyze
the QC sample after the analysis of the calibration standards
and immediately following the analysis of each set of spiked
matrix samples. If the sample results fell outside of the accep-
tance limits of 36–44 µg/L, the laboratory was directed to re-
analyze the sample; if the second analysis also failed, the labo-
ratory was asked to recalibrate their instrument. All QC results
were to be reported as part of the study data.
A fifth IMDL Youden concentration pair was included in the
reagent water matrix set to evaluate method performance of
hexavalent chromium concentrations close to the reported
MDL for EPA method 218.6. The concentration values of this
low-level Youden pair were 1.2 and 1.6 µg/L, which were 3 and
4 times higher than the MDL of 0.4 µg/L cited.
Each participating laboratory received the method, 18 sam-
ple spike solutions (10 solutions for spiking into reagent water
and 8 solutions for spiking into a water-of-choice), 1 known
calibration standard concentrate, 1 QC sample concentrate
with true values and acceptance limits, report forms, a ques-
tionnaire, and an instruction manual. Prior to distribution, the
test solutions were analyzed and contents were confirmed by
EMSL-Cincinnati against standards freshly prepared from neat
materials. The sample spiking concentrates, calibration con-
centrates, and QC sample concentrates were buffered to pH 9.2
with ammonium sulfate/ammonium hydroxide and packaged
in polypropylene bottles which had been previously acid-
cleaned and rinsed with deionized water. Because the buffer
solution was found to leach measurable levels of chromium
from Pyrex ampules, polypropylene bottles were used to store
samples. The collaborating laboratories were instructed to cali-
brate their analytical instruments by using the calibration stand-
ard concentrate provided, to analyze the spiked samples in strict
accord with the written method (4), and to complete the analy-
ses within 30 days of receipt of sample.
Treatment of Data
Upon receipt, each data set was screened for data points
which exceeded one-fifth to 5 times the true value. Calculations
for such data were checked, and if no error was found, the data
were retained and submitted with the other study data to statis-
tical processing.
Each laboratory submitted a questionnaire which was re-
viewed for adherence to study instructions and QC require-
ments. If a laboratory failed to comply with the study instruc-
tions, its data were removed from the study. Although there
were a few instances where QC sample results were outside the
acceptance limits, the study data were not removed.
The participating laboratories were instructed to report
background concentrations of Cr(VI) separately and not to sub-
mit “background-corrected” data. EMSL-Cincinnati per-
formed background corrections so the effect of background
concentration on spiked concentration could be evaluated.
The background-corrected data for the 4 highest Youden
pairs were grouped by water type and arranged into 8 subsets
as defined by the 8 different samples. Next, they were evalu-
ated using the U.S. EPA computer program, Interlaboratory
Method Validation Study (IMVS) designed for these studies
(Outler, E.C., & McCreery, J.H. (1982) Interlaboratory Method
Validation Study: Program Documentation, Battelle Columbus
Laboratories, Columbus, OH). IMVS replaced missing data
points with interpolated values, and “less than” and “nonde-
tect” values were converted to zero. Then outlier tests were ap-
plied. The first outlier test was Youden’s laboratory ranking
procedure (3) which rejected laboratories that had a consis-
tently higher or lower bias in their submitted data for a given
analyte compared with the other laboratories. If a bias was de-
termined, all 8 analyte values for that laboratory were rejected.
This procedure was applied to each analyte data set, for each
water type, at the 5% level of significance. Zeroes, interpolated
values, and negative numbers were removed before further
analyses. As a final outlier test, Thompson’s test for individual
outliers (5) was applied to the retained data for a specific water
type and sample at the 5% significance level. If an individual
data point was rejected on the basis of this test, it was removed
from the subset and the Thompson test was repeated on the
remaining data for that water type and sample.
The results from the IMDL Youden pair reagent water sam-
ples were evaluated separately from the other concentrations
due to limitations of the IMVS software. Data from any labo-
ratory identified by the ranking test as an outlier during evalu-
ation of the 8 higher concentration reagent water samples were
automatically excluded from the Youden pair before statistical
evaluation. Thompson’s test for individual outliers was then
applied to the remaining data points.
Summary statistics were calculated for the mean recovery
and overall method precision for each of the 8 higher concen-
tration levels and each sample of the IMDL Youden concentra-
tion pair. Single-analyst precision was calculated for each of the
5 concentration pairs. The IMVS computer program used the
summary statistics for the 8 higher concentration samples to
calculate relationships between mean recovery and true con-
centration, and between precision and mean recovery in the
form of linear regression equations, using the weighted least
squares technique [6; Edgell, K.W., & Jenkins, E.L. (1989)
“U.S. EPA Method Study 39,” EPA/600/4-88/034, Environ-
mental Monitoring Systems Laboratory, U.S. EPA, Cincinnati,
OH]. Coefficients of determination of the weighted regression
equations (CODw) were also calculated to evaluate the fit of the
regressions to the retained data sets. For these same data, IMVS
also tested for matrix effects between the reagent water data and
recovery of the spikes from the waters-of-choice. The IMDL
was calculated from the within-laboratory precision estimates
obtained from the IMDL samples and compared with the sin-
gle-analyst method detection limit reported for the method.
993.23 Dissolved Hexavalent Chromium in Drinking
Water, Groundwater, and Industrial Wastewater
Effluents—Ion Chromatographic Method
First Action 1993
(Applicable to determination of 1.2–960 µg/L hexavalent
chromium in drinking water, groundwater, and industrial
wastewaters)
Method Performance:
See Table 993.23 for method performance data.
(Caution: Hexavalent chromium is toxic and possibly car-
cinogenic. See “Appendix: Laboratory Safety” on special
chemical hazards of carcinogens)
A. Principle
After pH of filtered test sample is adjusted to 9–9.5, Cr(VI),
as CrO42−, is separated by ion chromatography (IC) using anion
exchange separator column. Cr(VI) is derivatized with
diphenylcarbazide in post-column reactor and detected as col-
ored complex at 530 nm.
B. Apparatus
(a) IC system.—Equipped with pump capable of 2000 psi
at constant flow of 1–5 mL/min and containing no metal parts
in sample, eluant, or reagent flow path. Plastic pressurized
eluant container, 1–2 L. Various sample loops, 50–250 µL.
Pressurized post-column derivatization reagent delivery mod-
ule with mixing tee and beaded mixing coil capable of constant
flow, 0.5 mL/min. Detector with low volume flow-through cell
containing no metal parts in contact with eluant flow path, ca-
pable of 530 nm. Recorder, integrator, or computer to receive
analog or digital signals for detector response (peak height or
area) as function of time. Chromatographic conditions: eluant
flow rate, 1.5 mL/min; post-column flow rate, 0.5 mL/min;
typical Cr(VI) retention time, 3.8 min.
(b) Columns.—(1) Guard column.—(For use with waste-
water samples.) Capable of removing strongly adsorbing or-
ganics and particles that would damage separator column
(Dionex IonPac NG1, Sunnyvale, CA, is suitable). (2) Sepa-
rator column—Packed with high capacity anion exchange
resin capable of separating CrO42− from other sample constitu-
ents (Dionex IonPac AS7 is suitable).
(c) Glassware.—Class A: volumetric flasks, graduated cyl-
inder, and calibrated pipettes.
(d) Syringes.—10 mL, male Luer-lock, disposable.
(e) Syringe cartridge filters.—0.45 µm, 7.3 cm diam.,
polysulfone filter (Gelman Acro 50A is suitable).
(f) Storage bottle.—1 L, high density polypropylene.
(g) Sample bottle.—125 mL, high density polypropylene.
(h) pH meter.—Capable of determining pH to accuracy of
± 0.03 unit.
C. Reagents
(a) Ammonium hydroxide (NH4OH).
(b) Ammonium sulfate [(NH4)2SO4].
(c) 1,5-Diphenylcarbazide (DPC).
(d) Methanol (CH3OH).—LC grade or equivalent.
(e) Sulfuric acid (H2SO4).—Concentrated (sp.gr. 1.84).
(f) Reagent water.—ASTM Type I water (ASTM D1193).
Suitable water may be obtained by passing distilled water
through mixed bed of anion and cation exchange resins.
(g) Sodium chromate tetrahydrate (Na2CrO4⋅4H2O).
(h) Stock solution Cr(VI).—Accurately weigh 4.501 g
Na2CrO4⋅4H2O, (g), to nearest 0.001 g, dissolve, and dilute to
volume with reagent water, (f), in 1 L volumetric flask. Transfer
to polypropylene storage bottle, B(f).
(i) Eluant.—250 mM (NH4)2SO4, 100 mM NH4OH. Dis-
solve ca 33 g (NH4)2SO4, (b), in 500 mL reagent water, (f), add
6.5 mL NH4OH, (a), and dilute to volume with reagent water
in 1 L volumetric flask.
(j) Post-column reagent.—2 mM DPC, 10% v/v CH3OH,
1N H2SO4. Dissolve ca 0.5 g 1,5-diphenylcarbazide, (c), in
100 mL CH3OH, (d). Add to 500 mL reagent water, (f), con-
taining 28 mL conc. sulfuric acid, with stirring. Dilute to vol-
ume with reagent water in 1 L volumetric flask. Stable 4–
5 days. Prepare as needed.
(k) Buffer solution.—Dissolve ca 33 g (NH4)2SO4, (b), in
75 mL reagent water, (f), and add 6.5 mL NH4OH, (a). Dilute
to 100 mL with reagent water.
D. Sample Collection, Preservation, and Storage
Filter sample through 0.45 µm filter, B(d). Wet syringe fil-
tration unit using portion of sample, then filter and collect re-
quired volume of filtrate. Add buffer, C(k), dropwise, to adjust
sample pH to 9.0–9.5, periodically checking pH with pH meter.
Ca 10 mL sample are sufficient for 3 analyses.
Ship and store samples at 4°. Warm to ambient temperature
prior to analysis. Analyze samples within 24 h of collection.
E. Calibration
Establish IC operating conditions, B(a). Prepare calibration
standards at 3 concentration levels, bracketing anticipated con-
centration range of samples. Dilute aliquots of stock standard,
C(h), with reagent water, C(f), in volumetric flasks. Adjust
calibration solutions to pH 9.0–9.5 with buffer solution, C(k),
prior to final dilution. Prepare low-level calibration standards
daily.
Plot analyte response (peak height or area) versus analyte
concentration to construct calibration curve. Coefficient of de-
termination (COD), R2, for curve should be ≥0.999. Do not
prepare calibration curve over range >2 orders of magnitude.
Verify continuing calibration, using laboratory blank and
calibration check standard (mid-level concentration) as surro-
gate samples every 10 analyses. If concentration of analyte de-
viates from true concentration by greater than ± 5%, recalibrate
and reanalyze previous 10 samples.
Verify calibration stock standards, C(h), every 3 months by
analyzing standard prepared from USEPA Certified Reference
Material, or equivalent, using acceptance limits of ± 10% of
established value. If sample results are outside acceptance lim-
its, perform second analysis. If second value exceeds accep-
tance limits, stop analyses, find source of problem, and recali-
brate instrument.
F. Procedure
(1) Equilibrate stored samples to ambient temperature prior
to analysis.
(2) Initiate instrument operating configuration and cali-
brate.
(3) Draw ca 3 mL sample into new, unused syringe, B(c),
and attach syringe filter, B(d). Discard first 0.5 mL through fil-
ter and load 10× sample loop volume to thoroughly flush loop.
If concentrations are higher than established linear dynamic
range, dilute samples using reagent water, C(f), and adjust pH
if necessary.
G. Calculations
Determine peak height or peak area counts of Cr(VI) in each
sample. Compare with calibration curve to determine sample
concentration in µg/L.
H. Quality Control
Minimum QC requirements:
(1) Determine method detection limit (MDL).—Spike 7
replicate portions of reagent water, C(f), at concentration 2–5X
estimated detection limit of Cr(VI), buffer to pH 9.0–9.5. Proc-
ess replicate portions through analytical method (F), then ana-
lyze. Calculate MDL as follows:
MDL = t × s
where t = Student’s t value, 99% confidence level, n-1 degrees
of freedom (t = 3.143 for 7 replicates) and s = standard devia-
tion of replicate analyses.
MDL is concentration that can be distinguished from back-
ground by analytical instrumentation used.
(2) Analyze method blank daily for contamination.
(3) Analyze 1 laboratory spiked water blank at 40 µg/L, for
each 10 samples. Sample recovery from test sample must be
within 36–44 µg/L, or corrective action should be taken and
documented before analyses continue.
(4) Analyze 1 spiked matrix sample for each 10 samples.
Spike concentration should be 40 µg/L or 2× background con-
centration, whichever is higher. Calculate percent recovery of
analyte spike. If results are not within 90–100%, reanalyze
spiked and unspiked samples to document sample matrix ef-
fect.
Ref.: J. AOAC Int. 77, 994 (1994)
Results and Discussion
Four of 8 data points from Laboratory 11, in the reagent
water data sets, exceeded the review limits of one-fifth to
5 times the true value. No calculation errors were found. The
reagent water data set for this laboratory was later identified by
the laboratory ranking outlier test as having a systematic low
bias and was removed from the study.
Quality Control Sample Results
Quality control (QC) samples, prepared at 40 µg/L, were
analyzed immediately after the calibration curve and after each
of the 2 spiked matrix sets. The QC results were evaluated
against acceptance limits of 36–44 µg/L (± 10%) to confirm the
accuracy of the calibration curve and to assess the quality of
each spiked matrix set. All 21 QC results following the calibra-
tion standard analyses were grouped within the acceptance lim-
its. However, 2 of 21 QC results following the reagent water
matrix and 3 of 21 QC results following the water-of-choice
matrix fell outside the acceptance limits.
Laboratory 4 reported unacceptable QC sample results from
both matrix sets. In each case, their QC result was high
(>110%). As a result, the collaborator analyzed additional cali-
bration standards (which also reflected enhanced instrument
signal), recalibrated, and recalculated the results of their QC
sample and matrix spike samples with this revised curve. After
recalibration, the adjusted QC results were found within the
acceptance limits; however, the laboratory ranking test still
identified the Laboratory 4 reagent water data set as having a
systematic high bias, averaging 126% recovery.
Laboratory 6 reported low QC sample results from each ma-
trix set. The collaborator decided not to reanalyze the QC sam-
ple or recalibrate the instrument. The laboratory ranking proce-
dure identified the data from both matrix sets as outliers with a
systematic low bias and the data were removed from the study.
Laboratory 15 reported an unacceptable QC sample result
after the water-of-choice matrix set. The collaborator recali-
brated their instrument with fresh standards, reanalyzed a QC
sample that met acceptance limits, and reanalyzed the entire set
of spiked water-of-choice matrix samples. The spiked matrix
water data passed the ranking test and were included in the
study.
Rejection of Outliers
No laboratory data sets were removed from the study prior
to outlier testing. For the entire study, the IMVS computer pro-
gram rejected 88 (27.0%) of the 326 data points submitted, ex-
clusive of “nondetect” responses. Ninety percent of rejected
data in this study came from the laboratory ranking test. In the
reagent water data set, the laboratory ranking test rejected
4 laboratories for having systematic low bias and 2 laboratories
for having systematic high bias. Data received from Laborato-
ries 16 and 22 were removed from the reagent water data set by
the laboratory ranking test despite mean Cr(VI) recoveries
across the 8 concentration levels of 89% and 92%, respectively.
In the matrix water data set, the laboratory ranking test re-
jected 3 laboratories for having systematic low bias and 2 labo-
ratories for having systematic high bias. All of these 5 labora-
tories had mean Cr(VI) biases greater than ±10% of the true
value.
Observations from previous U.S. EPA interlaboratory stud-
ies indicate that the laboratory ranking test in ASTM D-2777-
86 is very powerful in identifying laboratories with systematic
biases even if the submitted data are close to the expected val-
ues, e.g., Laboratories 16 and 22. This is particularly true when
there is a core set of retained data which are very precise as seen
in this study. However, the large percentage of rejected labora-
tories should not be equated with poor method performance.
On the contrary, this method exhibited good accuracy and pre-
cision.
The “detection-level” Youden pair reagent water samples
were evaluated separately from the IMVS computer program.
Before the statistics were calculated, the laboratories identified
as outliers by the laboratory ranking procedure were removed
from the main study reagent water data set. The majority of the
data labeled for removal by the laboratory ranking test were
already marked as “nondetect” data by the collaborators.
Thompson’s test for individual outliers was then performed at
the 95% confidence interval, which identified only 1 additional
outlier.
Method Performance
Dionex instrumentation, AS-7 columns, and 250 mM am-
monium sulfate–100 mM ammonium hydroxide eluant were
used by 18 of the 21 participating laboratories. One laboratory
used a Dionex instrument with a CS-5 column with 10 mM
pyrridine 2,6-dicarboxylic acid, and 148 mM NH4OH. The re-
maining 2 laboratories used Waters LC instruments, IC-Pak
Anion HC columns, and 25.0 mM ammonium sulfate/10.0 mM
ammonium hydroxide as the eluant. There were no apparent
differences in the returned data sets among the 3 types of instru-
mentation and conditions used by the 21 laboratories.
No laboratories reported background Cr(VI) concentrations
in their reagent water; however, Laboratories 6, 10, 14, and 15
did report background Cr(VI) concentrations in their matrix
water blanks. Laboratory 6 reported the highest background
Cr(VI) concentration of 8.51 µg/L in their drinking water and
background correction made no improvement in the data. In
fact, the entire Laboratory 6 drinking water data set was re-
jected by the laboratory ranking test. Laboratory 15 reported a
background concentration of 3.76 µg/L which was inconsistent
with the rest of the matrix water data results. Recovery correc-
tion, using the results for the apparently contaminated unspiked
sample, was judged to be inappropriate. Laboratories 10 and
14 reported background levels around 1.5 µg/L, which was
compatible with their spiked matrix water data. Background
corrections were made to the study data for these 2 laboratories
before statistical outlier testing. The background-corrected data
and summary statistics for the wastewater samples are pre-
sented in Table 1.
 A slightly high bias was observed in the reagent water re-
coveries across all 8 concentration levels (Table 2). This bias
was negligible for the 4 highest concentration levels. The aver-
age percent recoveries of 101–102% were well within the ex-
pectations of the method. However, the positive bias continued
to increase as the concentration level decreased until a percent
recovery of 125% was reached for the lowest concentration
level of 1.2 µg/L (Table 3). The data reported for the lowest
concentration pair appeared to be of 2 types: data with recover-
ies averaging around 106% and data with recoveries greater
than 130%.
An explanation for the 2 types of data was sought in the
questionnaires returned by each participating laboratory. It was
discovered that the laboratories could also be divided into
2 groups according to the approach used in the preparation of
the calibration curve. Calculation packages supported by
Dionex Instruments provide the option to calibrate the instru-
ment by fitting a least squares linear regression equation to a
series of standards. Such a regression can be useful over a nar-
row concentration range, but since it tends to be dominated by
the high concentration members of the data set, it becomes rela-
tively inaccurate the further it is extended, particularly if there
is any nonlinearity in the calibration curve. One group of labo-
ratories used the regression option to prepare a single calibra-
tion curve in the range 1–1000 µg Cr(VI)/L; the other group
used manual calibration curves or prepared shorter range cali-
bration curves by using the linear regression option, typically
1–100 µg Cr(VI)/L and 100–1000 µg Cr(VI)/L. This latter
group reported data much closer to the true value than the group
using a 3 orders of magnitude calibration curve (Table 4). The
broad-range linear regression produced relatively large errors
in the estimates of analyte concentration at the lowest concen-
tration range of the calibration curve. The statistical data (Ta-
ble 4) illustrate these errors and suggest, by the positive bias,
that calibration curves are not linear over 3 orders of magni-
tude, and that the positive bias in the reagent water data set is
correctable by proper calibration.
The overall standard deviation (sR) is the precision associ-
ated with measurements generated by a group of laboratories.
Single-analyst standard deviation (sr) is the precision associ-
ated with performance in an individual laboratory. The
weighted linear regression equations (Table 5) describe method
precision as a function of mean recovery. The CODw calculated
for these weighted equations show them to be representative of
the submitted data sets and acceptable for use in estimating the
precision at any concentration value within the range tested.
The between-laboratory method precision ranged 2–15%
for the individual concentration levels in reagent water and 4–
26% for the same concentrations in the waters-of-choice.
Above 100 µg Cr(VI)/L, the precision was less than 5% in
spiked reagent waters and less than 8% in spiked matrix waters.
Below 20 µg Cr(VI)/L, the precisions ranged from 10 to 15%
in spiked reagent water and 10 to 26% in spiked matrix water.
The within-laboratory precision ranged 1.5–7% for the spiked
reagent water matrix data and 3–11% for the spiked waters-of-
choice data.
Weighted least squares linear regression equations over this
concentration range are presented in Table 5. The coefficients
of determination (CODw) calculated for the weighted linear re-
gression equations (Table 5) were greater than 0.999, which
confirm the suitability of these equations for estimating the
mean recovery and precision at any concentration level within
the study range.
Effect of Water Type
A wide variety of matrix waters was used in this study. The
treated wastewaters included 1 primary effluent, 6 secondary
effluents, and 4 unspecified effluents. The untreated waste-
waters included flyash pond, laboratory, lift station, and animal
storage bin wastewaters. Other waters tested included
1 groundwater and 3 finished drinking water sources. The mis-
cellaneous matrixes included a seawater and a Toxicity Char-
acteristic Leaching Procedure (TCLP) leachate, both of which
were rejected by the laboratory ranking test. The laboratory us-
ing seawater had recoveries ranging 40–60% across all concen-
tration levels. This laboratory reported that the chromate ion
peak had split in the seawater matrix, apparently due to the in-
creased ionic strength of the sample matrix. The laboratory us-
ing TCLP leachate could not detect the 6.0 or 8.0 µg Cr(VI)/L
concentration of spike levels; had recoveries of <50% for the
16 and 20 µg Cr(VI)/L spike levels; and had recoveries 80–
90% for the spike levels 100–960 µg Cr(VI)/L. Their reagent
water data set was very good with no data removed as outliers.
Although this laboratory had difficulties with the TCLP
leachate, there is insufficient data to conclude that the matrix
alone was the cause of the poor data.
The reagent water and the water-of-choice data sets were
subjected to an analysis of variance (ANOVA) test to determine
the effect of water type on recovery and precision. The ANOVA
test detected a statistically significant matrix effect on spike
recovery at the 95% confidence interval for the combined
water-of-choice samples compared with the reagent water sam-
ples. The effect was observed as slightly lower mean recoveries
from the spiked matrixes at the 4 higher concentration levels,
compared with the spiked reagent water samples. As the con-
centration levels decreased, the mean recoveries from the
spiked matrixes decreased steadily to a low of 91% while the
mean recoveries from the spiked reagent water increased to
about 110%. The matrix effect on bias observed in this study is
considered to be of practical significance at the lower concen-
trations studied.
Method Detection Limit
U.S. EPA defines the method detection limit (MDL) as the
minimum concentration of a substance that can be measured
and reported with 99% confidence that the analyte concentra-
tion is greater than zero (7). Calculation of the MDL requires
the analyses of a minimum of 7 replicate spiked reagent water
samples at a concentration of 2–5 times the estimated MDL
reported in the method. In this study, the concentration levels
of the “detection-level” concentration pair, 1.2 and 1.6 mg/L,
were 3–4 times the single laboratory MDL of 0.4 µg/L cited for
U.S. EPA method 218.6. Of the 21 laboratories submitting data
for these samples, 5 reported “nondetect” at these spike levels
which indicates the closeness of the samples to the MDL value.
After outlier removal following AOAC guidelines (8), the re-
maining 13 paired analyses (see Table 3) were used to calculate
the within-laboratory standard deviation (sr) at the average ana-
lyte concentration of 1.4 µg/L. The calculated sr, 0.15 µg/L,
was used in the following equation to calculate MDL:
MDL = t(df, α = 0.99) [sr]
MDL = (2.65)(0.15)at 13 degrees of freedom
MDL = 0.40 µg/L
This interlaboratory MDL supports the single-laboratory
MDL reported in U.S. EPA method 218.6 and the number of
acceptable data points reported at the 1.2 µg/L concentration
level supports the applicability of this method to low level
Cr(VI) analyses, approaching 0.4 µg/L, as long as a short range
calibration curve is used.
Performance-Based QC Acceptance Limits
Since the weighted linear regression equations for precision
were found to be representative of the submitted data sets, they
can be used to derive performance-based QC limits for this
method. To establish performance-based QC limits for use with
a QC sample, estimates of the within-laboratory standard de-
viation were calculated for Cr(VI) spike level of 40 µg using
the reagent water weighted regression equations reported in Ta-
ble 5. At this concentration, the mean recovery (X) is estimated
to be 40.0 µg/L as the slight (3%) positive bias was attributed
to correctable calibration discrepancies. The within-laboratory
standard deviation (sr) at 40.0 µg/L is estimated to be
1.22 µg/L. The QC acceptance limits, at the 99% confidence
level, were calculated as:
QC acceptance limits = X ± 3sr
= 36.3–43.7 µg/L
These performance-based QC acceptance limits support the
continued use of the acceptance limits used in this study, 36–
44 µg/L (90–110% recovery). Eighteen of the 21 laboratories
had no trouble meeting the study QC acceptance limits of
±10%. One additional laboratory was able to meet the accep-
tance limits upon recalibration and reanalysis of the QC sam-
ple. The high quality of the returned data in this study supports
the applicability of these acceptance limits.
Conclusion
U.S. EPA method 218.6 was shown to be accurate and pre-
cise for the analysis of hexavalent chromium in waters and vari-
ous matrix waters in a collaborative study involving 21 labora-
tories. The equations presented for method recovery, overall
standard deviation, and single-analyst standard deviation can
be used to estimate method performance at any concentration
value within the study range.
The statistically significant matrix effects, identified by the
ANOVA test, were considered to be of practical significance for
the water-of-choice matrixes as a group. Recoveries of spikes
from matrix waters were lower at all concentration levels when
compared to recoveries of spikes from reagent water samples.
It is recommended that linear regression calibration curves
be limited to 1–2 orders of magnitude. Using least squares lin-
ear regression analysis to fit calibration data over 3 orders of
magnitude tends to produce very large biases at the lower con-
centration levels. The method was very accurate and precise for
chromium Cr(VI) concentration levels as low as 1.2 µg/L, as
long as a short range calibration curve (e.g., 1–100 µg/L) was
used to quantify the sample data.
The method study data were suitable for the development of
performance-based QC limits. It is recommended that users of
this method routinely test a QC sample prepared in reagent
water and compare the results with the performance-based ac-
ceptance limits derived from this study. The calculated QC ac-
ceptance limits using the IMVS estimates fully supported the
±10% limits imposed on the participants of this study.
The outlier procedures employed in this study, which re-
jected 27% of the submitted data, appear to have rejected a sig-
nificant amount of data that would be judged acceptable by
application of normal QC acceptance criteria, and should be
further evaluated.
Two laboratories that produced data of apparently good
quality were rejected by the laboratory ranking test. Although
the overall study rejection rate exceeds the 2/9 (22%) limit sug-
gested by AOAC guidelines (8), it does not mean that the
method or the study is flawed. Because of the rejection of these
2 laboratories, the conclusion should be made that the labora-
tory ranking outlier procedure can be a little too critical when-
ever the single-analyst standard deviation is generally so small.
It is recommended that research be conducted into the
unique characteristics of seawater and TCLP leachate in rela-
tion to Cr(VI) analyses using this method. The laboratories us-
ing these 2 matrixes produced low biased data which were re-
jected by the laboratory ranking test.
The MDL of the method was confirmed by the IMDL to be
0.40 µg/L. The use of interlaboratory single-analyst precision
to evaluate MDLs should continue. These studies provide a
broad-based assessment of MDLs and give a reliable estimate
of how well an analytical laboratory can be expected to perform
using the method. In addition to low concentration detection-
level studies in reagent water, studies should also be considered
where practical with other matrixes (i.e. wastewater, ground-
water, leachate, etc.) at these low levels.
Recommendation
On the basis of the results of this study it is recommended
that the ion chromatographic method for determination of dis-
solved hexavalent chromium in drinking water, groundwater,
and industrial waste water effluents be adopted first action by
AOAC.
Acknowledgments
The authors thank the following:
Elizabeth Arar, Technology Applications, Inc., Cincinnati, OH
 Timothy Arnold, American Electric Power, Columbus, OH
John Capito, San Diego Gas & Electric, San Diego, CA
Robert Dovi, Waste Management, Inc., Geneva, IL
Dominic Dragotta, DuPont, Newark, DE
Peter Grohse, Research Triangle Institute, Research Trian-
gle Park, NC
Peter Jackson, Waters, Milford, MA
Robert Joyce, Dionex Corp., Sunnyvale, CA
Barbara Mitchell, Tennessee Valley Authority, Muscle
Shoals, AL
Jack Northington, West Coast Analytical Services, Sante Fe
Springs, CA
William Pratt, ENSECO/RMAL, Arvada, CO
Raimund Roehl, California Department of Health Services,
Berkeley, CA
Christopher Scutt, DOFASCO, Inc., Hamilton, ON, Canada
John Sherwell, Radian Corp., Austin, TX
Chris Shoop, Tennessee Eastman Co., Kingston, TN
Thomas Surveski, Travelers Insurance Co., Hartford, CT
Dave Tucker, City of San Jose WPCP, San Jose, CA
Vincent White, Department of Environmental Resources,
Harrisburg, PA
F.W. Williams, Manville, Littleton, CO
William Wolf, Carolina Power & Light Co., New Hill, NC
John Stanley, Midwest Research Institute, Kansas City, MO
The authors thank Edward Katz for providing analytical
support for the study and John Ortman for IMVS computer
processing.
References
(1) Standard Methods for the Examination of Water and Waste-
water (1989), 17th Ed., APHA, AWWA, WPCF, Washington,
DC
(2) Annual Book of ASTM Standards (1990), “Chromium-Total”,
D 1687-86, Vol. 11.02, Water, American Society for Testing
and Materials, Philadelphia, PA
(3) Youden, W.Y., & Steiner, E.M. (1975) Statistical Manual of
the AOAC, AOAC, Arlington, VA, p.88
(4) Annual Book of ASTM Standards (1990) “Standard Practice
for Determination of Precision and Bias of Applicable Meth-
ods of Committee D-19 on Water”, ASTM D2777-86, Vol.
11.01, Water (1), American Society for Testing and Materi-
als, Philadelphia, PA
(5) Barnett, X., & Lewis, Y., (1984) Outliers in Statistical Data,
John Wiley and Sons, New York, NY
(6) Draper, N., & Smith, H. (1968) Applied Regression Analysis,
John Wiley and Sons, New York, NY
(7) Code of Federal Regulations, Chapter 40, Part 136, appendix
B, Revision 1.11, Government Printing Office, Washington,
DC
(8) “Guidelines for Collaborative Study Procedures to Validate
Characteristics of a Method of Analysis” (1989) J. Assoc.
Off. Anal. Chem. 72, 694-704
Table 993.23. Method performance for determination
of dissolved hexavalent chromium in drinking water,
groundwater, and industrial wastewater effluents by ion
chromatographic methoda
Mean
recovery,
Spike, µg/L µg/L sr sR RSDr, % RSDR, %

Reagent Water

1.2 1.50
1.6 1.87 0.15 0.57 9.0 33.8
6 6.68
8 8.64 0.53 1.06 6.9 13.9
16 17.4
20 21.4 0.77 2.28 4.0 11.8
100 101
140 143 3.8 4.3 3.1 3.5
800 818
960 966 12.7 21.2 1.4 2.4

Drinking water, ground water, and industrial wastewaters

6 5.63
8 7.31 0.55 1.20 8.2 17.8
16 15.1
20 19.8 1.85 1.85 10.4 10.4
100 98.9
140 138 3.3 4.8 2.8 4.0
800 796
960 944 27.1 66.6 3.1 7.6
a
Samples analyzed as Youden pairs.
Table 1. Background-corrected data and summary statistics for collaborative study to determine hexavalent
chromium in wastewater, µg Cr(VI)/L
True value

Lab. Matrix type 6.00 8.00 16.0 20.0 100 140 800 960

1 2° Effluent 6.42 8.26 15.6 20.6 103 138 773 930

2 Flyash pond 7.35 8.84 17.1 19.5 97.5 136 783 945
a
4 Seawater 2.77 3.45 8.88 8.88 5.05 83.2 459 558
b
5 Ground water 0.00b 2.36 8.53 21.0 101 142 798 952
a c c
6 Drinking water — 0.94 — — — 26.1 245 123
7 Plant effluent 7.48 9.74 18.0 21.8 99.9 148 798 906
8 Plant effluent 4.21 8.46 18.2 19.4 96.4 145 802 1002
a
9 Plant effluent 9.00 10.7 17.0 22.0 105 148 810 954
10 Animal bin 5.87 8.61 16.1 20.4 102 143 832 1019
b
11 Lab. wastewater 111b 4.76 13.9 b
10.3b b
186b 121 706 763
12 1° effluent 6.10 8.57 16.2 19.8 96.8 141 798 949
14 Cold mill waste 4.60 7.60 15.2 20.0 96.4 144 816 931
b
15 Lift station 3.28 4.64 9.14 12.2b b
69.3b 119 898 996
16 Clarifier effluent 4.73 7.18 15.0 18.8 96.7 136 756 910
17 Drinking water 5.86 7.58 15.0 18.8 98.3 135 778 927
18 2° effluent 5.78 77.70 16.7 20.4 106 147 846 1020
19 2° effluent 6.53 8.38 15.8 19.0 103 142 879 1060
a
20 TCLP leachate <2.7 2.7 5.04 9.18 81.3 119 698 863
21 2° effluent 5.46 7.87 15.8 19.0 100 134 830 959
22 2° effluent 5.18 6.43 16.0 18.1 87.7 131 649 840
a
23 Drinking water 7.01 9.34 18.6 21.5 116 162 967 1090
Mean (X) 5.63 7.31 15.1 19.8 98.9 138 796 944
Recovery, % 93.4 91.4 94.6 98.8 98.9 98.63 99.6 98.4
SR 1.17 1.91 2.70 1.01 4.36 8.39 60.6 72.1
RSDR, % 20.8 26.1 17.8 5.1 4.4 6.1 7.6 7.6
sr 0.55 1.85 3.31 27.1
RSDr, % 8.6 10.6 2.8 3.1
a
Data removed by Youden’s laboratory ranking test.
b
Data removed by Thompson’s individual outlier test.
c
Not detected.
Table 2. Raw data and summary statistics for collaborative study to determine hexavalent chromium in reagent
water, µg Cr(VI)/L
True value

Lab. 6.00 8.00 16.0 20.0 100 140 800 960

1 6.78 9.03 16.9 21.6 101 142 802 961

2 7.96 9.13 22.1 25.9 102 139 813 983
a
4 7.72 10.6 22.4 28.8 108 168 1001 1098
5 6.63 9.51 18.9 21.8 102 141 805 972
a b b
6 — 10.0 — — 70.9 98.9 289 328
7 8.96 10.4 18.9 21.5 104 144 782 948
8 5.49 7.38 16.1 18.3 93.9a 143 820 973
9 5.93 8.13 15.7 20.7 99.7 136 797 938
10 6.33 8.78 16.6 21.1 99.1 148 816 983
a
11 4.67 6.38 15.9 17.3 63.7 134 1.94 856
12 7.82 7.82 16.1 19.6 102 143 820 956
14 5.70 7.66 15.8 22.4 101 147 858 998
a
15 8.00 9.97 17.3 21.6 108 166 910 1050
a
16 1.93 7.38 15.5 19.2 99.4 140 775 934
17 6.28 8.23 15.8 19.7 102 141 807 962
18 5.83 7.62 16.2 20.1 97.1 142 816 938
19 6.05 7.87 15.9 20.6 103 149 849 1055c
20 8.00 11.3 15.4 19.1 99.4 131 796 965
21 6.03 8.09 22.3 26.8 134c c
191c 826 956
a
22 5.37 7.74 14.8 19.2 93.1 131 695 874
23 6.41 8.61 17.9 21.1 103 153 871 991

Mean (X) 6.68 8.64 17.4 21.4 101 143 818 966
Recovery, % 111 108 108 107 101 102 102 101
sR 1.03 1.10 2.25 2.31 1.91 5.52 24.3 18.5
RSDR, % 15.4 12.7 12.9 10.8 1.9 3.9 3.0 1.9
sr 0.53 0.77 3.76 12.7
RSDr, % 6.9 4.0 3.1 1.4
a
Data removed by Youden’s laboratory ranking test.
b
Not detected.
c
Data removed by Thompson’s individual outlier test.
Table 3. Raw data and summary statistics for
detection-level concentration Youden pair, µg Cr(VI)/L
Calibration True value, True value, Difference,
Lab curve range 1.20 µg/L 1.60 µg/L µg/L

1 long 1.69 2.14 0.45

2 short 1.33 1.63 0.30
a,b a,b a,b a,b
4 long — — —
5 long 2.21 3.17 0.96
a,b a,b b
6 long — 10.4b —
7 long 2.64 3.10 0.46
8 short 1.02 1.13 0.11
9 short 1.08 1.35 0.27
10 long 1.58 1.71 0.13
b
11 short 1.65b 13.6b —
12 long 1.44 1.68 0.24
c
14 long 3.80c a a
— —
a,b a,b a,b a,b
15 short — — —
b
16 long 0.68b 0.73b —
17 short 1.45 1.89 0.44
18 long 0.90 1.31 0.41
19 short 1.86 2.16 0.30
a a a a
20 long — — —
21 short 1.28 1.54 0.26
a,b a,b a,b a,b
22 long — — —
23 short 1.08 1.56 0.48
d
Mean 1.50 1.87 —
% Recovery 125 117 —
Overall sR 0.50 0.63 —
e
Single-analyst, sr — — 0.15
RSDR, % 33 34 —
f
RSDr, % — — 8.9
a
Not detected.
b
Laboratories rejected on basis of Youden laboratory ranking test
applied to data in 6–960 µg/L range were also rejected here.
c
Rejected data by Thompson’s individual outlier test.
d
Mean values calculated for 13 retained values only.
e
Single-analyst standard deviation, sr, calculated as standard
2 .
deviation of the differences/√
f
Single-analyst relative standard deviation calculated as sr / mean
of pair.
Table 4. Comparison of low-level Youden pair
summary statistics quantified using “short range”
versus “long range” calibration curves
Short range Long range
Statistic calibration curvea calibration curveb

True value,
µg/L 1.20 1.60 1.20 1.60
Number of
pairs 7 7 6 6
Mean
recovery 1.30 1.61 1.74 2.18
Recovery, % 108 101 145 136
Std deviation 0.29 0.34 0.61 0.78
RSD, % 24 21 35 36
a
Short range calibration curves typically 1–100 µg Cr(VI)/L.
b
Long range calibration curves were 1–1000 µg Cr(VI)/L.
Table 5. Weighted linear regression equations
calculated from collaborative study statistics for
chromium in reagent water and matrix watersa
Reagent water, Matrix water,
Statistic 6–960 µg/L 6–960 µg/L

Mean recovery X = 1.020C + 0.592 X = 0.989C − 0.411

Between-laboratory
standard deviation sR = 0.035X + 0.893 sR = 0.059X + 1.055
Within-laboratory
standard deviation sr = 0.021X + 0.375 sr = 0.041X + 0.393
a
X = mean recovery, µg/L; C = true value, µg/L; sR = reproducibility
standard deviation, µg/L; sr = repeatability standard deviation,
µg/L.