THE DETERMINATION
OF ORGANOPHOSPHORUS
PESTICIDES IN INDUSTRIAL
AND MUNICIPAL WASTEWATER
Method 614
Thomas A. Press ley
and
James E. Longbottom
Physical and Chemical Methods Branch
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268
January 1982
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI. OHIO 45268

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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPCRT NO.
EF'A-600/4-82- 004
2.
REPORT
3. RECIPIENT'S ACCESSIOf+NO.
PB82 15598 7
4. TITLIE AND SUBTITLE
The Determination of Organophosphorus Pesticides in
Industrial and Municipal Wastewater
Method 614
5. REPORT DATE
February 1982
6. PERFORMING ORGANIZATION CODE
RGANIZATION CC
033 01 5
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Thorras A. Press!ey and James E. Longbottom
9. PERFORMING ORGANIZATION NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
Environmental Monitoring and Supgprt Lab - Cincinnati
Office of Research and Development
U. S. Environmental Protection Agency
Cincinnati, Ohio 45268
ABEB1C
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Same as Above
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/6
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This is a gas chromatographic (GC) method applicable to the determination
of selected pesticides in municipal and industrial discharges as provided
under 40CFR 136.1. A sample is solvent extracted with 15%-methylene chloride
;n hexane using a separatory funnel. The extract is concentrated, then
analyzed by GC with a flame photometric or phosphorus/nitrogen detector.
The compounds included in the method scope are:
azinphos methyl, demeton, diazinon, dichlorofenthion, dioxathion, disulfoton,
ethion, malathion, parathion ethyl, and parathion methyl.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
3. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unci assi fied
•••'. .S
20. SECURITY CLASS (This page)
Unclassi fied
EPA Form 2220-1 (9-73)
/

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THE DETERMINATION OF ORGANOPHOSPHORUS PESTICIDES
IN INDUSTRIAL AND MUNICIPAL WASTEWATER
METHOD 614
1. Scope and Application
1.1	This method covers the determination of certain organophosphorus
pesticides. The	following parameters can	be determined by this
method:
Parameter	STORET No.	CAS No.
Azinphos methyl	39580	86-50-0
Demeton	39560	8065-48-3
Diazinon	39570	333-41-5
Disulfoton	39010	298-04-4
Ethion	--	563-12-2
Malathion	39530	121-75-5
Parathion ethyl	39540	56-38-2
Parathion methyl	39600	298-00-0
1.2	This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above in industrial and
municipal discharges as provided under 40 CFR 136.1. 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.
1.3	The method detection limit (MDL, defined in Section 15) for several
parameters are 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.
1.4	The sample extraction and concentration steps in this method are
essentially the same as in Method 617. Thus, a single sample may
be extracted to measure the parameters included in the scope of
both of these methods. When cleanup is required, the concentration
levels must be high enough to. permit selecting aliquots, as
necessary, in order to apply appropriate cleanup procedures. Under
Gas Chromatography, the analyst is allowed the latitude to select
chromatographic conditions appropriate for the simultaneous
measurement of combinations of these parameters (see Section 12).
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1.5	This method is restricted to use by or under the supervision of
analysts experienced in the use of gas chromatography and in the
interpretation of gas chromatograms. Each analyst must demonstrate
the ability to generate acceptable results with this method using
the procedure described in Section 8.2.
1.6	When this method is used to analyze unfamiliar samples for any or
all of the compounds above, compound identifications should be
supported by at least one additional qualitative technique. This
method describes analytical conditions for a second gas
chromatographic column that can be used to confirm measurements
made with the primary column. Section 14 provides gas
chromatograph/mass spectrometer (GC/MS) criteria appropriate for
the qualitative confirmation of compound identifications.
2. Summary of Method
2.1	A measured volume of sample, approximately 1-liter, is solvent
extracted with ~\5%> methylene chloride in hexane using a separatory
funnel. The extract is dried and concentrated to a volume of 10 mL
or less. Gas chromatographic conditions are described which permit
the separation and measurement of the compounds in the extract by
flame photometric or thermionic bead gas chromatography.
2.2	Method 614 represents an editorial revision of a previously
promulgated U.S. EPA method for organophosphorus pesticides J
While complete method validation data is not presented herein, the
method has been in widespread use since its promulgation, and
represents the state of the art for the analysis of such materials.
2.3	This method provides selected cleanup procedures to aid in the
elimination of interferences which may be encountered.
3. Interferences
3.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware and other sample processing apparatus that lead
to discrete artifacts or elevated baselines in gas chromatograms.
All reagents and apparatus 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 thoroughly
rinsing with the last solvent used in it. Follow by washing
with hot water and detergent and thorough rinsing with tap
and reagent water. Drain dry, and heat in an oven or muffle
furnace at 400°C for 15 to 30 min. Do not heat volumetric
ware. Thermally stable materials such as PCBs, might not be
eliminated by this treatment. Thorough rinsing with acetone
and pesticide quality hexane may be substituted for the
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heating. After drying and cooling, seal and store glassware
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
minimize 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
coextracted 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
sampled. The cleanup procedure in Section 11 can be used to
overcome many of these interferences, but 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
must 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 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 borosilicate or flint glass,
1-liter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
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,
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the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
5.2	Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1	Separatory funnel - 125-mL, 1000-mL and 2000-mL, with
TFE-fluorocarbon stopcock, ground glass or TFE stopper.
5.2.2	Drying column - Chromatographic column 400 mm long x 19 mm
ID with coarse fritted disc.
5.2.3	Chromatographic column - 400 mm long x 19 mm ID with coarse
fritted disc at bottom and TFE-fluorocarbon stopcock (Kontes
K-420540-0224 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. 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	Pipet, disposable - 140 mm long x 5 mm ID.
5.2.9	Vials - Amber glass, 10 to 15 mL capacity with
TFE-fluorocarbon lined screw cap.
5.3	Boiling chips - approximately 10/40 mesh. Heat at 400°C for 30
min or Soxhlet extract with methylene chloride.
5.4	Water bath - Heated, with concentric ring cover, capable of
temperature control (± 2°C). The bath should be used in a hood.
5.5	Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6	Gas chromatograph - Analytical system complete with gas
chromatograph suitable for on-column injection and all required
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accessories including syringes, analytical columns, gases, detector
and stripchart recorder. A data system is recommended for
measuring peak areas.
5.6.1	Column 1 - 180 cm long x 4 mm ID glass, packed with 3% 0V-1
on Gas Chrom Q (100/120 mesh) or equivalent. This column
was used to develop the method performance statements in
Section 15. Alternative columns may be used in accordance
with the provisions described in Section 12.1.
5.6.2	Column 2 - 180 cm long x 4 mm ID glass, packed with 1.5%
0V-17/1.95% QF-1 on Gas Chrom Q (100/120 mesh) or equivalent.
5.6.3	Detector - Phosphorus specific: Flame photometric (FPD)
(526 nm filter) or thermionic bead detector in the nitrogen
mode. These detectors have proven effective in the analysis
of wastewaters for the parameters listed in the scope. The
FPD was used to develop the method performance statements in
Section 15. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions
described in Section 12.1.
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	Acetone, hexane, isooctane, methylene chloride - Pesticide quality
or equivalent.
6.3	Ethyl ether - Nanograde, redistilled in glass if necessary. Must
be free of peroxides as indicated by EM Quant test strips.
(Available from Scientific Products Co., Cat. No. PI 126-8, and
other suppliers.) Procedures recommended for removal of peroxides
are provided with the test strips. After cleanup, 20 mL ethyl
alcohol preservative must be added to each liter of ether.
6.4	Acetonitrile, hexane-saturated - Mix pesticide quality acetonitrile
with an excess of hexane until equilibrium is established.
6.5	Sodium sulfate - (ACS) Granular, anhydrous. Heat treat in a
shallow tray at 400°C for a minimum of 4 h to remove phthalates
and other interfering organic substances. Alternatively, heat 16 h
at 450-500°C in a shallow tray or Soxhlet extract with methylene
chloride for 48 h.
6.6	Sodium chloride solution, saturated - Prepare saturated solution of
NaCl in reagent water and extract with hexane to remove impurities.
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6.7	Alumina - Woelm, neutral; deactivate by pipeting 1 mL of distilled
water into a 125-mL ground glass-stoppered Erlenmeyer flask.
Rotate flask to distribute water over surface of glass.
Immediately add 19.0 g fresh alumina through small powder funnel.
Shake flask containing mixture for 2 h on a mechanical shaker.
6.8	Florisil - PR grade (60/100 mesh). Purchase activated at 1250°F
and store in dark in glass container with ground glass stopper or
foil-lined screw cap. Before use activate each batch at least 16 h
at 130°C in a foil covered glass container.
6.9	Stock standard solutions (1.00 yg/yL) - Stock standard solutions
may be prepared from pure standard materials or purchased as
certified solutions.
6.9.1	Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material in pesticide quality isooctane or acetone and
dilute to volume in a 10-mL volumetric flask. Larger
volumes may be used at the convenience of the analyst. If
compound purity is certified at 96% or greater, the weight
may be used without correction to calculate the
concentration of the stock standard. Commercially prepared
stock standards may be used at any concentration if they are
certified by the manufacturer or by an independent source.
6.9.2	Transfer the stock standard solutions into
TFE-fluorocarbon-sealed screw cap vials. Store at 4°C and
protect from light. Frequently check stock standard
solutions for signs of degradation or evaporation,
especially just prior to preparing calibration standards
from them.
6.9.3	Stock standard solutions must be replaced after six months
or sooner if comparison with check standards indicates a
problem.
7. Calibration
7.1	Establish gas chromatographic operating parameters equivalent to
those indicated in Table 1. The gas chromatographic system may be
calibrated using either the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2	External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration
standards at a minimum of three concentration levels by
adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with
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isooctane or other suitable solvent. One of the external
standards should be representative of a concentration near,
but above, the method detection limit. The other
concentrations should correspond to the range of
concentrations expected in the sample concentrates or should
define the working range of the detector.
7.2.2	Using injections of 1 to 5 yL 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 each parameter. Alternatively, the ratio of the
response to the mass injected, defined as the calibration
factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation
of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place
of a calibration curve.
7.2.3	The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any
parameter 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 for that parameter.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
analytical behavior to the compounds of interest. The analyst must
further demonstrate 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 each parameter of interest 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 n'sooctane or other suitable solvent.
One of the standards should be representative of a
concentration near, but above, the method detection limit.
The other concentrations should correspond to the range of
concentrations expected in the sample concentrates, or
should define the working range of the detector.
7.3.2	Using injections of 1 fto 5 yL of each calibration standard,
tabulate the peak height or area responses against the
concentration for each: compound and internal standard.
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Calculate response factors (RF) for each compound as follows:
RF = (AsCis)/(Ais Cs)
where:
As = Response for the parameter to be measured.
A-js = Response for the internal standard.
C-js = Concentration of the internal standard in yg/L.
Cc = Concentration of the parameter to be measured in
yg/L.
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 may be used for
calculations. Alternatively, the results may be used to
plot a calibration curve of response ratios, As/A-jS
against RF.
7.3.3 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any parameter 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 for that compound.
7.4	The cleanup procedure in Section 11 utilizes Florisil
chromatography. Florisil from different batches or sources may
vary in adsorptive capacity. To standardize the amount of Florisil
which is used, the use of lauric acid value is suggested. This
procedure® determines the adsorption from hexane solution of
lauric acid, in mg, per g of Florisil. The amount of Florisil to
be used for each column is calculated by dividing this factor into
110 and multiplying by 20 g.
7.5	Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absense of interference 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
with this method. This ability is established as described
in Section 8.2.
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8.1.2	In recognition of the rapid advances occurring in
chromatography, the analyst is 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 acetone 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.
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4	Table 2 provides single operator recovery and precision for
diazinon, parathion methyl and parathion ethyl. Similar
results should be expected from reagent water for all
organophosphorus compounds listed in this method. Compare
these results to the values calculated in Section 8.2.3. If
the data are not comparable, 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?
that are useful in observing trends in performance.
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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.
Alternatively, the analyst may use four wastewater data
points gathered through the requirement for continuing
quality control in Section 8.4. The accuracy statements
should be updated regularly.''
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 spiked
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 parameter does not fall within the
control limits for method performance, the results reported for
that parameter 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 must demonstrate through
the analysis of a 1-1 iter 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 must be processed as a safeguard 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
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a
dissimilar column, specific element detector, or mass spectrometer
must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant
performance evaluation studies.
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices^ 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 plastic and other potential sources
of contamination.
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9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
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.
10.2	Add 60 mL 15% methylene chloride in hexane (V:V) to the sample
bottle, seal, and shake 30 s to rinse the inner walls. Transfer
the solvent to the separatory funnel and extract the sample by
shaking the funnel for 2 min with 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.
Drain the aqueous phase into a 1000-mL Erlenmeyer flask and collect
the extract in a 250-mL Erlenmeyer flask. Return the aqueous phase
to the separatory funnel.
10.3	Add a second 60-mL volume of 15% methylene chloride in hexane to
the sample bottle and repeat the extraction procedure a second
time, combining the extracts in the 250-mL Erlenmeyer flask.
Perform a third extraction in the same manner.
10.4	Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other
concentration 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 cm 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 hexane to complete the quantitative transfer.
10.6	Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
about 1 mL methylene chloridej to the top. Place the K-D apparatus
on a hot water bath, 80 to 85|°C, so that the concentrator tube is
partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathe;d with hot vapor. Adjust the vertical
position of the apparatus and1 the water temperature as required to
complete the concentration in; 15 to 20 min. At the proper rate of

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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 min.
10.7	Remove the Snyder column and rinse the flask and its lower joint
into the concentrator tube with 1 to 2 mL of hexane and adjust the
volume to 10 mL. A 5-mL syringe is recommended for this operation.
Stopper the concentrator tube and store refrigerated if further
processing will not be performed immediately. If the extracts will
be stored longer than two days, they should be transferred to
Teflon-sealed screw-cap bottles. If the sample extract requires no
further cleanup, proceed with gas chromatographic analysis. If the
sample requires cleanup, proceed to Section 11.
10.8	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 industrial and municipal
effluents. If particular circumstances demand the use of an
alternative cleanup procedure, the analyst must determine the
elution profile and demonstrate that the recovery of each compound
of interest for the cleanup procedure is no less than 85%.
11.2	Acetonitrile partition - The following acetonitrile partitioning
procedure may be used to isolate fats and oils from the sample
extracts. The applicability of this procedure to organophosphorus
pesticides is indicated in Table 3.
11.2.1	Quantitatively transfer the previously concentrated extract
to a 125-mL separatory funnel with enough hexane to bring
the final volume to 15 mL. Extract the sample four times by
shaking vigorously for 1 min with 30-mL portions of hexane-
saturated acetonitrile.
11.2.2	Combine and transfer the acetonitrile phases to a 1-liter
separatory funnel and add 650 mL of reagent water and 40 mL
of saturated sodium chloride solution. Mix thoroughly for
30 to 45 s. Extract with two 100-mL portions of hexane by
vigorously shaking for 15 s.
11.2.3	Combine the hexane extracts in a 1-liter separatory funnel
and wash with two 100-mL portions of reagent water. Discard
the water layer and pour the hexane layer through a drying
column containing 7 to 10 cm of anhydrous sodium sulfate
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into a 500-mL K-D flask equipped with a 10-mL concentrator
tube. Rinse the separatory funnel and column with three
10-mL portions of hexane.
11.2.4	Concentrate the extracts to 6 to 10 ml. in the K-D as
directed in Section 10.6. Adjust the extract volume to 10
mL with hexane.
11.2.5	Analyze by gas chromatography unless a need for further
cleanup is indicated.
11.3 Florisil column cleanup - The following Florisil column cleanup
procedure has been demonstrated to be applicable to the seven
organophosphorus pesticides listed in Table 3. It should also be
applicable to the cleanup of extracts for ethion.
11.3.1	Add a weight of Florisil (nominally 20 g) predetermined by
calibration (Section 7.4 and 7.5), to a chromatographic
column. Settle the Florisil by tapping the column. Add
anhydrous sodium sulfate to the top of the Florisil to form
a layer 1 to 2 cm deep. Add 60 mL of hexane to wet and
rinse the sodium sulfate and Florisil. Just prior to
exposure of the sodium sulfate to air, stop the elution of
the hexane by closing the stopcock on the chromatography
column. Discard the eluate.
11.3.2	Adjust the sample extract volume to 10 mL with hexane and
transfer it from the K-D concentrator tube to the Florisil
column. Rinse the tube twice with 1 to 2 mL hexane, adding
each rinse to the column.
11.3.3	Place a 500-mL K-D flask and clean concentrator tube under
the chromatography column. Drain the column into the flask
until the sodium sulfate layer is nearly exposed. Elute the
column with 200 mL of 6% ethyl ether in hexane (V/V)
(Fraction 1) using a drip rate of about 5 mL/min. Remove
the K-D flask and set aside for later concentration. Elute
the column again, using 200 mL of 15% ethyl ether in hexane
(V/V) (Fraction 2), into a second K-D flask. Perform a
third elution using 200 mL of 50% ethyl ether in hexane
(V/V) (Fraction 3) and a final elution with 200 mL of 100%
ethyl ether (Fraction 4), into separate K-D flasks. The
elution patterns for seven of the pesticides are shown in
Table 3.
11.3.4	Concentrate the eluates by standard K-D techniques (Section
10.6), using the water: bath at about 85°C (75°C for
Fraction 4). Adjust final volume to 10 mL with hexane.
Analyze by gas chromatography.
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11.4 Removal of sulfur (9) - Elemental sulfur will elute in Fraction 1
of the Florisil cleanup procedure. If a large amount of sulfur is
present in the extract, it may elute in all fractions. If so each
fraction must be further treated to remove the sulfur.
11.4.1	Add 1 or 2 boiling chips to the 10-mL hexane solution
contained in a concentrator tube. Attach a micro-Snyder
column and concentrate the extract to about 0.2 mL in a hot
water bath at 85°C. Remove the micro K-D from the bath,
cool, and adjust the volume to 0.5 mL with hexane.
11.4.2	Plug a disposable pipet with a small quantity of glass
wool. Add enough alumina to produce a 3-cm column after
settling. Top the alumina with a 0.5-cm layer of anhydrous
sodium sulfate.
11.4.3	Quantitatively transfer the concentrated extract to the
alumina microcolumn using a 100-yL syringe. Rinse the ampul
with 200-yL of hexane and add to the microcolumn.
11.4.4	Elute the microcolumn with 3 mL of hexane and discard the
eluate.
11.4.5	Elute the column with 5 mL of 10% hexane in methylene
chloride, and collect the eluate in a 10-mL concentrator
tube. Adjust final volume to 10 mL with hexane. Analyze by
gas chromatography.
12. Gas Chromatography
12.1	Table 1 summarizes the recommended operating conditions for the gas
chromatograph. Included in this table are estimated retention
times and method detection limits that can be achieved by this
method. Other packed columns, chromatographic conditions, or
detectors may be used if the requirements of Section 8.2 are met.
Capillary (open-tubular) columns may also be used if the relative
standard deviations of responses for replicate injections are
demonstrated to be less than 6% and the requirements of Section 8.2
are met.
12.2	Calibrate the system daily as described in Section 7.
12.3	If the internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
instrument. Mix thoroughly.
12.4	Inject 1 to 5 yL of the sample extract using the solvent-flush
technique.^ Record the volume injected to the nearest 0.05 uL,
and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of
extract may also be used.
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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 of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. 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.
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, yg/L = —(V-)(V ')—
where:
A = Amount of material injected, in nanograms.
V-j = Volume of extract injected in yL.
V-t = Volume of total extract in yL.
Vs = Volume of water extracted in mL.
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:
(M^s)
Concentration, ug/L = (a. )(RF)(V )
where:
As = Response for the parameter to be measured.
A-js = Response for the internal standard.
Is = Amount of internal standard added to each extract
in yg.
^o = 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.
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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 parameters must be labeled as suspect.
14. GC/MS Confirmation
14.1	It is recommended that GC/MS techniques be judiciously employed to
support qualitative compound identifications made with this
method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight
of the compound. The instrument must be capable of scanning the
mass range at a rate to produce at least 5 scans per peak but not
to exceed 7 s per scan utilizing a 70 V (nominal) electron energy
in the electron impact ionization mode. A GC to MS interface
constructed of all-glass or glass-lined materials is recommended.
A computer system should be interfaced to the mass spectrometer
that allows the continuous acquisition and storage on machine
readable media of all mass spectra obtained throughout the duration
of the chromatographic program.
14.2	Gas chromatographic columns and conditions should be selected for
optimum separation and performance. The conditions selected must
be compatible with standard GC/MS operating practices.
Chromatographic tailing factors of less than 5.0 must be
achieved.n
14.3	At the beginning of each day that confirmatory analyses are to be
performed, the GC/MS system must be checked to see that all
decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved J 2
14.4	To confirm an identification of a compound, the background
corrected mass spectrum of the compound must be obtained from the
sample extract and compared with a mass spectrum from a stock or
calibration standard analyzed under the same chromatographic
conditions. It is recommended that at least 25 nanograms of
material be injected into the GC/MS. The criteria below must be
met for qualitative confirmation.
14.4.1	All ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the
mass spectrum of the sample with agreement to plus or minus
10%. For example, if the relative abundance of an ion is
30% in the mass spectrum of the standard, the allowable
limits for the relative abundance of that ion in the mass
spectrum for the sample would be 20% to 40%.
14.4.2	The retention time of the compound in the sample must be
within 6 seconds of the same compound in the standard
solution.
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14.4.3 Compounds that have very similar mass spectra can be
explicitly identified by GC/MS only on the basis of
retention time data.
14.5	Where available, chemical ionization mass spectra may be employed
to aid in the qualitative identification process.
14.6	Should these MS procedures fail to provide satisfactory results,
additional steps may be taken before reanalysis. These may include
the use of alternate packed or capillary GC columns or additional
cleanup (Section 11).
15. Method Performance
15.1	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.13 The MDL concen-
trations listed in Table 1 were obtained using reagent water.^
15.2	In a single laboratory, Susquehanna University, using spiked tap
water samples, the average recoveries presented in Table 3 were
obtained. The standard deviation of the percent recovery is also
included in Table 3.^
References
1.	"Methods for Benzidine, Chlorinated Organic Compounds, Pentachlorophenol
and Pesticides in Water and Wastewater," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory - Cincinnati,
Ohio 45268, September 1978.
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, Aug. 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.	ASTM Annual Book of Standards, Part 31, D3086, Appendix X3,
"Standardization of Florisil Column by Weight Adjustment Based on
Adsorption of Laurie Acid," American Society for Testing and Materials,
Philadelphia, PA, p 765, 1980.
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7.	"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.
8.	ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
9.	Law, L. M. and D. F. Goerlitz, "Microcolumn Chromatographic Cleanup for
the Analysis of Pesticides in Water," Journal of the Association of
Official Analytical Chemists, 53, 1276, (1970).
10.	Burke, J. A., "Gas Chromatography for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the Association of Official Analytical
Chemists, 48, 1037 (1965).
11.	McNair, H.M. and Bonelli, E. J., "Basic Chromatography," Consolidated
Printing, Berkeley, California, p. 52, 1969.
12.	Eichelberger, J.W., Harris, L.E., and Budde, W.L. "Reference Compound to
Calibrate Ion Abundance Measurement in Gas Chromatography-Mass
Spectrometry," Analytical Chemistry, 47, 995 (1975).
13.	Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology, 15, 1426 (1981).
14.	McGrath, T. F., "Recovery Studies of Pesticides From Surface and
Drinking Waters," Final Report for U.S. EPA Grant R804294, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
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TABLE 1
CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
Parameter
Retention Time
(min.)
Column 1 Column 2
Method
Detection Limit
(yq/L)
Diazinon
1.8
1.8
0.012
Disulfoton
1.9
2.1
ND
Demeton
2.3
2.1
ND
Parathion methyl
2.5
3.7
0.012
Malathion
2.9
3.9
ND
Parathion ethyl
3.1
4.5
0.015
Ethion
6.8
9.1
ND
Azinphos methyl
14.5
29.9
ND
ND = Not determined
Column 1 conditions: Gas-Chrom Q (100/120 mesh) coated with 3% OV-1 packed
in a 1.8 m long x 4 mm ID glass column with nitrogen carrier gas at a flow
rate of 60 mL/min. Column temperature, isothermal at 200°C. A flame
photometric detector was used with this column to determine the MDL.
Column 2 conditions: Gas Chrom Q (100/120 mesh) coated with 1.5%
0V-17+1.95% QF-1 packed in a 1.8 m long x 4 mm ID glass column with nitrogen
carrier gas at 70 mL/min flow rate. Column temperature, isothermal at
215°C.
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TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION
Average Standard Spike Number
Percent Deviation Range	of Matrix
Parameter	Recovery	(%)	(ug/L) Analyses Types
Diazinon
94
5.2
0.04-40
27
4
Parathion methyl
95
3.2
0.06-60
27
4
Parathion ethyl
102
4.1
0.07-70
27
4
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TABLE 3
FLORISIL FRACTIONATION PATTERNS
AND ACETONITRILE PARTITION APPLICABILITY
FT ori si 1 Fractionaction Pattern Acetonitrile
Parameter	Percent Recovery by Fraction Partition
No. 1 No. 2 No. 3 No. 4 Applicability
Demeton	TOO ND
Disulfoton	TOO ND
Diazinon	100 Yes
Malathion	5 95 Yes
Parathion ethyl	100 Yes
Parathion methyl	100 Yes
Azinphos methyl	20 80 ND
Ethion	ND ND ND ND Yes
ND = Not determined
Florisil eluate composition by fraction
Fraction 1 - 200 mL of 6% ethyl ether in hexane
Fraction 2 - 200 mL of 15% ethyl ether in hexane
Fraction 3 - 200 mL of 50% ethyl ether in hexane
Fraction 4 - 200 mL of ethyl ether
21

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