EPA/600/2-79/166
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il Protection
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Municipal Environmental Research tPA-600 -'. 73 !
Laboratory November 1 979
Cincinnati OH 45268
Research and Development
Evaluation of
Protocols for
Pesticides and
PCB's in Raw
Wastewater
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EPA-600/2-79-166
November 1979
EVALUATION OF PROTOCOLS FOR PESTICIDES AND PCB'S IN RAW WASTEWATER
by
Alegria B. Caragay
Philip L. Levins
Arthur D. Little, Inc.
Cambridge, Massachusetts 02140
Contract No. 68-01-3857
Project Officer
Robert T. Williams
Wastewater Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
-------
DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publi-
cation. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor
does mention of trade names or commercial products constitute endorsement
or recommendation for use.
11
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FOREWORD
The Environmental Protection Agency was created because of increasing
public and government concern about the dangers of pollution to the health
and welfare of the American people. Noxious air, foul water, and spoiled
land are tragic testimony to the deterioration of our natural environment.
The complexity of that environment and the interplay between its components
require a concentrated and integrated attack on the problem.
Research and development is that necessary first step in problem solution
and it involves defining the problem, measuring its impact, and searching
for solutions. The Municipal Environmental Research Laboratory develops
new and improved technology and systems for the prevention, treatment, and
management of wastewater and solid and hazardous waste pollutant discharges
from municipal and community sources, for the preservation and treatment of
public drinking water supplies, and to minimize the adverse economic, social,
health, and aesthetic effects of pollution. This publication is one of the
products of that research; a most vital communications link between the
researcher and the user community.
The research and the results reported in this document confirm the appli-
cability of the standard EPA protocol to the evaluation of the priority
pesticides and PCB's in raw waste waters. The confirmation of this
procedure insures that high quality data for pesticides and PCB will be
obtained from the planned national surveys of priority pollutants in
publicly owned treatment plants.
Francis T. Mayo
Director
Municipal Environmental Research
Laboratory
111
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ABSTRACT
The general EPA protocol for screening industrial effluents for priority
pollutants (Federal Register 38, No. 75, Part II), has been tested for
its applicability to the analysis of the priority pollutant pesticides
and PCB's in raw waste water. For this study, samples of raw waste water
(RWW) before and after the grit chamber were obtained from the municipal
sewage treatment plant in Brockton, Massachusetts and the analysis was
performed on the samples as collected and after dosing with 1-30 ppb
of the priority pollutant pesticides and PCB's.
In testing and validating the protocol as adaped for raw waste water
samples, the experimental design was planned to allow the assessment of
losses associated with each step of the total procedure. The overall
procedure evaluated consists of the following steps: extraction with 15%
methylene chloride/hexane with centrifugation to break up the emulsion,
concentration by Kuderna-Danish evaporation, removal of interferences by
acetonitrile partition, chromatography on Florisil and Sephadex LH-20,
and sulfur removal by treatment with mercury. Samples were assayed by gas
chromatography using an electron capture detector. Compound confirmation
was done with GC/MS using GC conditions identical to those used for GC/ECD
analysis.
Recovery data have been obtained for alpha-, beta-, gamma- and delta-BHC,
aldrin, dieldrin, endrin, endrin aldehyde, heptachlor, heptachlor epoxide,
p,p'-DDD, p,p'-DDE, p,p'-DDT, endosulfan I, endosulfan II, endosulfan
sulfate, chlordane, Arochlors 1016, 1254 and 1260 when added to RWW at
levels of 1 to 30 ppb. The data obtained show that the Kuderna-Danish
evaporation step could be a significant source of sample loss unless the
evaporation process is carried out at a fast rate. Treatment with mer-
cury effectively cleans up the extracts with no significant loss of
pesticides. Sample clean up on Sephadex LH-20 is recommended as an alter-
native to the Florisil column clean up procedure, in as much as it offers
several advantages and performs essentially the same clean up function
as Florisil.
The method tested works well for part per billion determinations, however,
there is a need to assess its practical application to raw waste water in
as much as the pesticide levels normally found in most raw waste water
samples appear to be at parts per trillion level.
This report was submitted in fulfillment of Contract No. 68-01-3857, Task
5, by Arthur D. Little, Inc. under the sponsorship of the U.S.
Environmental Protection Agency. This report covers the period June 5,
1978, to October 1978, and work was completed as of November 1978.
iv
-------
CONTENTS
Foreword ill
Abstract , iv
Figures vi
Tables viii
1. Introduction 1
2. Conclusions and Recommendations 2
3. Approach and General Experimental Conditions 4
Procedure and Approach 4
Analytical Conditions and Grouping of Pesticides .. 4
4. Results and Discussion 19
Raw Waste Water Samples 19
Recovery After Extraction and KD Concentration .... 26
Separation Procedures for Clean Up 34
Effect of Storage on Sample Stability 69
Comparison of Data Derived from Two GC Units 77
Detection Limits and GC/MS Analysis 77
References 84
Appendices
A. Chemical Formula of Priority Pollutant Pesticides .... 85
Various Names for Pesticides 85
B. Procedure for the Determination of Priority Pollutant
Pesticides and PCB's in Raw Waste Water 87
Scope and Application 87
Summary and Principle of the Method 87
Interferences , 88
Apparatus and Materials 88
Reagents, Solvents, and Standards 89
Calibration 91
Quality Control 92
Sample Preparation 92
Extraction 92
Clean-up and Separation Procedures 94
Calibration of Standards 99
Calculation and Reporting Results 99
-------
FIGURES
Number Page
1 Diagrammatic representation of general approach and
methodology evaluated 5
2 Chromatogram of Mix 2 (M2) 8
3 Chromatogram of Mix 3 (M3) 9
4 Chromatogram of Chlordane (2.4 ng) 10
5 Chromatogram of Toxaphene (7.5 ng) 11
6 Chromatogram of Arochlors 1016 and 1242 12
7 Chromatogram of Arochlor 1254 (1.2 ng) 13
8 Chromatogram of Arochlor 1260 (1.8 ng) 14
9 Chromatogram of Mix 6 (M6) 15
10 Chromatogram of Mix 5 (M5) using Perkin Elmer 900, 180 cm x
2 mm ID column 17
11 Chromatogram of Mix 5 (M5) using Hewlett Packard Model 5840 A,
180 cm x 4 mm ID column 18
12 Chromatogram of extract of RWW before grit chamber (sample
6/8/78) 20
13 Chromatogram of extract of RWW after grit chamber (sample
6/8/78) 21
14 Chromatogram of extract of RWW before grit chamber (sample
7/11/78) 22
15 Chromatogram of extract of RWW after grit chamber (sample
7/11/78) 23
16 Chromatogram of extract of RWW before grit chamber (sample
7/25/78) 24
17 Chromatogram of extract of RWW after grit chamber (sample
7/25/78) 25
-------
Number Page
18 Chromatogram of 6% ether/petroleum ether fraction (sample
M2-9, 3 ug/liter) 37
19 Chromatogram of 15% ether/petroleum ether fraction (sample
M2-9, yg/liter) 38
20 Chromatogram of extract of RWW spiked with Mix 2 at 3 l_lg/
liter (sample M2-12) 46
21 Chromatogram of Sephadex LH-20 Fraction 5 (sample M2-12,
scheme 1) 47
22 Chromatogram of Sephadex LH-20 Fraction 6 (sample M2-12,
scheme 1) 48
23 Chromatogram of Sephadex LH-20 Fraction 7 (sample M2-12,
scheme 1) 49
24 Chromatogram of Sephadex LH-20 Fraction 8 (sample M2-12,
scheme 1) , 50
25 Chromatogram of Sephadex LH-20 Fraction 9 (sample M2-12,
scheme 1) 51
26 Chromatogram of Florisil Fraction in 6% ether/petroleum
ether before treatment with mercury (sample M3-6) 70
27 Chromatogram of Florisil Fraction in 6% ether/petroleum
ether after treatment with mercury (sample M3-6) 71
28 Chlordane in RWW extract before and after mercury treatment
(test level = 30 u g/liter) 74
29 Chromatogram of sample M5-1 on HP-5840A/column 2 80
30 Chromatogra'-" of RWW blank on HP-5840A/column 2 81
vii
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TABLES
Number Page
1 Comparison of Analytical Recovery: Sephadex LH-20 vs.
Florisil 3
2 List of Priority Pollutant Pesticides and PCB's 7
3 Sample Loss on KD: Recovery of Aldrin, Heptachlor Epoxide,
Dieldrin 27
4 Sample Loss on KD: Recovery of Arochlor 1254 28
5 % Recovery of Pesticides from Raw Waste Water 29
6 % Recovery of Pesticides from Raw Waste Water 30
7 % Recovery of Various Pesticides from RWW 31
8 Recovery of Arochlor 1254 from RWW 32
9 Recovery of Arochlor 1260 from RWW 32
10 Recovery of Pesticides and PCB's at Test Level of 30 jag/liter. 33
11 % Recovery of Pesticides: Acetonitrile Partition Experiments. 35
12 Effect of Acetonitrile Partition on Pesticide Recovery 36
13 Analytical Data for Florisil Fractions Expressed as %
Recovery 39
14 Analytical Data for Florisil Fractions Expressed as %
Recovery 40
15 Florisil Fractions: Analytical Recovery of Various Pesticides
and Arochlor 1260 41
16 Florisil Chromatography: Observed Elution Pattern of Priority
Pollutant Pesticides and PCB's 42
17 Chromatographic Separation on Sephadex LH-20 45
18 Analytical Data for Sephadex LH-20 Fractions: Sample M2-12,
3 ppb 52
viii
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Number Page
19 Recovery of Various Pesticides from RWW After Clean up on
Sephadex LH-20 53
20 Chromatographic Separation on Sephadex LH-20 54
21 Analytical Data for Sephadex LH-20 Fractions: Sample M6-1 ... 55
22 Analytical Data for Sephadex LH-20 Fractions: Sample M6-2 ... 56
23 Analytical Data for Sephadex LH-20 Fractions: Sample M6-3 ... 57
24 Analytical Data for Sephadex LH-20 Fractions: Sample M6-4 ... 58
25 Analytical Data for Sephadex LH-20 Fractions: Sample M6-5 ... 59
26 Analytical Data for Sephadex LH-20 Fractions: Sample M6-6 ... 60
27 Analytical Data for Sephadex LH-20 Fractions: Sample M6-7 ... 61
28 Analytical Data for Sephadex LH-20 Fractions: Sample M6-8 ... 62
29 Analytical Data for Sephadex LH-20 Fractions: Sample M5-1 ... 63
30 Analytical Data for Sephadex LH-20 Fractions: Sample M5-3 ... 64
31 Distribution of Sample Mass in Various Sephadex LH-20 Fractions
Fractions 65
32 Elution Pattern of Phthalate Esters on Sephadex LH-20 67
33 Recovery of Pesticides and PCB After KD Concentration of
Methanol/Toluene (50:50) Solution 68
34 Effect of Hg Treatment for Sulfur Removal on Pesticide
Recovery 72
35 Effect of Hg Treatment for Sulfur Removal on Pesticide
Recovery 73
36 Recovery of Chlordane from RWW 75
37 Recovery of Arochlor 1016 from RWW 75
38 Effect of Sample Storage on Pesticide Recovery 76
39 Pesticide Recovery from RWW: Analytical Data from PE-900/
Column 1 78
40 Pesticide Recovery from RWW: Analytical Data from HP-5840A/
Column 2 79
ix
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SECTION 1
INTRODUCTION
Publicly owned treatment works are regarded as one of the most signifi-
cant sources of environmental exposure to priority pollutants. However,
insufficient exposure data exists because current methodology does not
provide reliable and valid information due to analytical interferences
and lack of adequate methods. Thus, the Municipal Environmental Research
Laboratory, Environmental Protection Agency sponsored an overall program
on the development of protocols for the determination of priority pol-
lutants in raw waste water and sludges derived from municipal sewerage
treatment plants.
The experimental program described in this report deal specifically with
the evaluation of test protocols for only the priority pollutant pesti-
cides and PCB's in raw waste water. The main thrust of the study was
directed at evaluating and/or modifying the standard EPA protocol for
priority pollutant pesticides and PCB's. This standard protocol is
described in "Sampling and Analysis Procedures for Screening of Industrial
Effluents for Priority Pollutants," U.S. EPA Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio 45268, March 1977, revised April
1977 (Federal Register 38, No. 75, Part II).
-------
SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
The overall method for the priority pollutant pesticides and PCB's
as tested within the scope of this program, works well at the ppb
levels. The clean up methods tested show that the mercury treatment
is an effective sulfur removal procedure with minimal sample loss.
The Sephadex LH-20 and Florisil column chromatography procedure also
works well. Of these two methods, however, the Sephadex LH-20 offers
the advantages of providing a cheaper and a less tedious procedure
with better sample recoveries, as shown in Table 1, without sacri-
ficing the clean up action of the Florisil. Thus, the preferable
clean up sequence is sulfur removal followed by Sephadex LH-20
chromatography.
The issue which needs further resolution relates to the fact that it
appears that the actual amounts of the pesticides present in raw waste
water are in the ppt level. This information is based on the data
for the Brockton samples and other POTW samples obtained from Cincin-
nati facilities in a separate ADL program also for EPA.* Thus,
reliability of the method for ppt analysis needs further investigation
if a hazard is presented by ppt levels of pesticides in raw waste
water.
*EPA Contract No. 68-01-3857, Task 6, Publicly Owned Treatment
Water Works Study, 1978.
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SECTION 3
APPROACH AND GENERAL EXPERIMENTAL CONDITIONS
PROCEDURE AND APPROACH
The basic procedure which has been evaluated and verified as applicable
to raw waste water (RWW) samples consists of the following steps:
extraction of the pH7 RWW with 15% methylene chloride/hexane and centri-
fugation to break up the emulsion, sample concentration by Kuderna-
Danish evaporation, and analysis by gas chromatography with an electron
capture detector. Removal of interferences by four techniques, i.e.,
acetonitrile partition, chromatography on Florisil and on Sephadex LH-20,
sulfur removal by mercury treatment, was also investigated.
The experiments were designed such that GC analysis was carried out at
each appropriate interim stage in order to identify the various steps
in the procedure where sample loss could occur and the magnitude of
such losses. This interim monitoring was possible for samples spiked
with 30 yg/liter of the chlorinated priority pesticides (except chlordane
and toxaphene) because of their high sensitivity. The basic method-
ology and approach adapted is illustrated in Figure 1.
ANALYTICAL CONDITIONS AND GROUPING OF PESTICIDES
The primary analytical instrument used for these studies was a Perkin
Elmer Model 900 gas chromatograph with a Ni-63 electron capture detector.
A Spectra Physics System 1 computing integrator was generally used for
quantitation except for cases where the interferences cause erroneous
integration, in which case peak height or peak area measurement by
planimetry was used as necessary. The other operating conditions are
as follows:
Column 1: Glass 180 cm x 2-mm ID, packed with 1.5% SP 22.50/1.95%
SP 2401 on 100/120 mesh Supelcoport
Carrier gas: 95% Argon/5% methane at a column flow rate of 45 ml
per minute plus auxiliary flow rate of 25 ml per
minute through detector.
Column Temperature: 195 C
Detector Temperature: 310 C
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Injection Port Temperature: 295 C
Sample Aliquot Injected: 2 microliters
Unless otherwise specified, this instrumental setup was used for the
various analyses. At the operating conditions described above, the
sixteen pesticides, which are pure compounds (99%), give a chromatographic
resolution pattern indicated by the relative retention times (RRT) shown
in Table 2. Note that heptachlor epoxide was used as the reference
compound instead of the commonly used aldrin because heptachlor
epoxide appeared to be more stable than aldrin under the conditions
used for these studies. As the RRT values indicate, complete resolu-
tion of the sixteen pure pesticides is not achieved with the 180 cm
x 2-mm ID column used. Compound confirmation was done with GC/MS using
GC conditions identical to those used for GC/ECD analysis.
A Hewlett Packard Model 5840A equipped with an electron capture
detector and a glass column, 180 cm x 4 mm ID, packed with 1.5%
SP 2250/1.95% SP 2401 on 100/120 mesh Supelcoport (designated as
Column 2) was also used in a few experiments in this program. The
RRT values of the compounds of interest in this column are also shown
in Table 2; only two of the 16 pesticides are not resolved by this
column.
Two pesticide groupings designated as Mix 2 (M2) and Mix 3 (M3) were
used for most of this study. The composition of these two mixtures
is indicated in Table 2; each mixture consists of chromatographically
resolved peaks as shown in Figures 2 and 3. The other pesticides,
chlordane and toxaphene, and the PCB's, which are by themselves complex
mixtures, were each studied as single compounds. As a reference point,
representative chromatograms of chlordane, toxaphene, Arochlors 1016,
1242, 1254 and 1260 are shown respectively in Figures 4-8.
The chemical formula of the priority pollutant pesticides are illustrated
in Appendix A.
As a final permutation, two other mixtures designated as Mix 5 (M5) and
Mix 6 (M6) were also used. M5 contains all the sixteen pure pesticides
while M6 contains 12 pesticides and Arochlor 1260 in approximately
equal amounts. The GC/ECD chromatogram of M6, shown in Figure 9,
illustrates that when the Arochlor is present in the sample at concen-
trations equal to that of the pesticides, the typical Arochlor 1260
chromatographic pattern (see Figure 8) is hidden by the pesticide peaks.
Only the major Arochlor 1260 peak No. 10 (retention time = 9 minutes)
is discernible in the M6 reference standard solution; this peak cor-
responds to the peak No. 13 in Figure 9 and was used to estimate the
Arochlor 1260 content of the various M6 samples.
The Mix 5 (M5) samples which contain the sixteen pesticides were
analyzed using two different instruments (i.e., the Perkin Elmer
Model 900 and a Hewlett Packard Model 5840A gas chromatographs) and
glass columns of the same length but different internal dimensions
-------
TABLE 2. LIST OF PRIORITY POLLUTANT PESTICIDES AND PCB'S
Pesticides and PCB's
a-BHC
A-BHC (lindane)
3-BHC
Heptachlor
A-BHC
Aldrin
Heptachlor epoxide **
Endosulfan I
p,p' DDE
Dieldrin
Endrin
p,p! DDD
Endosullan II
p,p' DDT
Endrin aldehyde
Endosulfan sulfate
*
RRT
0.38
0.47
0.54
0.56
0.62
0.68
1.00
1.25
1.45
1.52
1.85
2.22
2.22
2.66
2.94
3.60
Composition of
mixtures used Column A
2 3 RRT
• x 0.37
• 0.46
x 0.52
• 0.56
• 0.60
x 0.67
• x 1.00
• x 1-25
. 1.43
x 1.53
• 1.85
• 2.21
x 2.21
• 2.62
• 2.92
• x 3.55
Chlordane
Toxaphene
Arochlors 1016
1221
1232
1242
1248
1254
1260
*The column was glass, IQQ cm x 2 ram ID packed with 1,5% SP 2250/1.95%
SP 2401 on 100/120 mesh Supelcoport.
**RRT reference point
A - Glass, 180 cm x 4 mm ID packed with same column packing as above.
-------
Figure 2. Chromatogram of mix 2 (M2).
-------
1 Q-BHC 220 pg
2 0-BHC 260
3 Aldrin 210
4 Heptachlor epoxide 240
5 Endosulfan I 400
6 Dieldrm 260
7 Endosulfanll 250
8 Endosulfan sulfate 30
Figure 3. Chromatogram of mix 3 (M3).
-------
Figure 4. Chromatogram of Chlordane (2.4 ng)
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Figure 6. Chromatograms of Arochlors 1016 and 1242.
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1 Q-BHC
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3 Heptachlor
4 /--BHC
Heptachlor epoxtde
DDE
8 Endrm
ODD
10. DDT
11 Endrin aldehyde
12 Endosulfan sulfate
13 Arochlor 1260
(Peak No 10)
Figure 9. Chromatogram of Mix 6 (M6).
15
-------
(2-mm ID vs 4-mm ID) packed with the same column packing described
earlier. Representative chromatograms obtained for these two conditions
are shown in Figures 10 and 11. In the 2-mm ID column (Figure 10), only
thirteen peaks are observed because of three pairs of unresolved pesti-
cides, while the 4-mm ID column gives complete resolution of the
sixteen pesticides with the exception of ODD and endosulfan II which
elutes together.
Various samples of raw waste water were spiked with these pesticide
mixtures or with single entities of the complex compounds at test levels
of 10 - 30 vg/liter and 1-5 yg/liter.
16
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2. X-BHC
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4. A-BHC
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7. Endosulfan I
8. DDE
Dieldrm
9. Endnn
10. ODD
Endosulfan 11
11 DDT
12. Endnn aldehyde
13. Endosulfan sulfate
200 pg
100
110
120
100
90
210
250
130
110
100
110
110
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Figure 10. Chromatogram of Mix 5 (M5) using Perkin Elmer 900,
180 cm x 2 mm ID column.
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SECTION 4
RESULTS AND DISCUSSION
RAW WASTE WATER SAMPLES
Samples were collected at the Municipal Sewerage Treatment Plant in
Brockton, Massachusetts. The treatment facilities are of the activated
sludge type, and include units for grit removal, comminution and screen-
ing, primary settling using longitudinal tanks, aeration using mechanical
aerators, final settling tanks of the circular type with provisions for
rapid removal of returned sludge, chlorination and sludge treatment
including thickening of the waste activated sludge by flotation process,
digesters, counter-current elutriation and vacuum filtration. Sludge
gas is used for heating the digesters, the digester filter building
and the operating building.
Brockton has small industries and many of the industries, which include
a tannery and a large laundry business, now empty into the sewerage
system. Of the current total population of 85,000, only 77,000 are
connected to the sewer system. The average flow is 12 million gallons
per day (MGD) with a maximum flow capacity of 30 MGD.
Samples of raw waste water (RWW), before and after the grit chambers,
were collected on 6/8/78, 7/11/78 and 7/25/78; these samples were used
in the various spiking and recovery experiments. Of these three samples,
the "cleanest" was the 6/8/78 samples which were collected soon after
a rainfall of 0.35 inches; the other two sampling dates were planned
such that there was no rainfall for at least five days before the
sampling date.
Various aliquots of the RWW samples were extracted to serve as blanks
and controls for the respective spiked samples. Typically, 0.5 liter
of RWW was extracted with 15% methylene chloride/hexane, concentrated
to 10.0 ml and a 2.0 microliter aliquot was analyzed by GC/ECD. Re-
presentative chromatograms of the "before" and "after" grit chamber
RWW samples collected during these three dates are shown in Figures 12
and 13 (6/8/78), 14 and 15 (7/11/78), and 16 and 17 (7/25/78). There
appears to be no significant difference between the "before" and "after"
the grit chamber samples. The chromatograms also illustrate that the
blank levels became progressively worse as the sampling period moved
deeper into the summer.
19
-------
Figure 12. Chromatogram of extract of RWW before
grit chamber (sample 6/8/78).
20
-------
-uh
\J
Column Temperature: 190 C
Figure 13. Chromatogram of extract of RWW after
grit chamber (sample 6/8/78).
21
-------
Sample 15, Before Grit RWW
Figure 14. Chromatogram of extract of RWW before
grit chamber (sample 7/11/78).
22
-------
Sample 18, After Grit RWW
Figure 15. Chromatogram of extract of RWW after
grit chamber (sample 7/11/78).
23
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The blank levels associated with the 7/25/78 RWW samples presented
serious interference problems for studies at the 1-5 ppb for the
early eluting pesticides such as the BHC's and even at 30 ppb levels
for chlordane and Arochlor 1016.
RECOVERY AFTER EXTRACTION AND KD CONCENTRATION
The initial experiments on extraction with 15% methylene chloride/hexane
and Kuderna-Danish concentration showed that the KD evaporation step
can be a significant source of sample loss unless proper measures are
adapted. Some data which indicate this loss are shown in Tables 3
and 4. The data in Table 3 also indicate the importance of the solvent
used to dissolve the pesticides and PCB's for spiking, i.e., a water
miscible solvent like methanol gave higher recoveries than hexane.
With respect to the KD concentration step, it has been determined that
a rapid evaporation rate must be maintained to avoid sample loss. The
rapid rate means that concentrating 150 ml of 15% methylene chloride/
hexane to 10 ml, including two rinse cycles with 20 - 30 ml hexane per
cycle, should take no more that 60 minutes. Good recoveries were
obtained when the necessary precautions were followed.
The data in Tables 5 to 9 show that extraction with 15% methylene
chloride/hexane gives adequate and good recoveries of the pesticides
and PCB's (Arochlor 1254 and 1260) from raw waste water at test levels
of 30 pg/liter. Tables 5 and 6 also show the comparative data when
the methylene chloride/hexane extract was analyzed before and after KD
concentration to 10.0 ml. The concentrated samples had to be rediluted
1 to 10 in order to be within the narrow linear working range* of
the GC/ECD used. It should be noted that for these early tests where
the 6/8/78 RWW samples were spiked with the pesticides at test levels
of 30 yg/liter, the necessary redilution step dilutes the waste water
interferences to insignificant levels and allows for the assay of these
samples without the need for sample clean up.
The various data show no significant loss associated with the KD con-
centration process when a rapid evaporation rate was used. The data
also shows that there is no statistically significant difference
between the recoveries obtained from the "before" and "after" the grit
chamber RWW samples. Thus, the four samples for each experiment set
were treated as replicate samples.
The data in Table 10 summarizes the results for the pesticides at test
levels of 30 yg/liter and show good recoveries of at least 75% for all
the pesticides cited except for heptachlor, aldrin and endrin aldehyde,
with respective recoveries of 66, 74 and 47%. The recoveries of endrin
aldehyde have been consistently low throughout these studies.
* Note that appropriate calibration curves were used for the quanti-
tative measurements,
26
-------
TABLE 3. SAMPLE LOSS ON KD; RECOVERY OF ALDRIN, HEPTACHLOR EPOXIDE, DIELDRIN
Sample: 1 liter distilled water spiked with pesticides at 30-50 yg/liter
Aldrin Heptachlor epoxide Dieldrin
Sample Code MC/Hex KD MC/Hex KD MC/Hex KD
1
2
3
4
(a)
(a)
(b)
(b)
49
48
97
91
10
32
NA
NA
82
81
101
99
13
50
NA
NA
66
77
101
95
8
52
NA
NA
(a) A solution of the pesticides in hexane was used for spiking.
(b) A solution of the pesticides in methanol was used for spiking.
NA Not analyzed.
27
-------
TABLE 4. SAMPLE LOSS ON KD: RECOVERY OF AROCHLOR 1254
Sample: 1 liter distilled water spiked with 30 yg Arochlor 1254
Quantitative Data Sample 5 Sample 6
Based on MC/Hex KD MC/Hex KD
Peak 1 61 26 82 51
Peak 4 62 27 85 51
Peak 7 56 27 82 53
Peak 10 56 29 80 54
28
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30
-------
TABLE 7. % RECOVERY OF VARIOUS PESTICIDES FROM RWW
(Samples analyzed after KD concentration of 15% MC/hexane extract)
Pesticide
ct-BHC
A-BHC
Heptachlor
A-BHC
Heptachlor epoxide
Endosulfan I
DDE
Endrin
DDD
DDT
Endrin aldehyde
*
Endosulfan sulfate
Added
yg/liter
28
30
34
30
29
22
36
30
32
30
4
4
Before
M2-5
94
88
63
77
100
103
87
110
92
98
40
56
grit RWW
M2-6
90
88
59
91
88
92
73
104
98
78
35
70
After
M2-7
94
97
80
101
100
104
90
106
91
109
53
75
grit RWW
M2-8
87
87
62
98
81
97
84
101
87
105
59
84
*The reported values have been corrected for a component which behaves like
endosulfan sulfate present in the raw waste water at ca. 1 yg/liter.
31
-------
TABLE 8. RECOVERY OF AROCHLOR 1254 FROM RWW
(Sample size:
Sample code
1254-1*
1254-2*
1254-3**
1254-4**
Average
a
0.5 liter of RWW spiked
Peak selected for
6 7
69
65
69
71
69
2.6
* RWW, before grit chamber.
** RWW, after grit chamber.
TABLE 9. RECOVERY OF AROCHLOR
91
91
87
91
90
2.
at 30 yg/liter)
quantitation
10
89
85
82
81
84
0 3.6
1260 FROM RWW
(Sample size:
Sample code
1260-1*
1260-2*
1260-3**
1260-4**
Average
a
0.5 liter of RWW spiked
Peak selected for
7 8
87
80
67
83
79
8.7
83
80
64
75
75
8
at 30 yg/liter)
quantitation
10
62
87
61
73
71
.2 12.1
* RWW, before grit chamber.
** RWW, after grit chamber.
32
-------
TABLE 10. RECOVERY OF PESTICIDES AND PCB's AT TEST LEVEL OF 30 yig/LITER
(Data based on analysis after KD
Pesticide & PCB
a-BHC
X-BHC
B-BHC
Heptachlor
A-BHC
Aid r in
Heptachlor epoxide
Endosulfan I
DDE
Dieldrin
Endrin
DDD
Endosulfan II
DDT
Endrin aldehyde**
Endosulfan sulfate**
Arochlor 1254
Arochlor 1260
concentration of Mc/hexane
Average recovery*
85
90
78
66
92
74
89
95
84
80
105
92
93
98
47
87A
90
79
extract)
a
6.7
4.7
2.5
9.5
10.7
3.1
7.3
6.2
7.4
0.5
3.8
4.5
4.3
13.8
11.1
21. OA
2.0
8.7
* Each value is based on the average of 4 samples except for a-BHC,
heptachlor epoxide, endosulfan I and endosulfan sulfate which are
based on 8 samples.
** The samples were spiked at a test level of 4 yg/liter.
A The raw waste water samples contain a component with a retention time
equal to endosulfan sulfate. The average value was estimated to be
1 yg/liter with a standard deviation of 0.7 or an RSD of 70%; the value
for each sample was corrected for this blank value.
33
-------
SEPARATION PROCEDURES FOR CLEAN UP
The various clean up procedures evaluated are discussed below. The
silica gel column clean up procedure suggested in the standard EPA
method for separating PCB's from pesticides was not tried because of
time and budget limitations and the fact that the data reported indi-
cate that the procedure does not effectively separate the chlorinated
pesticides from the PCB's for all practical purposes.
Acetonitrile Partition
A few experiments were carried out to determine the recovery of some
pesticides when the clean up procedure described in Appendix B is used.
The data in Tables 11 and 12 indicate that there is no significant loss of
any of the twelve pesticides tested. The Table 12 data should be compared
with individual sample data in Table 7. Observe that the recovery for
endrin aldehyde has been consistently low.
Chromatography on Florisil
Sample clean up using the standard Florisil column procedure does not
effectively eliminate the major interference from raw waste water which
elutes within the first three minutes; this so called "hump" is not pro-
nounced in the raw waste water samples collected on 7/25/78. (See Figures
16 and 17). The chromatograms shown in Figures 18 and 19 illustrate the
components which elute in the 6% and the 15% ether/petroleum either frac-
tions, respectively. The "hump" which elutes in the 6% ether fraction can
mask the early eluting pesticides (the BHC's, aldrin, heptachlor and
heptachlor epoxide) and PCB's (Arochlors 1016, 1221, 1232,. 1242) which
would all elute in the 6% fraction. In a later experiment discussed in
the following section, it was observed that the "hump" can be eliminated by
treatment with mercury to remove sulfur.
Several extracts of raw waste water spiked with various pesticides
at levels of 3, 10 and 30 ug/liter together with Arochlor 1260, at
10 ug/liter were chromatographed on Florisil. The 6%, 15%, and 50%
ether/petroleum ether fractions were collected, concentrated by KD
and analyzed by gas chromatography. The recovery data for these
samples are shown in Tables 13 to 15 For the M6 samples reported
in Table 15, the sample size chromatographed is only one-half of
the total extract (0.5 liter RWW in 10 ml) spiked with 10 ug/liter.
Thus, a total of 2 to 3 pg of each pesticide and Arochlor 1260 was
applied to the column. In addition, the 6% fraction was treated
with mercury prior to GC analysis. The data obtained show that
30 - 40% losses of the pesticides and Arochlor 1260 are observed.
Endrin aldehyde and endosulfan sulfate (refer to Table 15) show very
low values.
The general elution pattern from Florisil is shown in Table 16-
Most of the pesticides elute in the 6% fraction. Others, such as
endosulfan I, endosulfan II and dieldrin are split in two fractions.
34
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35
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TABLE 12. EFFECT OF ACETONITRILE PARTITION ON PESTICIDE RECOVERY*
Pesticide
a-BHC
A-BHC
Heptachlor
A-BHC
Heptachlor epoxide
Endosulfan I
DDE
Endrin
ODD
DDT
Endrin aldehyde
Endosulfan sulfate
Before
M2-5
90
93
101
107
98
91
80
94
83
95
30
95
grit RWW
M2-6
87
90
83
89
92
89
73
92
84
74
35
90
After
M2-7
90
96
95
107
98
97
82
98
90
93
35
115
grit RWW
M2-8
90
96
97
101
98
94
82
97
82
97
35
110
* These data were obtained on spiked RWW samples which have gone
through methylene chloride/hexane extraction, KD concentration,
acetonitrile partition and a second KD concentration. Compare
with Table 7 data.
36
-------
Figure 18. Chromatogram of 6% ether/petroleum ether fraction
(sample M2-9,3 pg/liter).
37
-------
Figure 19. Chromatogram of 15% ether/petroleum ether fraction
(sample M2-9,3 yg/liter).
38
-------
TABLE 13. ANALYTICAL DATA FOR FLORISIL FRACTIONS EXPRESSED AS % RECOVERY*
Pesticide
a-BHC
A-BHC
Heptachlor
A-BHC
Heptachlor epoxide
Endosulfan I
DDE
Endrin
ODD
DDT
Endrin aldehyde
Endosulfan sulfate
M2-9,
6% Ether
74
72
Masked
—
60
50
52
—
82
60
—
—
3 ppb
15% Ether
—
—
—
112
1
—
—
88
—
—
25
91**
M2-5,
6% Ether
91
86
103
—
73
41
52
—
81
77
—
—
30 ppb
15% Ether
—
—
—
98
0.6
39
—
84
—
—
39
79**
* % Recovery = (yg Found v yg Added to RWW) x 100
** This value has not been corrected for blanks.
39
-------
TABLE 14. ANALYTICAL DATA FOR FLORISIL FRACTIONS EXPRESSED AS % RECOVERY*
Pesticide
a-BHC
B-BHC
Aldrin
Heptachlor epoxide
Endosulfan I
Dieldrin
Endosulfan II
Endosulfan sulfate
M3-5,
6% Ether
68
59
Masked
43
32
15
—
—
3 ppb
15% Ether
—
—
—
—
5
36
16
—
M3-6,
6% Ether
80
111
Masked
57
58
27
6
—
3 ppb
15% Ether
—
5
—
—
5
63
70
85**
* % Recovery = (yg Found * yg Added to RWW) x 100
** This value has not been corrected for blanks.
40
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TABLE 16. FLORISIL CHROMATOGRAPHY: OBSERVED ELUTION PATTERN
OF PRIORITY POLLUTANT PESTICIDES AND PCB's
Major proportion found in
Compound 6% fraction 15% fraction 50% fraction
a-BHC X
A-BHC X
3-BHC X
Heptachlor X
A-BHC X
Aldrin X
Heptachlor epoxide X
Endosulfan IX X
DDE X
Dieldrin X X
Endrin X
ODD X
Endosulfan II XX
DDT X
Endrin aldehyde X
Endosulfan sulfate ? ? X
PCB's X
42
-------
The results obtained show that the 6%, 15% and 50% fractions should
all be analyzed since some of the pesticides elute in these fractions.
Sephadex LH-20
This is the main clean up procedure evaluated in this study because
it offered the following advantages:
a) the method eliminated the interference due to the "hump"
derived from raw waste water;
b) it separated the "mass" of the samples from the pesticides;
c) the solvent volumes of each fraction are small and avoid the
additional concentration step required for the Florisil column
procedure.
The development of the Sephadex LH-20 fractionation scheme followed a
course whereby column bed volumes (1.9 x 7 cm, 1.9 x 14 cm, 1.9 x 19 cm)
and single solvent or solvent combinations compatible with an electron
capture detector were tried. Based on a few quick tests and initial
observations, the column dimension of 1.9 x 19 cm and the solvent
system methanol/toluene (1:1) was chosen for evaluation.
The gel was allowed to swell in methanol for at least overnight, and
packed as a slurry in methanol into a glass column plugged with a
small wad of precleaned glass wool. The column was tapped while pack-
ing and more gel was added as needed to fill the column to a height of
19 cm (i.e., column bed volume = 54 ml). The packed column was then
washed first with 150 ml methanol followed by at least 150 ml of
methanol/toluene (1:1). This treatment cleans the Sephadex and pre-
pares it for sample application. During the course of these studies,
four such columns were packed and reused repetitively, employing the
precaution that after the completion of every sample elution, the
column was rewashed with at least 150 ml of methanol/toluene (1:1)
before a new sample was applied.
Basically two fractionation schemes, henceforth referred to as
Fractionation Schemes 1 and 2, were tested. The main difference was
that Scheme 1 consisted of collecting 5 ml fractions vs. 10 ml frac-
tions for Scheme 2, In addition, for Scheme 1, the samples were
concentrated in a nitrogen stream to 1 - 2 ml before loading to the
column. In Scheme 2, 5.0 ml aliquots of the RWW extracts which have
been concentrated to 10 ml were loaded to the column. It is not clear
at this point whether or not the 2 vs. 5 ml of sample exerts a real
difference in the fractionation scheme, but it is duly noted in as
much as the sample solution is in hexane which causes some shrinkage
of the gel. The advantage of using a 5 ml sample aliquot (vs. 2 ml)
is that it minimizes the possibility of sample loss during further con-
centration of the sample extract.
43
-------
The elution pattern for Fractionation Scheme 1 is described in Table
17. The nature of the clean up which can be achieved by this technique
is shown by the chromatograms in Figures 20 to 25. Figure 20 is the
chromatogram of a concentrated RWW extract (Sample M2-12) containing
3 ppb of a dozen pesticides (Mix 2). Note that the region from 0 to 3
minutes is masked by the "hump" characteristic of the RWW extract.
When this sample was subjected to a Sephadex clean up procedure, five
fractions (5 to 9) were observed to contain the pesticides and the
main early eluting "hump" was eliminated. The chromatograms of these
five fractions are shown, respectively, in Figures 21 to 25. Quanti-
tative analysis of these fractions reveal a recovery and pesticide
distribution pattern in the various fractions as typified by the data
shown in Table 18. Although most of the pesticides elute_in
Fractions 6 and 7, some separation of certain pesticides do occur such
as A-BHC which elutes mainly in Fraction 8.
By using this fractionation scheme, eight samples of raw waste water
extracts were precleaned and the pertinent fractions were analyzed.
The recoveries obtained for sixteen pesticides are summarized in
Table 19. The data reflects the overall recovery from the extraction
through the entire clean up process. It is probable that most of the
sample loss occurred during the auxiliary sample concentration from
10 to 2 ml, under a nitrogen stream, just prior to sample loading on
the Sephadex.
In subsequent experimental runs, two modifications were introduced;
i.e., the auxiliary sample concentration step was eliminated and 10 ml
fractions were collected in order to minimize the number of fractions
which must be analyzed. This revised fractionation scheme is shown in
Table 20.
Eight samples (M6 series) were fractionated in this fashion and analyzed.
The samples subjected to the Sephadex clean up process were extracts of
raw waste water spiked with 10 yg/liter of each of twelve pesticides
plus Arochlor 1260. The extracts were initially analyzed after KD con-
centration and then half of each sample was subjected to the Sephadex
clean up procedure. (The other half of four samples were chromatographed
on Florisil; see Table 15 data). The individual data for these eight
samples are shown in Tables 21 to 28.
Two other samples (M5 series), which are extracts of raw waste water
spiked with sixteen pesticides at test levels ranging from 1 to
10 yg/liter, were also fractionated on Sephadex. LH-20. Fractions 4
and 5 were desulfurized by mercury treatment and analyzed by gas
chromatography. The GC instrument used for this analysis was the
Hewlett Packard Model 5840A. The data for these two samples are shown
in Tables 29 and 30.
After GC analysis, the Sephadex LH-20 fractions were transferred to a
tared aluminum pan, the solvent was allowed to evaporate by air drying,
and the pans were weighed. Table 31 shows the data for the seven
fractions of four samples and indicates that most of the sample mass
44
-------
TABLE 17. CHROMATOGRAPHIC SEPARATION ON SEPHADEX LH-20
Fractionation scheme 1
Sample volume: 1 - 2 ml in hexane
Elluent: methanol/toluene (1:1)
Sequential
Fraction
no.
1
2
3
4
5
6
7
8
9
10
11
12
effluent volume
cut-off, ml
0-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
Color of
fraction
Colorless
Colorless
Yellow
Dk. Yellow
Lt. Yellow"
Colorless
,
1
Colorless
Colorless
Colorless
Colorless
Colorless
Colorless
Observations
At the end of this fraction, three
zones are observed — a broad yellow
zone at the front followed by a
translucent and an opaque zone.
Yellow zone begins to elute
*
Translucent band also begins to
elute.
Opaque zone elutes.
Pesticides and PCB's elute
! these fractions.
in
45
-------
Figure 20. Chromatogram of extract of RWW spiked with
Mix 2 at 3 yg/liter (sample M2-12).
46
-------
Figure 21. Chromatogram of Sephadex LH-20 Fraction 5
(sample M2-12, scheme 1).
47
-------
Figure 22. Chromatogram of Sephadex LH-20 Fraction 6
(sample M2-12, scheme 1).
48
-------
Figure 23. Chromatogram of Sephadex LH-20 Fraction 7
(sample M2-12, scheme 1).
49
-------
I
Figure 24. Chromatogram of Sephadex LH-20 Fraction
(sample M2-12, scheme 1).
50
-------
X16
Figure 25. Chromatogram of Sephadex LH-20 Fraction 9
(sample M2-12, scheme 1).
51
-------
a
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*>
-------
TABLE 19. RECOVERY OF VARIOUS PESTICIDES FROM RWW AFTER CLEAN UP ON SEPHADEX
LH-20
(Fractionation scheme 1)
Pesticide
a-BHC
X-BHC
3-BHC
Heptachlor
A-BHC
Aldrin
Heptachlor epoxide
Endosulfan I
DDE
Dieldrin
Endrin
DDD
Endosulfan II
DDT
Endrin aldehyde
Endosulfan sulfate
Average /
30 ppb
**
64
56
96
73
72
46
**
80
**
64
62
76
66
76
82
38
I
*
» recovery
3 ppb
52**
54
85
62
64
44
62
59**
42
60
58
72
64
46
41
**
79
*Each value is based on the average of two independent samples.
**Average based on four independent samples.-
53
-------
o
CN
I
pa
HJ
X!
w
w
CO
o
M
H
p:
PH
w
C/3
u
M
pa
K
o
o
w
u
o
CN
CN
cu
cu
o
CO
c
o
•H
4J
TO
c
o
•H
4-1
o
TO
CU
C
TO
c
-H
i
3
O
>
ex,
B
TO
CU
C
O
4-1
O
ta
TO
W
M-l
O
(-1
o
rH
O
o
cu
B
3
o
TO
•H 4-1
4-> C
C CU
CU 3
3 rH
ey M-I
CU MH
c/3 CU
Observations
Three zones migrate down the column — a broad yellow zone
followed by distinct translucent and opaque zones.
CO
fa
0
•H
4-J
u
TO
M
Q
O
CO
1
o
CN
j Translucent and opaque zones elute.
3
0
rH
i— l
0)
o
^j-
i
o
CO
V Pesticides and PCB's elute in these fractions.
en
en
QJ
rH
r_4
O
rH
O
o
i_O
1
o
-3"
en
en
cu
rH
}_<
o
rH
O
u
o
vO
1
0
m
CO
CO
QJ
rH
S-J
o
rH
O
u
o
[•*•*
1
o
%D
4J
O
54
-------
TABLE 21. ANALYTICAL DATA* FOR SEPHADEX LH-20 FRACTIONS: SAMPLE M6-1
Pesticide
a-BHC
A-BHC
Heptachlor
A-BHC
Heptachlor epoxide
Endosulfan I
DDE
Endrin
ODD
DDT
Endrin aldehyde
Endosulfan sulfate
Arochlor 1260
Fraction 4
0.3
—
20.7
—
0.8
5.8
30.3
7.9
—
11.2
8.0
3.3
Fraction 5 Fraction 6
89.4
87.3 3.8
28.0
63.4 9.2
88.4
79.5
29.9
80.7
93.0 1.4
43.7
23.9
96.9
Total
90
91
49
73
89
85
60
89
94
55
32
100
*Values are expressed as percent of the original amount added to RWW
found in each fraction.
55
-------
TABLE 22. ANALYTICAL DATA* FOR SEPHADEX LH-20 FRACTIONS: SAMPLE M6-2
Pesticide
a- BHC
A- BHC
Heptachlor
A-BHC
Heptachlor epoxide
Endosulfan I
DDE
Endrin
DDD
DDT
Endrin aldehyde
Endosulfan sulfate
Arochlor 1260
Fraction 4
0.3
—
20.7
—
2.6
6.3
23.8
12.5
4.6
15.7
—
4.4
Fraction 5
83.7
81.4
27.7
59.6
86.0
68.9
22.8
71.6
92.9
45.5
27.9
82.9
Fraction 6
2.0
6.1
—
20.8
—
—
7.4
—
2.1
—
—
2.5
Total
86
87
48
80
89
75
54
84
100
61
28
90
*Values are expressed as percent of the original amount added to RWW
found in each fraction.
56
-------
TABLE 23. ANALYTICAL DATA* FOR SEPHADEX LK-20 FRACTIONS: SAMPLE M6-3
Pesticide
a-BHC
A-BHC
Heptachlor
A-BHC
Heptachlor epoxide
Endosulfan I
DDE
Endrin
ODD
DDT
Endrin aldehyde
Endosulfan sulfate
Arochlor 1260
Fraction 4
24.4
6.9
51.7
—
50.0
62.6
66.0
61.4
46.4
43.7
13.8
32.6
Fraction 5
65.4
78.5
8.0
90.0
21.3
26.8
19.1
29.2
44.3
18.9
21.4
69.5
Fraction 6 Total
90
2.4 88
60
90
71
89
85
91
91
63
35
108
*Values are expressed as percent of the original amount added to RWW
found in each fraction.
57
-------
TABLE 24. ANALYTICAL DATA* FOR SEPHADEX LH-20 FRACTIONS: SAMPLE M6-4
Pesticide
a-BHC
A-BHC
Heptachlor
A-BHC
Heptachlor epoxide
Endosulfan I
DDE
Endrin
ODD
DDT
Endrin aldehyde
Endosulfan sulfate
Arochlor 1260
Fraction 4
16.7
5.8
22.7
—
15.5
26.8
37.0
26.9
25.5
17.5
11.6
19.9
Fraction 5 Fraction 6
71.1
75.4 3.1
50.0
77.7 2.3
58.1
59.0
37.7
56.3
62.4 0.4
42.0
21.7
72.1
Total
88
84
73
80
74
86
75
83
88
60
33
102
*Values are expressed as percent of the original amount added to RWW
found in each fraction.
58
-------
TABLE 25. ANALYTICAL DATA* FOR SEPHADEX LH-20 FRACTIONS: SAMPLE M6-5
Pesticide
a-BHC
A-BHC
Heptachlor
A-BHC
Heptachlor epoxide
Endosulfan I
DDE
Endrin
ODD
DDT
Endrin aldehyde
Endosulfan sulfate
Arochlor 1260
Fraction 4
—
—
6.3
—
0.4
5.3
13.3
2.6
1.4
6.3
4.0
2.2
Fraction 5
85.0
87.7
39.3
66.5
81.8
81.0
48.5
80.3
83.0
43.3
ND
99.9
Fraction 6
—
2.5
—
5.8
0.9
0.5
1.0
—
—
—
—
—
Total
85
90
46
72
83
87
63
83
84
50
102
*Values are expressed as percent of the original amount added to RWW
found in each fraction.
59
-------
TABLE 26. ANALYTICAL DATA* FOR SEPHADEX LH-20 FRACTIONS: SAMPLE M6-6
Pesticide
a-BHC
A-BHC
Heptachlor
A-BHC
Heptachlor epoxide
Endosulfan I
DDE
End r in
ODD
DDT
Endrin aldehyde
Endosulfan sulfate
Arochlor 1260
Fraction 4
2.4
1.4
24.0
—
8.1
23.7
30.9
20.8
15.6
19.4
—
10.1
Fraction 5 Fraction 6
82.1
89.6 6.5
31.3
65.8 17.7
73.3
70.0
40.1 1.7
73.5
83.3
45.2
2.5
95.0
Total
84
97
55
84
81
94
73
94
99
65
105
*Values are expressed as percent of the original amount added to RWW
found in each fraction.
60
-------
TABLE 27. ANALYTICAL DATA* FOR SEPHADEX LH-20 FRACTIONS: SAMPLE
Pesticide
a-BHC
X-BHC
Heptachlor
A-BHC
Heptachlor epoxide
Endosulfan I
DDE
End r in
ODD
DDT
Endrin aldehyde
Endosulfan sulfate
Arochlor 1260
Fraction 4
__
—
11.1
—
7.0
11.6
21.4
9.8
6.8
11.5
5.6
5.6
Fraction 5 Fraction 6
87 . 7
87.7
36.9
68.6 7.0
84.9
84.0
46.7
85 . 2
81.8
43.6
8.7
85.2
Total
88
88
48
76
92
96
68
95
89
55
14
91
*Values are expressed as percent of the original amount added to RWW
found in each fraction.
61
-------
TABLE 28. ANALYTICAL DATA* FOR SEPHADEX LH-20 FRACTIONS: SAMPLE M6-8
Pesticide
ct-BHC
X-BHC
Heptachlor
A-BHC
Heptachlor epoxide
Endosulfan I
DDE
End r in
ODD
DDT
Endrin aldehyde
Endosulfan sulfate
Arochlor 1260
Fraction 4
28.5
22.5
26.3
11.4
37.8
42.2
45.2
43.5
32.9
22.7
0
44.6
Fraction 5
44.0
49.8
10.0
63.9
31.8
36.2
21.0
37.9
42.6
23.2
3.7
46.0
Fraction 6 Total
None found 7 3
in this 72
fraction 46
75
70
79
66
81
76
46
4
91
*Values are expressed as percent of the original amound added to RWW
found in each fraction.
62
-------
TABLE 29. ANALYTICAL DATA* FOR SEPHADEX LH-20 FRACTIONS: SAMPLE M5-1
Pesticide
a-BHC
A-BHC
B-BHC
Heptachlor
A-BHC
Aid r in
Heptachlor
epoxide
Endosulfan I
DDE
Dieldrin
Endrin
ODD + Endosulfan II
DDT
Endrin aldehyde
Endosulfan sulfate
Fraction 4
65.3
50.6
12.5
94.0
10.8
76.2
86.8
91.5
78.8
95.7
88.5
49.7
40.6
37
90
Fraction 5
22.4
44.6
75.9
2.6
83.8
1.9
8.2
4.0
—
—
—
23.3
6.0
*
*
Total
88
95
88
97
95
78
95
95
79
96
86
73
47
37
90
*These peaks were masked by artifacts.
63
-------
TABLE 30. ANALYTICAL DATA* FOR SEPHADEX LH-20 FRACTIONS: SAMPLE M5-3
Pesticide
a-BHC
A-BHC
B-BHC
Heptachlor
A-BHC
Aldrin
Heptachlor
axpoxide
Endosulfan I
DDE
Dieldrin
Endrin
ODD + Endosulfan II
DDT
Endrin aldehyde
Endosulfan sulfate
Fraction 4
60.7
41.2
14.2
71.5
10.3
74.0
70.2
74.4
73.2
72.4
66.2
61.4
49.4
28
68.9
Fraction 5
31.6
52.6
76.0
14.3
83.9
11.5
18.8
17.0
—
27.2
11.7
24.5
16.5
Masked*
A
Total
92
94
90
86
94
87
89
91
73
100
78
86
66
28
69
*These peaks were masked by artifacts.
-------
TABLE 31. DISTRIBUTION OF SAMPLE MASS IN VARIOUS SEPHADEX LH-20 FRACTIONS
*
Fraction no.
1
2
3
4
5
6
7
M6-1
2.8
1.9
4.5
13.5
1.1
0.4
0.7
M6-2 M6-7
0.4
1.0
5.5
14.3
1.4
0.6
0.9
0.5
0.6
2.7
9.1
0.9
0.9
0.7
M6-8
1.0
1.2
2.5
10.5
0.5
0.9
0.8
*These values represent the weight of the total residue in each fraction
which were obtained by drying each fraction in a tared, aluminum weighing
pan.
65
-------
elutes in Fraction 4. Test for sulfur in a continuous series of seven
fractions reveal the presence of sulfur in Fractions 3, 4 and 5, with
the most amount found in Fraction 4.
The general trend of the data (in Tables 21 to 30) indicates that the
pesticides elute primarily in Fractions 4 and 5, although there is
some "bounce" in the relative distribution between these two fractions.
Fraction 3 does not contain pesticides, but Fraction 6 has been found
to contain a relatively small amount. The data are most encouraging
in that the pesticides reproducibly elute in the relative retention
volume (V , defined as the ratio of the elution volume to the total
volume of the gel) range of 0.56 to 0.93 which corresponds to Fractions
4 and 5.
The elution pattern of phthalate esters which are common interferences
in pesticide assay was also determined. Fifty micrograms of diethyl-,
dibutyl-, and dioctyl phthalate was added to 5.0 ml of hexane and the
sample was chromatographed on Sephadex LH-20. The six fractions of
three replicate samples were analyzed by gas chromatography. Diethyl
phthalate was hidden in the solvent response, but the data for dibutyl
and dioctyl phthalates, summarized in Table 32, illustrates that the
phthalates elute ahead of the pesticides and PCB's; i.e., Fractions
2 and 3 contain the phthalate esters.
Experiments were also conducted to determine the effect of concen-
trating combined fractions by KD. For this study, a few pesticides
and Arochlor 1016 was added to 30 ml of methanol/toluene (1:1) at
levels shown in Table 33. Six replicate samples were concentrated
by KD to 5 - 10 ml, and the samples were analyzed. The data in
Table 33 show that a loss of 20 to 30% generally occur; in a few
cases, less than 50% recovery are indicated.
The procedure recommended and described in Appendix B calls for
collecting together Fractions 1 to 3 (30 ml), Fractions 4 and 5 (20 ml),
and Fraction 6 (10 ml). The first 30 ml fraction is discarded, the
second 20 ml fraction is analyzed, preferably avoiding KD concentra-
tion, and the third 10 ml fraction is collected separately as an
insurance, but avoids further dilution of the major pesticide frac-
tions .
The Sephadex LH-20 clean up method achieves the same purpose as the
Florisil clean up and provides the following relative advantages:
a) the method is simple and fast,
b) avoids cumbersome distillation of solvents and sorbent clean up
associated with the Florisil method, and gives better recovery
because it eliminates one other concentration step.
66
-------
TABLE 32. ELUTION PATTERN OF PHTHALATE ESTERS ON SEPHADEX LH-20
Fraction no.
1
2
3
4
5
6
Total
Fractionation scheme 2
Average % found in fraction*
Dibutyl phthalate
—
10
50
18
—
—
78%
Dioctyl phthalate
—
36
51
—
—
—
87%
*Averages of three independent determinations
67
-------
TABLE 33. RECOVERY OF PESTICIDES & PCB AFTER KD CONCENTRATION OF METHANOL/
TOLUENE (50:50) SOLUTION
Pesticide and PCB
a-BHC
A -BHC
Heptachlor Epoxide
Endosulfan 1
DDE
End r in
ODD
DDT
Arochlor 1016
^g
1
3
2
3
2
3
3
3
15
Tnt-al
added
.4
.0
.9
.1
.8
.0
.2
.0
.0
//I
79
80
88
81
84
99
84
89
84
#2
49
*
80
64
91
91
82
70
88
-------
Sulfur Removal
Metallic mercury was used to remove sulfur interferences. The
method was applied directly to extracts of raw waste water, to the
6% ether/petroleum ether fraction, and to the Sephadex LH-20 fractions.
Initial experiments were performed on a 6% ether/petroleum ether trac-
tion which showed a "hump" in the chromatogram and had a sulfur
like odor. A 2 ml aliquot of this fraction was treated with two to
three drops of metallic mercury. A black precipitate formed and the
mercury was immediately separated from the solvent layer by centri-
fugation and decantation and an aliquot of the sample analyzed.
The comparative chromatograms of the fractions before and after treat-
ment with mercury are shown in Figures 26 and 27 respectively, which
clearly show that the "hump" is sulfur related.
In view of these observations, subsequent experiments were conducted
to determine the effect of this mercury treatment on the recovery
of the pesticides. The data shown in Tables 34 and 35 for various
fractions from Florisil and Sephadex LH-20 before and after treatment
with mercury, show virtually no loss for sixteen pesticides.
Experiments were also conducted to test the effect of the sulfur re-
moval procedure directly on the concentrated raw waste water extracts.
As noted earlier, the 7/25/78 raw waste water samples showed signifi-
cant levels of the early eluting "hump" which masked chlordane and
Arochlor 1016, even at test levels of 30 yg/liter (see Figure 28 for
sample chromatogram). These samples were again treated with mercury
and analyzed. The results obtained for four replicate samples for
chlordane and Arochlor 1016 are shown respectively in Tables 35 and
36. The average recoveries were 104% for chlordane and 74% for
Arochlor 1016.
EFFECT OF STORAGE ON SAMPLE STABILITY
Three liter aliquots of the "before" the grit chamber and "after" the
grit chamber raw waste water samples were spiked with Mix 6 at a test
level of 10 yg/liter. Each sample was mixed thoroughly and homogenized
in a blender. Immediately after the sample preparation, duplicate
0.5 liter aliquots of the two samples were extracted and concentrated
to 10 ml. (Samples M6-1, M6-2, M6-3, M6-4). The remaining portion of
the spiked samples were stored at 40 F for 48 hours and again duplicate
0.5 liter aliquots of both samples were processed similarly (Samples
M6-5, M6-6, M6-7 and M6-8).
The data summarized in Table 38 illustrates that the pesticides and
Arochlor 1260 are stable under these storage conditions. Of the
twelve pesticides, only endrin aldehyde and possibly heptachlor
appear to be significantly lower in the stored samples.
69
-------
Figure 26. Chromatogram of Florisil Fraction in 6% ether/petroleum
ether before treatment with mercury (sample M3-6).
70
-------
Figure 27. Chromatogram of Florisil Fraction in 6% ether/petroleum
ether after treatment with mercury (sample M3-6).
71
-------
TABLE 34. EFFECT OF Hg TREATMENT FOR SULFUR REMOVAL ON PESTICIDE RECOVERY
ng Found
M3-5, 6% ether fraction M3-6, 6% ether fraction
Pesticide Untreated Hg treated Untreated Hg treated
a-BHC 950 800 1100 1000
g-BHC 960 (?) 490 1800 1750
Aldrin Masked 350 Masked 780
Heptachlor epoxide 630 440 1200 1050
Endosulfan I 800 950 1400 1200
Dieldrin 250 270 440 530
Endosulfan II — — 90 110
72
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TABLE 36. RECOVERY OF CHLORDANE FROM RWW*
Single code
C -1
C -2
C -3
C -4
RWW source
Before Grit
Before Grit
After Grit
After Grit
yg/liter
Added Found
29.6 30.6
29.6 32.6
29.6 31.2
29.6 29.4
Percent recovery
103
110
105
99
Average 104
.a 4.6
^Quantitative analysis of these samples was possible only after sulfur
removal.
TABLE 37. RECOVERY OF AROCHLOR 1016 FROM RWW*
Single code
1016-1
1016-2
1016-3
1016-4
RWW source
Before Grit
Before Grit
After Grit
After Grit
yg/liter
Added Found
30
30
30
30
24.0
23.8
18.0
23.2
Percent recovery
80
79
60
77
Average.
74
9.4
^Quantitative analysis of these samples was possible only after sulfur
removal.
75
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As another estimate of the degree of variability or agreement which can
be expected, four samples of raw waste water extracts were analyzed
using two different GC units. The response of the Perkin Elmer 900
equipped with a 180 cm by 2-mm ID glass column (Column 1) used for most
of these studies was compared with that of the Hewlett Packard 5840A
equipped with a 180 cm by 4-mm ID glass column (Column 2) . Two
advantages are provided by the latter set up in that the Hewlett
Packard 5840A has a higher sensitivity than the Perkin Elmer 900 and
the 4-mm ID column gives better resolution of the sixteen pesticides;
i.e., only DDD and endosulfan II overlap vs. three pairs of unresolved
components with the 2-mm ID column.
The four samples were spiked with a mixture of sixteen pesticides at
individual concentrations ranging from 0.6 to 10 yg/liter. The samples
were extracted, concentrated, treated with mercury and analyzed; the
samples were rediluted by a factor of 4 before analysis. Four cali-
bration standards were used to calibrate the two analytical instruments,
The results obtained are shown in Tables 39 and 40. in
PE-900/Column 1 (Table 39) , average recoveries less than 75% were
obtained for combined B-BHC and heptachlor, combined DDE and dieldrin,
and aldrin; the endrin aldehyde appeared to be present at trace levels.
The HP-5840A/ Column 2 data (Table 40) , show average recoveries greater
than 80% for all pesticides except endrin aldehyde with an average
recovery of 35%. The RWW interference peak, which behaves like endo-
sulfan sulfate in Column 1, shows up to be a different component
partially resolved from endosulfan sulfate (RT = 21 minutes) in
Column 2. Chromatograms on Column 2 of a spiked RWW extract and a
corresponding blank are shown in Figures 29 and 30 respectively.
DETECTION LIMITS AND GC/MS ANALYSIS
The concentration range evaluated in this program was 1 to 30 yg/liter
and the data obtained for the spi-ked samples show that the method is
applicable to ppb analysis. However, the level of what appears to
be some pesticides found in the raw waste water samples are in the
parts per trillion (ppt) level.
The data obtained from extracts of the 7/25/78 raw waste water are
shown in Table 41. These extracts, in duplicate, were treated with
mercury to eliminate the major sample interference and analyzed by
the PE-900 and the HP-5840A. The sample peaks which appear to conform
to the indicated pesticides are shown in Table 41. xhe data based on
the two columns with identical substrates but different dimensions
indicate the presence of A-BHC, DDD and/or endosulfan II. GC/MS
analysis of a similar sample indicated only a BHC, most likely the
A-BHC. The estimated concentration of the A-BHC by the GC/ECD analysis
is 0.02 to 0.3 yg/liter. The uncertainties associated with these
values would negate any assumption that the GC/MS analysis can be used
for confirmation at the ppt level.
77
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TABLE 41. ANALYTICAL DATA ON RAW WASTE WATER SAMPLES
Estimated Concentration, yg/liter*
Pesticide PE-900/Column 1 HP 5840 A/Column 2
A-BHC 0.02 0.30
3-BHC 0.18
A-BHC 0.03
Aldrin 0.01
DDD ")
f '0.90* 0.80**
Endosulfan II J
Endosulfan sulfate 1.0
* Values reported are an average of two samples.
** DDD and endosulfan II are not resolved.
32
-------
There is no doubt that the sensitivity of the electron capture detec-
tor is more than adequate for doing pesticide analysis on "clean"
samples at the ppt level. However, there are practical and economic
issues involved in doing ppt analysis in raw waste water by GC/ECD
because of the interference problems. The standard protocol requires
identification based on KRT on two columns and GC/MS confirmation.
Since the GC/MS cannot be used at ppt levels, one alternative would
be to use a third column. On the basis of using three columns for
identification, and depending on the extent of the sample clean up
necessary, a rough estimate of the cost per sample to do the ppt
analysis is shown below.
One stage (sulfur removal) $125/sample
Two stages (sulfur removal and Sephadex) 250
Two stages (sulfur removal and Florisil) 325
Three stages (sulfur removal and Florisil, 750
and Silica gel)
It is noted that for this task, the GC/ECD procedure was used for
quantitative and qualitative analysis of the pesticides and PCB's
in the raw waste water extracts subjected to the concentration and
clean-up procedures discussed in the preceding sections. Compound
confirmation was done with GC/MS using GC conditions identical
to those used for GC/ECD analysis; the column was 1.5% SP-2250/
1.95% SP-2401 on 100/120 mesh Supelcoport. GC/MS calibration was
done by the use of standard solutions of pesticides and PCB's. It
has been established that 10 ng of each pesticide, except endrin and
DDT, which requires 30 ng, must be injected into the system for proper
GC/MS confirmation; a sample injection of 2-yL is used. Thus, each RWW
sample extract was further concentrated to a smaller volume based on
the GC/ECD quantitative results prior to GC/MS analysis. Using this
approach, it was found that the RWW samples collected at the Brockton
Sewerage Treatment Plant contained BHC, most likely X-BHC. It is fur-
ther noted that in a separate task, also for EPA, where approximately
200 RWW water samples from various sites in the U.S. have been collected,
21 samples showed pesticides tentatively identified by the GC/ECD method;
of these 21 samples, only 5 samples have been confirmed to contain pest-
icides by GC/MS analysis. The pesticides found in these samples at 1
to 3 yg/liter include heptachlor epoxide, heptachlor, and aldrin.
83
-------
REFERENCES
1. "Method for Organic Pesticides in Water and Wastewater," Environmental
Protection Agency, National Environmental Research Center, Cincinnati,
Ohio 45268, 1971.
2. Monsanto Methodology for Arochlors - Analysis of Environmental Materials
for Biphenyls, Analytical Chemistry Method 71-35, Monsanto Company, St.
Louis, Missouri 63166, 1970.
3. "Method for Polychlorinated Biphenyls in Industrial Effluents," Environ-
mental Protection Agency, National Environmental Research Center,
Cincinnati, Ohio 45268, 1973.
4. "Method for Organophosphorus Pesticides in Industrial Effluents,"
Environmental Protection Agency, National Environmental Research Center,
Cincinnati, Ohio 45268, 1973.
5. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," Chapter 6, Section 6.4, U.S. Environmental Protection
Agency, National Environmental Research Center, Analytical Quality
Control Laboratory, Cincinnati, Ohio 45268, 1973.
6. "Pesticide Analytical Manual," U.S. Dept. of Health, Education and
Welfare, Food and Drug Administration, Washington, B.C.
7. "Analysis of Pesticide Residues in Human and Environmental Sa,mples," U.S.
Environmental Protection Agency, Perrine Primate Research Laboratories,
Perrine, Florida 33157, 1971.
8. Mills, P.A., "Variation of Florisil Activity; Simple Method for
Measuring Adsorbent Capacity and its Use in Standardizing Florisil
Columns," Journal of the Association of Official Analytical Chemists.
51, 29 (1968).
9. Goerlitz, D. F. and Brown E., "Methods for Analysis of Organic Substances
in Water," Techniques of Water Resources Investigations of the U.S.
Geological Survey, Book 5, Chapter A3, U.S. Department of the Interior,
Geological Survey, Washington, D.C. 20402, 1972, pp. 24-40.
10. Steere, N.V., editor, "Handbook of Laboratory Safety," Chemical Rubber
Company, 18901 Cranwood Parkway, Cleveland, Ohio 44128, 1971, pp. 250-254,
11. Mills, P.A., et al, Journal of the Association of Official Analytical
Chemists, 55, 39 (1972).
84
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APPENDIX A
CHEMICAL FORMULA OF PRIORITY POLLUTANT PESTICIDES
VARIOUS NAMES FOR PESTICIDES
FORMULA
BHC's
Heptachlor
Heptachlor Epoxide
Aldrin
Dieldrin
1,2,3,4,5,6—Hexachlorocyclohexane,
Benzene hexachloride,
P:99%
MW = 290
a — isomer — Alpha BHC
0 - isomer - Beta BHC
6 - isomer - Delta BHC
7 — isomer — Lindane, Gamma-BHC, Streunex, Tri-6,
Aparasin®, Aphtiria, Ben-Hex®, HCH®,
Lorexane®, Streunex®, HGI®
LD50= 125mg/kg
1,4,5,6,7,8,8-Heptachloro-3a,4,7,7a-tetra-
hydro-4,7-methanoindene
E3314®, Velsicol 104®, Drinox®
P:99% -» LD50 = 195 mg/kg
MW = 373
1,4,5,6,7,8,8-Heptachloro-2,3-epoxy-3a,4,7,
7a-tetrahydro-4,7-methanoindan
P:99%
MW = 389
1,2,3,4,10,10-Hexachloro-1,4,4a,5,8,8a-
hexadrydro-endo-exo-1,4:5,8-dimethano-
naphthalene, Aldrin®
1,2,3,4,10,10-Hexachloro-1,4,4a,5,8,8a-
hexahydro-1,4, endo-exo-5,8-dimethylnaphthalene
P:99% LD50 = 55 mg/kg
MW = 365
1,2,3,4,10,10-Hexachloro-6,7-epoxy-1,4,
4a,5,6,7,8,8a-octahydro-endo-exo-1,4:5,
8-dimethanonaphthalene
Compound 497®, HEOD, Dieldrin, Octalox®
P:98.5% LD50 = 10-102 mg/kg
MW = 383
Cl
Cl
Cl
Cl
85
-------
Endrin
1,2,3,4,10,10-Hexachloro-6,7-epoxy-1,4,4a
5,6,7,8,8a-octahydro-1,4-endo-endo-5,8-
dimethanonaphthalene
Endrm, Experimental insecticide 269®
P:98% LD50= 7.5-17 mg/kg
MW = 383
• Cl
p, p'-DDD 1,1-Bis[p-chlorophenyl] 2,2-dichloroethane
IDE, Rothane®, Dichlorodiphenyl
dichloroethane, 2,2-bis[p-chlorophenyl] -
1,1-dichloroethane
P:96% LD50 = 3400 mg/kg
MW = 320
p,p' - DDE
p,p' - DDT
Endosulfans
Chlordane
Cl
1,1 Dichloro 2,2, bis [p-chlorophenyl] ethyleneO.1 g/1.0g
P:99%
MW=318
1,1,1-Trichloro-2,2-bis [p-chlorophenyl] ethane 1g/10g
P:99% LD50 = 250 mg/kg
MW = 354
1,4,5,6,7,7-Hexachloro-5-norbornene-2,3- 1g/10g
dimethanol cyclic sulfite
Chlorthiepm®, Malix®, Hoe 2671®, Thiodan®,
Cyclodan®, Beosit®, Thimul®, Thifor®,
6,7,8,9,10,10-Hexachloro-1,5,5a, 6,9,9a-hexahydro-6,9-
methano-2,4,3 benzo-dioxathiepin 3-oxide
P:98.9% LD50 = 80-110 mg/kg
MW = 407
1,2,4,5,6,7,8,8-0ctachloro-3a,4,7,7a- 1g/10g
tetrahydro-4,7-methanoindene
CD-68®, Toxichlor®, Velsicol® 1068, Octachlor®,
Octa-Klor®
1,2,4,5,6,7,8,8-Octachloro-4,7-methane-
3a,4,7,7a-tetrahydromdane
P:95% LD50 = 452-590 mg/kg
MW = 409.75
Toxaphene Polychloro bicyclic terpenes
Campheclor, Chlorinated camphene®,
Gemphene®, Penphene®, Phenacide®,
Phenetox®, Synthetic 3956®, Toxakil®,
Toxaphene®, Hercules 3956®, Alltex®
P:65%CI LD50 = 90 mg/kg
Cl
M
-------
APPENDIX B
PROCEDURE FOR THE DETERMINATION OF PRIORITY POLLUTANT PESTICIDES AND PCB' s IN
RAW WASTE WATER
SCOPE AND APPLICATION
1. This method covers the determination of the various priority pollutant
pesticides and PCB's in raw waste water.
2. The method is based on the standard EPA method for these priority pollu-
tants in industrial effluents and has been tested for the determination
of the following pollutants: alpha-, beta-, delta-, gamma-BHC, aldrin,
dieldrin, endrin, endrin aldehyde, heptachlor, heptachlor epoxide, p,p'-
DDD, p,p'-DDE, p,p'-DDT, endosulfan I, endosulfan II, endosulfan sulfate,
chlordane, and the arochlors.
3. For a sample size of 1 liter, the working limit of the method is estimated
to be 1 yg/1 liter for all the pesticides except for chlordane whose
working limit is estimated to be 5 yg/liter, the PCB's also have a working
limit of 5 yg/liter.
4. For samples containing high levels of the PCB's relative to the chlori-
nated pesticides (i.e., for example, a 20 to 1 ratio) differentiation
of the pesticides from the PCB's is not possible. In this case, con-
firmation by GC/MS is necessary.
SUMMARY AND PRINCIPLE OF THE METHOD
1. The priority pollutant pesticides and PCB's are extracted from pH7 raw
waste water with 15% methylene chloride/hexane; centrifugation is used
to break up the emulsion. The extract is concentrated to 5 to 10 ml in a
Kuderna-Danish evaporator. Sample clean-up and removal of interferences
is carried out by a combination of one or more of the following methods:
acetonitrile partition to remove fatty acids, treatment with mercury to
remove sulfur and chromatographic clean-up techniques using either
Florisil or Sephadex LH-20. Detection and measurement is accomplished
using an electron capture detector (or alternatively a microcoulometric
or electrolytic conductivity detector), Identification is made from
relative retention times and gas chromatographic patterns obtained
through the use of two or more unlike columns. Results are reported in
micrograms per liter.
87
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INTERFERENCES
1. Solvents, reagents, glassware, and other hardware used may yield discrete
artifacts and/or elevated baselines which can cause misinterpretation of
gas chromatograms. All of these materials must be demonstrated to be
free from intereferences under the conditions of the analysis. Specific
selection of reagents and purification of solvents by distillation in
all glass systems may be required.
2. The interferences in raw waste water are high and varied and often pose
great difficulty in the accurate and precise measurement of chlorinated
pesticices and the PCB's. Separation and clean-up procedures are
generally required to eliminate these interferences; however, such
techniques may result in some loss of the organo-chlorine compounds.
For this reason, great care should be exercised in the selection and use
of sample clean-up methods. While it is not possible to describe
procedures for overcoming all of the interferences that my be encountered
in municipal raw waste water, four clean-up procedures which have been
evaluated for raw waste water are described in the section.
3. The PCB's, when present in high concentrations (ie., 10 ]jg/liter or
greater), will act as severe interferences for the priorLty pesticides.
And conversely, the priority pollutant pesticides will interfere with
the PCB's.
4. Phthalate esters, certain organophosphorus pesticides, and elemental
sulfur will interfere when using an electron capture detector. These
materials do not interfere when the microcoulometric or electrolytic
conductivity detectors are used in the halogen mode.
APPARATUS AND MATERIALS
1. Gas chromatograph - equipped with glass lined injection port,
2. Detector options:
Electron capture - radioactive (tritium or nickel 63)
Microcoulometric titration
Electrolytic conductivity.
3. Recorder - potentiometric strip chart (10 in.) compatible with the
detector.
4. An electronic integrator or some other suitable method for measuring peak
areas.
5. Gas chromatographic column, glass (180 cm long x 4-mm ID) packed with
one of the following substrates:
1.5% SP2250/1.95% SP2401 on 100/120 mesh Supelcoport,
3% OV-1 on 100/120 Supelcoport or Gas Chrom Q.
5% OV-210 on 100/120 mesh Supelcoport or Gas Chrom Q,
88
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6% QFl/4% SE-30 on 100/120 mesh Supelcoport or Gas Chrom Q.
6. Kuderna-Danish (KD) glassware (Kontes)
Snyder column - three ball (macro) and two ball (micro)
Evaporative flasks - 100 ml
Receiver ampuls - 10 ml, graduated
Ampul stoppers
7. Chromatographic column - Chromaflex (400 mm long x 19 mm ID) with coarse
fritted plate on bottom and Teflon stopcock; 250 ml reservoir bulb at top
of column with flared out funnel shape at tope of bulb - a special order
(Kontes K-420540-9011).
8. Chromatographic column - pyrex (approximately 300 mm long x 19 mm ID)
with Teflon stopcock.
9. Micro syringes - 10, 25, 50 and 100 yl.
10. Separatory funnels - 125 ml, 1000 ml and 2000 ml with Teflon stopcock,
11. Blender - high speed, glass or stainless steel cup.
12. Test tubes - 8 ml with Teflon lined screw caps.
13. Graduated cylinders - 100, 250, and 1000 ml.
14. Assorted glassware.
15. Florisil - PR grade (60 - 100 mesh); purchase activated at 1250°F and
store in the dark in glass containers with glass stoppers or foil<-lined
screw caps. Before'use, pre-clean and activate each batch overnight at
130°C in foil-covered glass container. Determine lauric-acid value
(see section of Determination of Lauric-acid value). The following
apparatus are needed for this determination:
Buret - 25 ml with 1/10 ml graduations,
Erlenmeyer flasks - 125 ml narrow mouth and 25 ml, glass stoppered,
Pipet - 10 and 20 ml transfer,
Volumetric flask - 500 ml.
16. Sephadex LH-20 (Pharmacis Fine Chemicals). The dry bead diameter is
25 - 100 microns.
17. Boiling chips, Teflon.
REAGENTS, SOLVENTS, AND STANDARDS
1. Ferrous Sulfate - (ACS) 30% solution in distilled water,
2. Potassium Iodide - (ACS) 10% solution in distilled water.
89
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3. Sodium Chloride - (ACS) saturated solution in distilled water (pre-
rinse NaCl with hexane).
4. Sodium Hydroxide - (ACS) 10 N in distilled water.
5. Sodium Sulfate - (ACS) granular, anhydrous (pre-cleaned and conditioned
at 400°C for 4 hours).
6. Mercury - (ACS).
7. Sulfuric Acid - (ACS) mix equal volumes of concentrated H SO, with
distilled water.
8. Methylene Chloride - nanograde, redistill in glass if necessary.
9. Hexane - nanograde, redistill in glass if necessary.
10. Acetonitrile - nanograde, redistill in glass if necessary.
11. Methanol - nanograde, redistill in glass if necessary.
12. Toluene - nanograde, redistill in glass if necessary.
13. Petroleum Ether (boiling range 30° - 60°C) - nanograde, redistill in
glass if necessary.
14. Diethyl Ether - nanograde, redistilled in glass if necessary.
Must contain 2% alcohol and be free of peroxides by following test:
to 10 ml of ether in glass-stoppered cylinder previously rinsed with
ether, add one ml of freshly prepared 10% KI solution. Shake and let
stand one minute. No yellow color should be observed in either layer.
Decompose ether peroxide by adding 40 g of 30% ferrous suLfate solution
to each liter of solvent. CAUTION: reaction may be vigorous if the
solvent contains a high concentration of peroxides.
Distill deperoxidized ether in glass and add 2% ethanol.
15. Reagents and solvents for determination of Laurie Acid Value,
Alcohol, ethyl - USP or absolute, neutralized to phenolphthalein.
Hexane - distilled from all glass apparatus.
Laurie acid - purified, CP.
Laurie acid solution - transfer 10.000 g lauric acid to 500 ml volu-
metric flask, dissolve in hexane, and dilute lo 500 ml (1 ml = 20 mg) .
Phenolphthalein indicator - dissolve 1 g in alcohol and dilute to 100 ml.
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Sodium hydroxide - dissolve 20 g NaOH (pellets, reagent grade) in water
and dilute to 500 ml (IN). Dilute 25 ml IN NaOH to 100 ml with water
(0.05N). Standardize as follows: weigh 100 - 200 mg lauric acid into
125 ml Erlenmeyer flask. Add 50 ml neutralized ethyl alcohol and 3
drops phenolphthalein indicator; titrate to permanent end point.
Calculate mg lauric acid/ml 0.05N_ NaOH (about 10 mg/ml).
16. Pesticides and PCB standards - Reference Grade Analytical Standards
including the following:
Alpha-BHC
Beta-BHC
Delta-BHC
Gamma-BHC
Aldrin
Dieldrin
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
p,p'-ODD
p,p'-DDE
p,p'-DDT
Endosulfan I
Endosulfan II
Endosulfan sulfate
Chlordane
Arochlors 1016, 1221, 1232, 1242, 1248, 1254, 1260
CALIBRATION
1. Gas chromatographic operating conditions are considered acceptable if
the response to dicapthon is at least 50% of full scale when < 0.06 ng
is injected for electron capture detection and < 100 ng is injected for
91
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microcoulometric or electrolytic conductivity detection; or alternatively,
for an electron capture detector, the response of 0.05 ng of aldrin is
at least 50% of full scale. For all quantitative measurements, the
detector noise level should be less than 2% of full scale.
2. Standards are injected frequently as a check on the stability of
operating conditions.
3. The elution order and retention ratios of various organochlorine
pesticides are provided in Table 1-B as a guide.
QUALITY CONTROL
1. Duplicate and spiked sample analyses are recommended as quality control
checks. When the routine occurrence of a pesticide is being observed,
the use of quality control charts is recommended.
SAMPLE PREPARATION
1. Blend the sample if suspended matter is present and adjust pH to near
neutral (pH 6.5 - 7.5) with 50% sulfuric acid or 10 N sodium hydroxide.
2. Quantitatively transfer a 1 liter aliquot of homogenized sample into a
2 liter separatory funnel.
EXTRACTION
1. Add 60 ml of 15% methylene chloride in hexane (v:v) to the sample in the
separatory funnel and shake vigorously for two minutes.
2. Allow sample to stand for at least 15 minutes and draw the bottom
aqueous layer into a one liter Erlenmeyer flask, retaining any emulsion
layer with the top organic solvent layer. Transfer the top organic
solvent layer into a 250 ml centrifuge bottle.
3. Centrifuge the organic solvent layer at 1000 rpm for 5 - 30 minutes to
break up emulsion. With the aid of a disposable pipet, draw the clear
top organic layer, pass it through a column containing 3-4 inches of
anhydrous sodium sulfate, and collect it in a 500 ml KD flask equipped
with a 10 ml ampul.
4. Return both the aqueous layers in the Erlenmeyer flask and in the centri-
fuge bottle back into the separatory funnel. Rinse both Erlenmeyer
flask and centrifuge bottle with 60 ml of 15% methylene chloride/hexane,
transfer these solvent rinsings into the separatory funnel and repeat
the extraction procedure a second time including centrifugation step and
combine the clear organic extract with the first extract in the K-D
assembly.
5. Repeat the extraction process in the manner a third time.
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TABLE 1-B. RETENTION TIME OF PRIORITY POLLUTANT PESTICIDES RELATIVE TO
HEPTACHLOR EPOXIDE
Pesticide
a-BHC
A-BHC (lindane)
g-BHC
Heptachlor
A-BHC
Aldrin
Heptachlor epoxide
Endosulfan I
p,p' DDE
Dieldrin
Endrin
p , p ' ODD
Endosulfan II
p,p' DDT
Endrin aldehyde
Endosulfan sulfate
1.5% SP2550*
1.95% SP2401
0.37
0.46
0.52
0.56
0.60
0.67
1.00
1.25
1.43
1.53
1.85
2.21
2.21
2.62
2.92
3.55
1.5% OV-17**
1.95% QF-1
0.35
0.45
—
0.53
—
0.65
1.00
1.27
1.45
1.56
1.90
2.26
2.33
2.71
—
—
A
5% OV-210
0.33
0.42
—
0.45
—
0.52
1.00
1.28
1.09
1.55
1.84
1.94
2.38
2.11
—
—
A
3% OV-1
0.27
0.34
—
0.61
—
0.78
1.00
1.27
1.56
1.51
1.70
2.04
1.76
2.73
—
—
* Glass column 180 cm x 4 mm ID, solid support 100/120 mesh Supelcoport,
column temperature 200°C, Argon/Methane Carrier flow rate at 60 ml/minute.
** Same conditions as (1) except solid support is 100/120 mesh Gas-Chrom Q.
A Glass column 180 x 4 mm ID, solid support 100/120 Gas-Chron Q, column
temperature 200°C, Argon/Methane Carrier flow rate at 70 ml/minute.
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6. Add a piece of Teflon boiling chip and concentrate the extract in the
KD evaporator on a hot water bath. This evaporation step should be
conducted at a fast rate. It should take no more than 60 minutes to
concentrate the combined extracts, including two sequential rinsings of
the KD assembly with 25 ml of hexane and evaporation to 5.0 ml.
(Tests have shown that a slow evaporation rate yields poor recoveries.)
7. Analyze by gas chromatography. Perform any sample dilution or clean-up
necessary as indicated by this initial GC analysis.
CLEAN-UP AND SEPARATION PROCEDURES
1. Interferences in the form of distinct peaks and/or high background in the
initial gas chromatographic analysis, as well as the physical character-
istics of the extract (color, cloudiness, viscosity) and background
knowledge of the sample will indicate whether clean-up is required. When
these interfere with measurement of the pesticides, or affect column life
or detector sensitivity, proceed as directed below.
2. Sulfur removal - the procedure described below eliminates a common
interference found in raw waste water. Tests have shown that the method
can be applied directly to the concentrated extract or to the respective
column fractions with quantitative recovery of the priority pollutant
pesticide and PCB's. However, the analyst must demonstrate quantitative
recovery of specific pesticides and PCB's at least once.
Take a 2 ml aliquot of the sample extract or column fraction and
transfer to a centrifuge tube with ground-glass stopper.
With the aid of a disposable pipet, add 1 drop of metallic mercury
to the centrifuge tube and shake gently. Observe for the formation
of a black precipitate which indicates the presence of sulfur. Let
stand for 2 minutes only, centrifuge quickly and immediately transfer
this solution into a second tube, avoiding transfer of any mercury
or black precipitate. Repeat this process two to three times but
do not allow the sample solution to stay in contact with the mercury
for longer than 5 minutes.
Analyze by gas chromatography.
3. Acetonitrile partition - this procedure is used to isolate fats and oils
from the sample extracts. Tests have shown that the priority pollutant
pesticides and PCB's are quantitatively recovered by this procedure;
however, the analyst must demonstrate the efficicency of the partitioning
for specific pesticides and PCB's at least once.
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 vigor-
ously for one minute with 30 ml portions of hexane-saturated
acetonitrile.
94
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Combine and transfer the acetonitrile phases to a 1 liter separatory
funnel and add 650 ml of distilled water and 40 ml of saturated
sodium chloride solution. Mix thoroughly for 30-45 seconds.
Extract with two 100 ml portions of hexane by vigorously shaking
about 15 seconds.
Combine the hexane extracts in a 1 liter separatory funnel and
wash with two 100 ml portions of distilled water. Discard the
water layer and pour the hexane layer through a 3-4 inch anhydrous
sodium sulfate column into a 500 ml KD flask equipped with a 10-ml
ampul. Rinse the separatory funnel and column with three 10-ml
portions of hexane.
Concentrate the extracts to 5-10 ml in the KD evaporator in a hot
water bath.
Analyze by gas chromatography unless a need for further clean-up
is indicated.
4. Florisil column adsorption chromatography
Clean-up of Florisil and column preparation:
Clean-up the Florisil before use by washing the Florisil with
boiling water (distilled, or preferably filtered through a
Milli Q unit or equivalent); 200 g of Florisil requires three
washes with 1 liter of boiling water each time. Allow most of
the water to drain and dry the Florisil at 130°C for 4 hours.
Then calcine the Florisil by heating in a muffle furnace at
675°C for 25 hours. Store in a dessicator and keep protected
from light.
Determine lauric acid value as described below.
Just before use, weigh the amount needed based on the lauric
acid value in a 125 ml beaker, cover with foil and heat again
for 1 hour at 130°C. Immediately after removing from the oven,
pack the Florisil into the Chromaflex column with gentle
tapping to let the Florisil settle and proceed as directed
under column chromatography procedure.
Determination of lauric acid value - a rapid method for determining
adsorptive capacity of Florisil is based on adsorption of lauric
acid from hexane solution (6)(8). An excess of lauric acid is used
and amount not adsorbed is measured by alkali titration. Weight
of lauric acid adsorbed is used to calculate, by simple proportion,
equivalent quantities of Florisil for batches having different
adsorptive capacities. The procedure is as follows:
Transfer 2.000 g Florisil to 25 ml glass stoppered Erlenmeyer
flasks. Cover loosely with aluminum foil and heat overnight
at 130°C. Stopper, cool to room temperature, add 20.0 ml
95
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lauric acid solution (400 mg), stopper, and shake occasionally
for 15 minutes. Let adsorbent settle and pipet 10.0 ml of
supernatant into 125 ml Erlenmeyer flask. Avoid inclusion of
any Florisil.
Add 50 ml neutral alcohol and 3 drops indicator solution;
titrate with 0.0514 to a permanent end point.
Calculate amount of lauric acid adsorbed on Florisil as
follows:
Lauric acid value = mg lauric acid/g Florisil =
200 - (ml required for titration x mg lauric acid/ml
0.05:N NaOH).
To obtain an equivalent quantity of any batch and multiply by
20 g. The pattern of elution of pesticides are indicated
below and must be independently verified by the analyst.
Column chromatography procedure
Adjust the sample extract volume to 10 ml with hexane.
After settling the Florisil by tapping the column, add about
one-half inch layer of anhydrous granular sodium sulfate to
the top. Allow Florisil to cool.
Pre-elute the column, after cooling, with 50-60 ml of petroleum
ether. Discard the eluate and just prior to exposure of the
sulfate layer to air, quantitatively transfer the sample
extract into the column by decantation and subsequent petroleum
ether washings. Adjust the elution rate to about 5 ml per
minute and, separately, collect up to three eluates in 500 ml
KD flasks equipped with 10 ml ampuls. (See eluate composition
below). Perform the first elution with 200 ml of 6% ethyl
ether, and the second elution with 200 ml of 15% ethyl ether
in petroleum ether. Perform the third elution with 200 ml of
50% ethyl ether - petroleum ether and the fourth elution with
200 ml of 100% ethyl ether.
Concentration the eluates to 6-10 ml in the KD evaporator in
a hot water bath.
Analyze by gas chromatography.
Eluate composition by using an equivalent quantity of any batch of
Florisil as determined by its lauric acid value, the pesticides
will be separated into the eluates indicated on the next page.
96
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6% Eluate
Aldrin DDT Pentachloronitrobenzene
BHC Heptachlor Strobane
Chlordane Heptachlor epoxide Toxaphene
ODD Lindane Trifluralin
DDE Methoxychlor PCB's
mirex
15% Eluate 50% Eluate
Endosulfan I Endosulfan II
Endrin Captan
Dieldrin
Dichloran
Phthalate esters
Certain thiophqsphate pesticides will occur in each of the above
fractions as well as the 100% fraction. For additional information
regrading eluate composition, refer to the FDA Pesticide
Analytical Manual (6).
5. Chromatography on Sephadex LH-20
This is suggested as an alternative to the Florisil column clean-up
procedure for the removal of phthalate interferences and other
high molecular weight interferences. Limited tests have shown that
the phthalates elute earlier than the priority pollutant pesticides
and PCB's. Tests have also shown quantitative recovery of these
chlorinated compounds using this chromatographic clean-up procedure.
However, the analyst must demonstrate the qualitative recovery of
the priority pollutant pesticides and PCB's with the procedure
described.
Preparation of chromatographic column
Allow the Sephadex LH-20 beads to swell in methanol for at
least on over night period. Approximately 13-14 g of dry
beads are needed to pack one column.
Plug the effluent end of a glass column (300 mm x 19 mm ID)
with a small wad of pre-cleaned glass wool. Mark the glass
column at a point 19 cm above the glass wool plug. Fill the
column half-way with methanol, gently swirl the swollen gel
to make a fairly thick slurry in methanol, open column stop-
cock and pack the column with enough gel to fill the column
exactly to the 19 cm mark. To prepare a well packed column,
it is advisable to add the gel slurry uninterrupted and employ
gentle tapping as the gel settles. A well packed column should
be free of cracks and air bubbles. (The total bed volume is
54 ml.)
97
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To clean the gel, wash the column bed with at least 200 ml
of methanol and collect a 20 ml effluent. Analyze effluent
by gas chromatography. If the analysis shows presence of
interferences, wash column with more methanol until the
column effluent is free of interferences; if the effluent is
"clean," proceed to the next step.
Wash the column gel bed with 100 ml of methanol/toluene (1:1).
After this conditioning cycle, adjust column flow-rate to 1.0
ml per minute and collect 20 ml of effluent. Analyze effluent
by gas chromatography. This effluent should, be "free" of
interferences.
Prepare column for sample application by allowing the solvent
to drain until the solvent level reaches the top level of the
gel bed. Close column stopcock.
Chromatographic separation
A sample size of 5 ml in hexane is carefully added to the
column minimizing agitation of the gel by carefully pouring
solution either down the wall of the column or down a glass
rod.
Position a 50 ml graduated tube beneath the column and open
stopcock. When the sample level reaches the top of the gel
bed, rinse the glass rod and/or wall of the column with 2-3
ml of methanol/toluene (1:1). Let the rinse solvent reach the
level of the gel bed again before gradually adding 50 ml of
methanol to toluene to the column. Maintain column flow rate
at 1 ml per minute and collect a total of 30 ml of effluent.
(Fraction 1.) This fraction contains the phthalates and other
interferences with molecular weights greater than the priority
pollutant pesticies and PCB's/
In another graduated tube, collect the next 20 ml of effluent
(Fraction 2.) This fraction contains priority pollutant
pesticides and PCB's.
Collect the next 10 ml of effluent. (Fraction 3.) This
fraction serves as assurance that the pesticide and PCB's
are completely eluted but avoids dilution of the preceding
fraction.
Analyze fractions 2 and 3 by gas chromatography.
Addendum notes on Sephadex LH-20
The elution volume cut off for the three fractions were based
on studies which showed that for a column gel bed of the given
dimensions, at least 80% of the total pesticides and PCB's
98
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elute in the relative retention volme (V ) range of .055-0.93,
where V is defined as:
r
_ Elution Volume
r Total bed volume
These V range correspond to the elution volume sequence of
30-50 ml (Fraction 2).
Limited experiments here also show that Fraction 3 (V range =
0.93-1.1) may contain a small amount, a 10-20% of the pesticide
sample. However, collecting fractions 2 and 3 together is not
recommended because of the dilution effect and the observed
losses associated with KD concentration. Tests have shown
that when 1 y.g of chlorinated pesticide and 15 Ug of PCB were
added to 30 ml of methanol/toluene (1:1) and then concentrated
to 10 ml by KD evaporation, losses of as much as 30-50% of
these chlorinated components can occur.
CALIBRATION OF STANDARDS
1. Prepare a calibration curve for the specific pesticides and PCB's of
interest to cover a linear response range. Use at least three different
concentrations.
2. Calculate the calibration constant (k) by the nanograms of components.
CALCULATION AND REPORTING RESULTS
1. Determine the pesticide concentration as described below expressing
results in micrograms per liter which is numerically equal to nanograms
per ml.
,, . A x Vt
micrograms/liter =
k x Vi x Vw
where:
A = Peak area of sample
k = Calibration constant, area per nanogram
Vi = Value of extract injected (yl)
Vt = Value of total extract (yl)
Vw = Value of water extracted (ml)
99
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing}
REPORT NO
EPA-600/2-79-166
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
EVALUATION OF PROTOCOLS FOR PESTICIDES AND PCB'S IN
RAW WASTEWATER
5. REPORT DATE
November 1979 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7 AUTHOR(S)
Alegria B. Caragay
Philip L. Levins
8. PERFORMING ORGANIZATION REPORT NO.
9 PERFORMING ORGANIZATION NAME AND ADDRESS
Arthur D. Little, Inc.
20 Acorn Park
Cambridge, Massachusetts 02140
10. PROGRAM ELEMENT NO.
1BC611, SOS #5, Task AI-04
11. CONTRACT/GRANT NO.
68-01-3857
12 SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory-Gin., OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
FINAL REPORT 6/78-10/78
14. SPONSORING AGfENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
Project Officer: Robert T. Williams (513) 684-7626
16 ABSTRACT
The general EPA protocol for screening industrial effluents for priority
pollutants (Federal Register 38, No. 75, Part II), has been tested for its applica-
bility to the analysis of the priority pollutant pesticides and PCB's in raw
wastewater. Raw wastewater from the municipal sewage treatment plant in Brockton,
Massachusetts was dosed with 1-30 ppb of the priority pollutant pesticides and PCB's.
The overall procedure evaluated consists of the following steps: extraction
with 15% methylene chloride/hexane with centrifugation to break up the emulsion,
concentration by Kuderna-Danish evaporation, removal of interferences by acetonitrile
partition, chromatography on Florisil and Sephadex LH-20, and sulfur removal by
treatment with mercury. Samples were assayed by gas chromatography using an electron
capture detector.
The data obtained show that the Kuderna-Danish evaporation step could be a
significant source of sample loss unless the evaporation process is carried out at a
fast rate. Treatment with mercury effectively cleans up the extracts with no signifi
cant loss of pesticides. Sample clean-up on a Sephadex LH-20 is recommended as an
alternative to the Florisil column clean-up procedure. The method tested works well
for parts per billion determination.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Pesticides
Extraction
Chemical Analysis
Centrifuging
DDT
Dieldrin
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Arochlors
Polychlorinated Biphenyl
Priority Pollutants
07C
18 DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
110
20 SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (Rev. 4-77)
100
AUSGPO: 1980-657-146/5503
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