600-4-85-021
DETERMINATION OF BENDIOCAR3 IN INDUSTRIAL
AND MUNICIPAL WASTEWATERS
J.S. Warner, T.M. Engel and P.J. Mondron
Bactelle Columbus Laboratories
Columbus, Ohio 43201
Contract No. 68-03-2956
Project Officer
Thomas Prassley
Physical and Chemical Methods Branch
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45263
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45263
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring and
Support Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents necessarily
reflect the view and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
ii
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FOREWORD
Environmental measurements are required to determine the quality
of ambient waters and the character of waste effluents. The
Environmental Monitoring and Support Laboratory - Cincinnati, conducts
research to:
• Develop and evaluate methods to measure the presence and
concentration of physical, chemical, and radiological
pollutants in water, wastewater, bottom sediments, and solid
waste.
• Investigate methods for the concentration, recovery, and
identification of viruses, bacteria and other microbiological
organisms in water; and, to determine the responses of aquatic
organisms to water quality.
• Develop and operate an Agency-wide quality assurance program
to assure standardization and quality control of systems for
monitoring water and wastewater.
• Develop and operate a computerized system for instrument
* automation leading to improved data collection, analysis, and
quality control.
This report is one of a series that investigates the analytical
behavior of selected pesticides and suggests a suitable test procedure
for their measurement in wastewater. The method was modeled after
existing EPA methods being specific yet as simplified as possible.
Robert L. Booth, Director
Environmental Monitoring and Support
Laboratory - Cincinnati
ill
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ABSTBACT
A method was developed for the determination of bendiocarb in
wastewaters. The method development program consisted_of a literature
review; determination of extraction efficiency for the compound from
water into methyLane chloride; development of a deactivated Florisil
cleanup procedure; and determination of suitable liquid chromatographic
analysis conditions.
The final method was applied to a relevant industrial wastewater in
order to determine the precision and accuracy of the method. The
wastewater was spiked wijrh _the compound at levels of 8.0 ug/L and
80 ug/L. Recovery for bendiocarb at the 8 ug/L level was 65 ± 24
percent. Recovery at the 80 ug/L level was 70 ± 4 percent. The method
detection limit (MDL) for bendiocarb in distilled water was 1.8 ug/L.
The MDL in wastewaters may be higher due to interfering compounds.
This report was submitted in partial fulfillment of Contract No.
68-03-2956 by Battelle Columbus Laboratories under the sponsorship of
the U.S. Environmental Protection Agency. This report covers the
period from February 1, 1982 to April 30, 1982.
iv
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CONTENTS
Foreword
Abstract ............................... iv
Figures ............................... vi
Tables .............................. • vii
1. Introduction ....... ' ................. 1
2. Conclusions ......... . ............... 2
Extraction and Concentration .............. 2
Cleanup ...... . ................. 2
Chrooatography ............ „ ......... 2
Validation Studies ................... 2
3 . Experimental ........ ................ 3
Extraction and Concentration .............. 3
Cleanup ... .................. ... 3
Chromatography ..................... 3
Validation Studies ................... 4
4. Results and Discussion ................... 5
Extraction and Concentration .............. . 5
Cleanup ........................ 5
Chromatography ..................... 5
Validation Studies ................... 8
References .............................. 10
Appendix
A. Bendiocarb Method 639 .................... 11
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FIGURES
Number Page
1 HPLC-UV Chromatogram of 10 ng of Bendiocarb 6
2 HPLC-OV Chromatogram of 600 ag of Bendiocarfa 7
3 Analytical Curve for Bendiocarb 9
vi
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TABLES
Number Page
1 Analytical Curve Data for Bendiocarb 8
vii
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SECTION 1
INTRODUCTION
Bendiocarb (I) is an insecticide acting by cholinesterase
inhibition and is effective as a contact and stomach poison. The acute
oral LD50 for rats is 180 rag/kg, which indicates moderate toxicity.
(I)
The CAS registry number for bendiocarb is 22781-23-3 and the CAS name
for bendiocarfa is 2,2-dimethyl-l,3-benzodioxol-4-yl methylcarbamate.
It has a melting point of 129-130 C. A synonym used for bendiocarb is
"Ficam". A GC method of analysis using a modified alkali flame
detector has been reported (L) as well as gas chromatographic.(GC)
pyrolysis and GC derivatization methods (2). It has been reported that
bendiocarb is thermally unstable and an high performance liquid
chromatographic (HPLC) method has been developed using reverse phase
chromatography (3).
Bendiocarb can be extracted from water with methylene chloride.
The selected approach to its determination in water included separatory
funnel extraction from water with mehylene chloride, cleanup using
Florisil chromatography, and analysis using HPLC with ultraviolet
detection (DVD). Standard concentration techniques using
Kuderna-Danish (K-D) equipment were used. The final method is included
in Appendix A of this report.
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SECTION 2
CONCLUSIONS
EXTRACTION AND CONCENTRATION
Bendiocarb can be extracted from water into methylene chloride
with greater than 85 percent recovery using separatory funnel
techniques. Use o£ K-D concentration equipment to perform extract
concentrations did not significantly affect compound recoveries.
CLEANUP
Bendiocarb elutes from deactivated Florisil with greater than 80
percent recovery. This was an effective cleanup procedure for a
relevant wasCewater sample.
CHROMATOGRAPHY
Two HPLC columns, Spherisorb-ODS and Lichrosorb RP-2, were found
to be acceptable for this application. The Spherisorb-ODS column»was
used as the primary column. The Lichrosorb RP-2 column was designated
as the alternate column.
VALIDATION STUDIES
Recoveries of bendiocarb from distilled water in the 4 to
1000 yg/L concentration range averaged greater than 80 percent. The
analytical curve constructed from this data was linear. The MDL in
distilled water was 1.8 ug/L. Recoveries of bendiocarb from a relevant
industrial wastewater at the 8 and 80 ug/L levels were 64 i 24 and
70 ± 4 percent, respectively.
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SECTION 3
EXPERIMENTAL
Studies were performed to determine if extractions with separatory
funnels, cleanup by Florisil adsorption chromatography, concentration
using K-D equipment, and analysis using HPLC with UVD would be
applicable techniques for the determination of bendiocarb in water.
EXTRACTION AND CONCENTRATION
Extraction of bendiocarb from water by separatory funnel
techniques was studied. One liter of distilled water was used. The
distilled water was spiked with bendiocarfa at the 20 Ug/L and 100 ug/L
levels. The samples were adjusted to pH 7 by the addition of 1 N
suIfuric acid or 1 £ sodium hydroxide and extracted three times with
60 mL each of methylene chloride. These studies were done in
duplicate. The extracts were dried by passing them through 10 cm of
anhydrous granular sodium sulfate and concentrated to two mL. The
samples were solvent exchanged with acetonitrile by concentrating the
sample to five mL after the addition of 15 mL of acetonitrile and
analyzed by HPLC.
CLEANUP
A 10-gram Florisil column (deactivated with water saturated methylene
chloride) was prepared and eluted with 100 mL of 50 percent methylene
chloride in petroleum ether. The bendiocarb, 20 yg dissolved in one mL of
methylene chloride, was added to the top of the column. The column was
eluted with 50 mL portions of 50 percent methylene chloride in petroleum
ether (Fl), methylene chloride (F2), five percent acetone in methylene
chloride (F3), 15 percent acetone in methylene chloride (F4), and 25
percent acetone in methylene chloride (F5). Each fraction was
concentrated to 1 mL. The fractions were solvent exchanged to
acetonitrile by reconcentrating them to 2 mL after the addition of 10 mL
of acetonitrile.
CHROMATOGRAPHY
Two reversed phase HPLC columns were evaluated for the determination
of bendiocarb: Spherisorb-ODS and Lichrosorb RP-2.
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VALIDATION STUDIES
The MDL for bendiocarb was determined by analyzing seven replicate
distilled water samples spiked at the 4 yg/L concentration level. The
sample extracts were cleaned up using the Florisil cleanup procedure
prior to analysis. The amounts recovered were determined by external
standard calibration and the MDLs were calculated from these data.
Distilled water was also spiked in duplicate at the 4, 20, 100,
250, and 1000 yg/L concentration levels and recoveries of the
bendiocarfa were determined as described earlier. An analytical curve
was generated by plotting the amount spiked into the samples versus the
amount recovered from the samples.
A relevant industrial wastewater (obtained from a bendiocarb manu-
facturing site) was used for wastewater validation studies. Seven replicates
of the wastewater were analyzed to determine the background levels. The
wastewater was spiked with bendiocarfa at the-8 and 80 yg/L concentration
levels, processed and analyzed. Seven replicate extractions were performed
at each concentration level.
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SECTION 4
RESULTS AND DISCUSSION
EXTRACTION AND CONCENTRATION
Bendiocarb -as extracted from water with greater than 35 percent
recovery using separatory funnel techniques. Recovery of bendiocarb from
water using separatory funnels were 32 and 96 percent at 20 ug/L and 91
and 93 percent at 100 jig/L. These data are the results of duplicate
analyses.
CLEANUP
Bendiocarb eluted from deactivated Florisil in the 5 percent acetone
in methylene chloride fraction (F3). Recovery of 20 yg of bendiocarb was
82 percent.
CHROMATOGRAPHY
Both the Spherisorb-ODS and Lichrosorb RP-2 columns were satisfactory
for the determination of bendiocarb. The Spherisorb-ODS column was chosen
as the primary column. The following conditions were used:
Column: Spherisorb-ODS, 5 micron,
250 x 4.6 mm
Solvent.: 40 percent acetonitrile/
60 percent water
Flow: 1 mL/min
Detector: UV (§254 nm
Injection Volume: 5 uL
Retention Time: 9.3 min
A chromatogram obtained under these conditions is shown in Figure 1.
The Lichrosorb RP-2 column was chosen as the alternate column. The
following conditions were used:
Column: Lichrosorb RP-2, 5 micron,
250 x 4.6 mm
Solvent: 50 percent acetonitrile/
50 percent water
Flow: 1 mL/min
Detector: UV (§254 nm
Injection Volume: 5 uL
Retention Time: 6.0 min
A chromatogram obtained under these conditions is shown in Figure 2.
5
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•
•H
•
o\
*•>
-
^^A.
2.
I x
1 Y
0 4.0 6.0
Bendlocarb
*
8.0 10.0 12.0 14.0 16.0 18.0 20.0
Retention Time, Min.
Figure 1. HPLC-UV Chromatogram of 10 ng of Bendiocarb (Column 1)
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Bendiocarb
"I •-• . -i~|—r-i—i • | --•-
4.0 6.0 8.0
H.O
16.0
10.0 12.0
Retention Time, Min.
Figure 2. HPLC-UV Chromatogram of 600 ng of Bendiocarb (Column 2)
' I • ' ' • 1
18.0 20.0
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VALIDATIONS STUDIES
Recovery of bendiocarb from distilled water at the 4.0 ug/L level
was 3.5 ± 0.6 ug/L. The MDL in distilled water was calculated to be
1.8 ug/L. Recoveries of bendiocarb from distilled water at the 4, 20,
100, 250, and 1000 ug/L levexs were 3.5, 17, 82, 200, and 810 yg/L,
respectively. These data were the averages of duplicate analyses.
Individual data points are given in Table 1. The resultant analytical
curve is shown in Figure 3.
TABLE 1. ANALYTICAL CURVE DATA FOR BENDIOCARB
Concentration, Amount Recovered,
Ug/L ug/L(a)
4 3.3, 3.7
20 16, 18
100 78, 86
250 190, 210
1000 770, 850
(a) Results of duplicate analyses.
Recoveries of bendiocarb from a relevant wastewater at the 8 and
80 ug/L levels were 65 ± 24 percent and 70 ± 4 percent, respectively.
These data were the averages of seven replicate analyses.
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1000 r
900 -
800 •
700
Amount Recovered,
600 •
500 -
400 •
300 •
200 •
100 '
100 200 300 400 500 600 700 800 900 1000
Amount Spiked ug/L
Figure 3. Analytical Curve for Bendiocarb
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REFERENCES
1. Richie, A.R., S.R. Solly, and M.H. Clear. The Application
of a Thermionic Detector to the Determination of Nitrogen-
Containing Compounds on Thin-Layer Chromatograms. J. ChromaEogr.,
150(2):557-561, 1978.
2. Whitcoak, R.J., J.B. Reary, and K.C. Overton. Bendiocarb.
In: Analytical Methods for Pesticides and Plant Growth Regulators,
Voll. 10, G. Zweig and J. Sherma, eds. Academic Press, New York,
San Francisco, and London, 1978. pp. 3-17.
3. Zehner, J.M., R.A. Simonaitis, and R.E. Bny. High Performance
Liquid Chromatographic Determination of Bendiocarb on Wool.
J. Assoc. Off. Anal. Chem.. 63(l):47-48, 1980.
10
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DETERMINATION OF BENDIOCARB IN MUNICIPAL AND INDUSTRIAL
WASTEWATERS BY LIQUID CHROMATOGRAPHY
METHOD 639
1. Scope and Application
1.1 This method covers the determination of bendiocarb pesticide. The
following parameter can be determined by this method:
Parameters CAS No.
Bendiocarb 22781-23-3
1.2 This is a high performance liquid chromatographic (HPLC) method
applicable to the determination of the compound listed above in
municipal and industrial discharges as provided under 40 CFR 136.1.
When this method is used to analyze unfamiliar samples for the
compound above, compound identifications should be supported by at
least one additional qualitative technique. This method describes
analytical conditions for a second liquid chromatographic column
that can be used to confirm measurements made with the primary
column.
1.3 The method detection limit (MDL, defined in Section 15) for
bendiocarb is listed in Table 1. The MDL for a specific
wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
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1.4 Any modification of this method beyond those expressly permitted,
shall be considered a major modification subject to application and
approval of alternate test procedures under 40 CFR 136.4 and 136.5.
1.5 This method is restricted to use by or under the supervision of
analysts experienced in the use of liquid chromatography and in
the interpretation of liquid chromatograms. Each analyst must
•
demonstrate the ability to generate acceptable results with this
method using the procedure described in Section 8.2.
2. Summary of Method
2.1 A measured volume of sample, approximately 1 liter, is solvent ex-
tracted with methyl ene chloride using a separatory funnel. The
methylene chloride extract is dried and exchanged to acetonitrile
during concentration to a volume of 2 ml or less. Liquid
chromatographic conditions are described which permit the
separation and measurement of the compounds in the extract by
HPLC-UV. (1) '
3. Interferences
3.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware that lead
to discrete artifacts or elevated baselines in liquid
chromatograms, ATI of these materials must be routinely
demonstrated to be free from interferences under the conditions of
the analysis by running laboratory reagent blanks as described in
Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. (2) Clean all
glassware as soon as possible after use by rinsing with the
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last solvent used in it. This should be followed by
detergent washing with hot water and rinses with tap and
disttlled water. It should then be drained dry, and heated
in a muffle furnace at 400°C for 15-30 minutes. Some thermally
stable materials such as PCBs may not be eliminated by this
treatment. Solvent rinses with acetone and pesticide
quality hexane may be substituted for the muffle furnace
heating. After drying and cooling, glassware should be
sealed and stored in a clean environment to prevent any
accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high purity reagents and solvents helps to mini-
mize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coex-
tracted from the sample. The extent of matrix interferences will
vary considerably from source to source, depending upon the nature
and diversity of the industrial complex or municipality being
sampled. Unique samples may require additional cleanup approaches
to achieve the MDL listed in Table 1.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the
lowest possible level by whatever means available. The laboratory
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is responsible for maintaining a 'current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified
in this method. A reference file of material data handling sheets
should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are
available and have been identified (3-5) for the information of the
analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber glass, 1-liter or 1-quart volume,
fitted with screw caps lined with Teflon. Foil may be
substituted for Teflon if the sample is not corrosive. If
amber bottles are not available, protect samples from light.
The container and cap liner must be washed, rinsed with
acetone or methylene chloride, and dried before use to
minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
silicone rubber tubing may be used. Before use, however,
the compressible tubing should be thoroughly rinsed with
methanol, followed by repeated rinsings with distilled water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow -
proportional composites.
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5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 2000-mL, with Teflon stopcock.
5.2.2 Drying column - Chromatographic column 400 mm long x 10 mm
ID with coarse frit.
5.2.3 Chromatographic column - 400 mm long x 19 mm ID with 250 mL
reservoir at the top and Teflon stopcock (Kontes K-420290 or
equivalent).
5.2.4 Concentrator tube, Kuderna-Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked
at the volumes employed in the test. A ground glass stopper
is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish - 500-mL (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2.6 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.7 Snyder column, Kuderna-Danish - two-ball micro (Kontes
K-569001-0219 or equivalent).
5.2.8 Vials - Amber glass, 5 to 10 mL capacity with Teflon lined
screw-cap.
5.3 Boiling chips - approximately 10/40 mesh carborundum. Heat to
400°C for 4 hours or extract in a Soxhlet extractor with methylene
chloride.
5.4 Water bath - Heated, capable of temperature control (_+2°C). The
bath should be used in a hood.
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5.5 Balance - Analytical, capable of accurately weighing to the
nearest 0.0001 g.
5.6 Liquid chromatograph - Analytical system complete with liquid
chromatograph and all required accessories including syringes,
analytical columns, detector and strip-chart recorder. A data
system is recommended for measuring peak areas.
5.6.1 Pump - Isocratic pumping system, constant flow.
5.6.2 Column 1 - Reversed-phase column, 5 micron Spherisorb-ODS,
250 x 4.6 mm or equivalent.
5.6.3 Column 2 - Reversed-phase column, 5 micron Lichrosorb RP-2,
250 x 4.6 mm or equivalent.
5.6.4 Detector - Ultraviolet absorbance detector, 254 nm.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6,2 Methylene chloride, methanol, acetonitrile, distilled-in-glass
quality or equivalent.
6.3 Sodium sulfate (ACS) Granular, anhydrous; heated in a muffle
furnace at 400°C overnight.
6.4 Sodium hydroxide, 1N_ - Prepare by adding 4 g of sodium hydroxide
to distilled water and diluting to 100 ml.
6.5 Sulfuric acid, 1N_ - Prepare by adding 2.8 ml of concentrated sulfuric
acid to distilled water and diluting to 100 ml.
6.6 Florisil - PR grade (60/100 mesh). Purchase activated at 1250°F
and store in a brown glass bottle. To prepare for use, place 150 g
in a wide-mouth jar and heat overnight at 160-170°C. Seal tightly
with Teflon or aluminum foil-lined cap and cool to room temperature.
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6.7 Stock standard solution (1.00 yg/uL) - Stock standard solution
can be prepared from pure standard materials or purchased as
certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing
about 0.0100 grams of pure material. Dissolve the material
in distilled-in-glass quality methanol and dilute to volume
in a 10-mL volumetric flask. Larger volumes can be used at
the convenience of the analyst. If compound purity is
certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock
standard. Commercially prepared stock standards can be used
at any concentration if they are certified by the
manufacturer or by an independent source.
6.7.2 Transfer the stock standard solution into Teflon-sealed
screw-cap bottles. Store at 4°C and protect from light.
Stock standard solution should be checked frequently for
signs of degradation or evaporation, especially just prior
to preparing calibration standards from them.
6.7.3 Stock standard solution must be replaced after six months
or sooner if comparison with quality control check standards
indicates a problem.
7. Calibration
7.1 Establish liquid chromatographic operating parameters equivalent to
those indicated in Table 1. The liquid chromatographic system can
be calibrated using the external standard technique (Section 7.2)
or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
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7.2.1 For the compound of interest, prepare calibration standards
at a minimum of three concentration levels by adding volumes
of one or more stock standards to a volumetric flask and
diluting to volume with acetonitrile. One of the external
standards should be at a concentration near, but above, the
method detection limit. The other concentrations should
»
correspond to the expected range of concentrations found in
real samples or should define the working range of the
detector.
7.2.2 Using injections of 2 to 5 uL of each calibration standard,
tabulate peak height or area responses against the mass
injected. 'The results can be used to prepare a calibration
curve for bendiocarb. Alternatively, the ratio of the
response to the mass injected, defined as the calibration
factor (CF), can be calculated at each standard concentration.
If the relative standard deviation of the calibration factor
is less than 10% over the working range, linearity through
the origin can be assumed and the average calibration factor
can be used in place of a calibration curve.
7.£.3 The working calibration curve or calibration factor must be
verified on each working day by the measurement of one or
more calibration standards. If the response for
bendiocarb varies from the predicted response by more
than ±10%, the test must be repeated using a fresh
calibration standard. Alternatively, a new calibration
curve or calibration factor must be prepared.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
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analytical behavior to bendiocarb. The analyst must further
deomonstrate that the measurement of the internal standard is
not affected by method or matrix interferences. Due to these
limitations, no internal standard applicable to all samples
can be suggested.
7.3.1 Prepare calibration standards at a minimum of three
concentration levels for bend.iocarb by adding volumes
of one or more stock standards to a volumetric flask.
To each calibration standard, add a known constant
amount of one or more internal standards, and dilute to
volume with acetonitrile. One of the standards should be at
a concentration near, but above, the method detection limit.
The other concentrations should correspond to the expected
range of concentrations found in real samples, or should
define the working range of the detector.
7.3.2 Using injections of 2 to 5 uL of each calibration standard,
tabulate the peak height or area responses against the
concentration for bendiocarb and internal standard.
Calculate response factors (RF) as follows:
RF • (AsC1s)/(A1sCs)
where:
As = Response for the compound to be measured.
A-jS » Response for the internal standard.
C-jS » Concentration of the internal standard in ug/L.
Cs * Concentration of the compound to be measured in
ug/L.
19
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If the RF value over the working range is constant, less
than, 10% relative standard deviation, the RF can be assumed
*
to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to
plot a calibration curve of response ratios, AS/A^S
against RF.
7.3.3 The working calibration curve or RF must be verified on each
working day by the measurement of one or more calibration
standards. If the response for bendiocarb varies from the
predicted response by more than _+10%, the test must be
repeated using a fresh calibration standard. Alternatively,
a new calibration curve must be prepared.
7.4- Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from the
reagents.
8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on
performance. The laboratory is required to maintain performance
records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
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with this method. This ability is established as described
in Section 8.2.
*
8.1.2 In recognition of the rapid advances occurring in chromato-
graphy, the analyst fs permitted certain options to improve
the separations or lower the cost of measurements. Each
time such modifications to the method are made, the analyst
is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each
* compound to be measured. Using stock standards, prepare a
quality control check sample concentrate in methanol 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipet, add 1.00 ml of the check sample concentrate
to each of a minimum of four 1000-mL aliquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
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Wastewater background corrections must be made before R and
s caJculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the
recovery and single operator precision expected for the
method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst
must review potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method
performance as follows:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in Section 8.2.3. The UCL
and LCL can be used to construct control charts (6) that are
useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method is defined as R ^ s.
The accuracy statement should be developed by the analysis
of four aliquots of wastewater as described in Section
8.2.2, followed by the calculation of R and s. Alternately,
the analyst may use four wastewater data points gathered
through the requirement for continuing quality control in
22
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Section 8.4. The accuracy statements should be updated
with,this method. This ability is established as described
*
regularly. (6)
8.4 The laboratory is required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10% of all samples or one sample
per month, whichever is greater. One aliquot of the sample must be
spiked and analyzed as described in Section 8.2. If the recovery
for a particular compound does not fall within the control limits
for method performance, the results reported for that compound in
all samples processed as part of the same set must be qualified as
described in Section 13.3. The laboratory should monitor the
frequency of data so qualified to ensure that it remains at or
below 5%.
8.5 Before processing any samples, the analyst should demonstrate
though the analysis of a 1-liter aliquot of reagent water that all
glassware and reagents interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a
laboratory reagent blank should be processed as a safequard against
laboratory contamination^
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
23
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doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as liquid chromatography with a
•
dissimilar column, must be used. Whenever possible, the laboratory
should perform analysis of standard reference materials arrt
participate in relevant performance evaluation studies.
9. Samples Collection. Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices (7) should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of Tygon and other potential sources of
contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with IN sodium hydroxide or
IN sulfuric acid immediately after sampling.
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel. Check the pH of the sample with wide
range pH paper and adjust to 7 with 1 N sodium hydroxide or 1 N
H2S04.
10.2 Add 60 ml of methylene chloride to the sample bottle, seal, and
shake 30 seconds to rinse the inner walls. Transfer the solvent to
24
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the separatory funnel and extract the sample by shaking the funnel
for 2 mln wath periodic venting to release excess pressure. Allow
*.
the organic'layer to separate from the water phase for a minimum of
10 min. If the emulsion interface between layers is more than one
third the volume of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool,
centrifugation, or other physical methods. Collect the methylene
chloride extract in a 250 ml Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedures second time,
collecting the extract. Perform a third extraction in the same
manner and collect the extract.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL
concentrator tube to a 250-mL evaporative flask. Other concentra-
tion devices or techniques may be used in place of the K-D if the
requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about
10 on of anhydrous sodium sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to
30 ml of methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse
the column with 30 to 40 ml of methylene chloride.
10.6 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a macro Snyder column. Prewet the Snyder column by adding
25
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about 1 ml methylene chloride to the top. Place the K-D apparatus
on a hot water bath, 60 to 65°C, so that the concentrator tube is
partially immersed in the hot water, and the entire lower rounded
r
surface of the flask is bathed with hot vapor. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 min. At the proper rate of
distillation, the balls of the column will actively chatter but the
chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 ml, remove the K-D apparatus and allow
it to drain.and cool for at least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 ml of methylene
chloride. A 5-mL syringe is recommended for this operation. Add 1
or 2 clean boiling chips and attach a two-ball micro-Snyder column
to the concentrator tube. Prewet the micro-Snyder column with
methylene chloride and concentrate the solvent extract as before.
When an apparent volume of 0.5 ml is reached, or the solution stops
boiling, remove the K-D apparatus and allow it to drain and cool
for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract
to 1.0 ml with methylene chloride. Stopper the concentrator tube
and store refrigerated if further processing will not be performed
immediately. If the extract is to be stored longer than two days,
transfer the extract to a screw capped vial with a Teflon-lined
26
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cap. If the sample extract requires no further cleanup, proceed
with solvent exchange to acetonitrile as described beginning in
Section IK5. If the sample requires cleanup, proceed to Section 11
10.9 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the water to a 1000-mL graduated
cylinder. Record the sample volume to the nearest 5 ml.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. The cleanup procedure recommended in this method
has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use
of additional cleanup, the analyst must demonstrate that the
recovery of each compound of interest is no less than 65%..
11.2 Slurry 10 g of Florisil in 100 ml of methylene chloride which *has
been saturated with reagent water. Transfer the slurry to a
chromatographic column. Wash the column with 100 ml of
methylene chloride. Use a column flow rate of 2 to 2.5 mL/min
throughout the wash and elution profiles
11.3 Add the extract from Section 10.8 to the head of the column. Allow
the solvent to elute from the column until the Florisil is almost
exposed to the air. Elute the column with 50 mL of methylene
chloride. Discard this fraction.
11.4 Elute the column with 50 ml of 5% acetone in methylene chloride.
Collect this fraction in a K-D apparatus. Concentrate the column
fraction to 1 ml as described in Sections 10.6 and 10.7.
27
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11.5 Add 10-mL of acetonitrile to the concentrate along with 1 or 2
clean boiling chips. Attach a three-ball micro-Snyder column to
*»
the concentrator tube. Prewet the micro-Snyder column with
acetonitrile and concentrate the solvent extract to an apparent
volume of 1 mL. Allow the K-D apparatus to drain and cool for 10
minutes.
11.6 Transfer the liquid to a 2-mL volumetric flask and dilute to the
mark with acetonitrile. Mix thoroughly prior to analysis. If the
extracts will not be analyzed immediately, they should be
transferred to Teflon sealed screw-cap vials and refrigerated.
Proceed with the liquid chromatographic analysis.
12. Liquid Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph. Included in this table are estimated
retention times and method detection limits that can be achieved by
this method. An example of the separations achieved by Column 1
and Column 2 are shown in Figures 1 and 2. Other columns,
chromatographic conditions, or detectors may be used if the
requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in
Section 7.
12.3 If an internal standard approach is being used, the analyst must not
add the internal standard to sample extracts until immediately before
injection into the instrument. Mix thoroughly.
12.4 Inject 2 to 5 uL of the sample extract by completely filling the
sample valve loop. Record the resulting peak sizes in area or peak
height units.
28
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12.5 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time
«_
variations,bf standards over the course of a djy. Three times the
standard deviation of the retention time for a compound can be used
to calculate a suggested window size; however, the experience of
the analyst should weigh heavily in the interpretation of
chromatograms.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
13. Calculations
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
(A)(Vt)
Concentration, ug/L *
(Vi)(Vs)
where:
A 3 Amount of material injected, in nanograms.
Vj * Volume of extract injected in uL. '
Y£ =» Volume of total extract in uL.
Vs » Volume of water extracted in ml.
29
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13.1.2 If the internal standard calibration procedure was used,
calculate the concentration in the sample using the response
*.
factor (RF) determined in Section 7.3.2 as follows:
(AS)(IS)
Concentration, ug/L - (AIS)(RF)(VQ)
where:
As » Response for the compound to be measured.
A-JS = Response for the internal standard.
Is = Amount of internal standard added to each
extract in ug.
V0 = Volume of water extracted, in liters.
13.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
report, all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 8.3,
data for the affected compounds must be labeled as suspect.
14. Method Performance
14.1 The method detection limit (MDL) is defined as the minimum
concentration of a substance that can be measured and reported with
99% confidence that the value is above zero.(8) The MDL
concentrations listed in Table 1 were obtained using reagent water.
(1) Similar results were achieved using representative
wastewaters.
14.2 This method has been tested for linearity of recovery from spiked
reagent water and has been demonstrated to be applicable over the
concentration range from 10 x MDL to 1000 x MDL.
30
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14.3 In a single laboratory, Battelle Columbus Laboratories, using
spiked wastewater samples, the average recoveries presented in
Table 2 were obtained. Seven replicates of each of two different
wastewaters were spiked and analyzed. The standard deviation of
the percent recovery is also included in Table 2. (1)
31
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REFERENCES
1. "Development of Methods for Pesticides in Wastewaters," Report for EPA
Contract 68-03-2956 (In preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation," American Society
for Testing and Materials, Philadelphia, PA, p. 679, 1980.
3. "Carcinogens - Working with Carcinogens," Department of Health, Education,
and Welfare, Public Health Service, Center for Disease Control, National
Institute for Occupational Safety and Health, Publication No. 77-206,
August, 1977.
4. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206 (Revised,
January, 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
6. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio 45268,
March, 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia,
PA, p. 76, 1980.
8. Glaser,- J. A. et al, "Trace Analysis for Wastewaters," Environmental
Science and Technology, 15, 1426 (1981).
32
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TABLE 1. CHROMATOGRAPH1C CONDITIONS AND METHOD DETECTION LIMITS
Retention Time (min.) Method Detection Limit
Parameter Column 1 Column 2 (ug/L)
Bendiocarb 9.3 6.0 1.8
Bendiocarb
Column 1 conditions: Spherisorb-ODS, 5 micron, 250 x 4.6 mm; 1 mL/min. flow;
40/60 acetonitrile/water.
Column 2 conditions: Lichrosorb RP-2, 5 micron, 250 x 4.6 mm; 1 mL/min. flow;
50/50 acetonitrile/water.
33
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TABLE 2. SINGLE LABORATORY ACCURACY AND PRECISION(a)
Parameter
Bendiocarb
Average
Percent
Recovery
65
70
Relative
Standard
Deviation,
%
35.6
5.7
Spike
Level
(ug/L)
8
80
Number
of
Analyses
7
7
*
Matrix
Type(b)
1
1
(a) Column 1 condition-s were used.
(b) 1 = Relevant industrial wastewater.
34
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Ul
2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0
Retention Time, Mln.
FIGURE 1. HPLC-UV CHROMATOGRAM OF 10 ng OF BENDIOCARB (COLUMN 1)
-------
Bendlocarb
14.0
T | r—i -ii \ ' • • • i
16.0 18.0 20.0
Retention Time, Mln.
FIGURE 2. HPLC-UV CHROMATOGRAM OF 600 ug OF BENDIOCARB (COLUMN 2)
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse tie fore completing
1. REPORT NO.
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
, REPORT DATE
( Determination of Bendiocarb in Industrial and
Municipal Wastewaters
6. PERFORMING ORGANIZATION COOS
7. AUTHOH(S)
J.S. Warner, T.M. EngeT and P.J. Mondron
3. PERFORMING ORGANIZATION REPORT NO,
10. PROGRAM ELEMENT NO.
CBEB1C
9. PERFORMING ORGANIZATION NAME AND AOOHSS5
Battelle Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
11. CONTRACT/GRANT NO.
68-03-2956
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Monitoring and Support Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final Report 4/82 - 11/82
14. SPONSORING AGENCY CODE
EPA 600/6
1S. SUPPLEMENTARY NOTES
16. ABSTRACT
A oechod was developed for the determination of bendiocarb in
wastewaters. The method development program consisted of a literature
review; determination of extraction efficiency for the compound from
water into methylene chloride; development of a deactivated Florisil
cleanup procedure; and determination of suitable liquid chromacographic
analysis conditions.
The final method was applied to a relevant industrial wastewater in
order to determine the precision and accuracy of-the method. The
wastewater was spiked with the compound at levels of 8.0 ug/L and
80 ug/L. Recovery for bendiocarb.'at the 3 ug/L level was 65 * 24
percent. Recovery at the 80 ug/L level was 70 ± 4 percent. The method
detection limit (MDL) for bendiocarb in distilled 'water was 1.3 ug/L.
The MDL in wastewaters may be higher due to interfering compounds.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lOENTIFIERS/OP6N ENDED TERMS C. COSATI 1-icid/CfOUp
ia. oisrHiauriON STATEMENT
Distribute to Public
19. SECURITY CLASS I IHit Reporti
Unclassified
21 NO Cf-
37
20. SECURi TY CLASS i Hit
Unclassi fied
22
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