Single-Laboratory Evaluation of Method
8080 -Organochlorine Pesticides and
PCBs (Polychlorinated Biphenyls)
Acurex Corp., Mountain View, CA
Prepared for
Environmental Monitoring Systems Lab,
Las Vegas, NV
Aug 87
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
••
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PB87-232591
EPA/600/4-87/022
August 1987
SINGLE-LABORATORY EVALUATION OF METHOD 8080 —
ORGANOCHLORINE PESTICIDES AND PCBs
by
Viorica Lopez-Avila, Sarah Schoen, and June Mi lanes
Acurex Corporation
Mountain View, California 94039
Contract Number 68-03-3226
Project Officer
U< Werner F. Beckert
& Quality Assurance Division
f>; Environmental Monitoring Systems Laboratory
[^ Las Vegas, Nevada 89193-3478
fs
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89193-3478
REPRODUCED BY
INFORMATION SERVICE
SPRINGFIELD, VA. 221 61
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing/
1. REPORT NO.
EPA/600/4-87/022
2.
ECIPIENTS ACCESSION NO.
PB87 2 32 5 9 I/AS
4. TITLE AND SUBTITLE
SINGLE-LABORATORY EVALUATION OF METHOD 8080
ORGANOCHLORINE PESTICIDES AND PCBs
5. REPORT DATE
August 1987
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Viorica Lopez-Avila, Sarah Schoen, and
June Milanes
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Acurex Corporation
Mountain View, CA 9A039
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
Contract No. 68-03-3226
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas. NV 89193-3478
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/07
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The design, execution and results of the improvement and single-laboratory evalua
tion of SW-846 Method 8080, "Organochlorine Pesticides and PCBs", are described. In
this study, the application of this method to the analysis of aqueous and solid
samples for organochlorine. pesticides and PCBs was examined. Deficiencies of the
present method were corrected by substituting a silica gel extract cleanup procedure
for the original Florisil procedure (which results in a better separation of the
pesticides from the PCBs) and by using capillary gas chromatographic columns in place
of the packed columns (which provides a better gas chromatographic separation). A
limited evaluation of the sample extraction methods 3510, 3520, 3540 and 3550 preceded
this study. Ruggedness testing of some of the more important parameters of the
modified method was conducted to determine the effect of critical parameters on
method performance. Method performance parameters, Including precision, accuracy and
detection limit data, are presented and discussed. The limitations of the method are
described and suggestions are included on how to put this method to best use. In
addition to the revised protocol, the report includes an extensive literature review
covering analytical methods for the determination of organochlorine pesticides and
polychlorinated biphenyls in water, soil, sediment and sludge samples. The revised
method is recommended for use as part of the Agency's monitoring program.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COS AT I Field/Group
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report I
UNCLASSIFIED
21. NO. OF PAGES
253
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (R«». 4-77) PREVIOUS EDITION is OBSOLETE
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NOTICE
The information in this document has been funded wholly or in part by the
United States Environmental Protection Agency under Contract Number 68-03-3226
to the Acurex Corporation, Mountain View, California. It has been subject to
the Agency's peer and administrative review, and it has been approved for
publication as an Environmental Protection Agency document. Mention of trade
names or commercial products does not constitute endorsement or recommendation
for use.
ii
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PREFACE
This is the final report for Work Assignments 5 and 8, EPA Contract
68-03-3226, "Evaluation of GC Method for Organochlorine Pesticides and PCBs,"
conducted at Acurex Corporation, Project Nos. 7902 and 8001. The project was
directed by Dr. Viorica Lopez-Avila. The work was performed in the
Environmental Systems Division.
This report was written by Dr. Viorica Lopez-Avila. Technical support
for the project was provided by Dr. Sarah Schoen and Mr. June Mi lanes.
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ABSTRACT
Method 8080 was developed for the determination of certain organochlorine
pesticides (OCRs) and polychlorinated biphenyls (PCBs) in liquids and solids.
Liquid samples are extracted according to Method 3510 (separatory funnel) or
Method 3520 (continuous liquid-liquid extractor) and solid samples according
to Method 3540 (Soxhlet extraction) or Method 3550 (sonication), the extracts
are concentrated, fractionated on Florisil and the fractions analyzed by
gas chromatography (GO on packed columns.
The Method 8080 protocol has been evaluated in a single laboratory on actual
and simulated wastes. It was found that the Florisil cleanup method is
problematic when both OCRs and PCBs are present; a cleanup and fractionation
on deactivated silica gel is more advantageous. Sulfur in extracts can be
removed with tetrabutylammonium sulfite. Toxaphene and chlordane pose special
problems because of their multiple-peak responses. The use of capillary
columns instead of packed columns in the GC analysis is advantageous because
better separations are obtained for complex samples containing combinations
of OCRs, PCBs, and other organics.
The Method 8080 protocol was revised, and several features were added or
changed, including the cleanup and fractionation procedures which now specify
deactivated silica gel, and the use of capillary columns instead of packed
columns. The revised protocol was evaluated by using extracts of environmental
samples spiked with the substances of interest of known concentrations. The
precision and accuracy results indicate that the revised Method 8080 can be
reliably applied to the determination of organochlorine pesticides and PCBs
in liquid and solid matrices.
Included in this report as an appendix is an extensive-literature review
covering analytical methods for the determination of organochlorine pesticides
and PCBs in water, soil, sediment and sludge samples.
IV
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TABLE OF CONTENTS
Section Page
Preface iii
Abstract iv
Figures vii
Tables x
1. Introduction 1
2. Conclusions 4
3. Recommendations 6
4. Literature Review 7
5. Method 8080 Evaluation 10
5.1 Experimental 11
5.2 Results 11
6. Method Improvement 18
6.1 Extraction 18
6.1.1 Extraction of Aqueous Samples 18
6.1.2 Extraction of Soil Samples 22
6.1.3 Ruggedness Test for Method 3550 26
6.2 Extract Cleanup 29
6.2.1 Silica Gel Cleanup -' 29
6.2.2 Alumina Cleanup 36
6.2.3 Silica Gel/Celite 48
6.2.4 Florisi 1/Charcoal 49
6.2.5 Sulfur Cleanup 54
6.3 Gas Chromatography 55
6.4 Recommendations for Method Revision 73
7. Revised Method 8080 — Evaluation Studies 74
7.1 Reproducibility Study for Gas Chromatographic
Analysis 74
7.2 Method Precision and Accuracy 80
7.3 Method Detection Limit 97
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CONTENTS (Continued)
Section Page
References 100
Appendices
A. Literature Review 102
B. Method 8080 - Organochlorine Pesticides and PCBs 191
C. Method 3510, 3520, 3540, 3550 Protocols 221
VI
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FIGURES
Number Page
1 GC/EC chromatograms of the Fieri si 1 Fractions I, II, and
III of liquid waste No. 1 extract, analyzed on the 1.8 m x
2 mm ID 1.5-percent OV-17/1.95-percent OV-210 column;
isothermal at 190°C 14
2 GC/EC chromatograms of the Florisil Fractions I, II, and
III of liquid waste No. 1 extract, spiked with the
organochlorine pesticides and PCBs, and analyzed on the
1.8 m x 2 mm ID 1.5-percent OV-17/1.95-percent OV-210
column; isothermal at 190°C 15
3 GC/EC chromatograms of the Florisil Fractions I, II, and
III of liquid waste No. 1 extract, analyzed on the 30 m x
0.25 mm ID DB-5 fused-silica capillary column.
Temperature program: 100°C (hold 2 min) to 160°C at
15°C/min, then at 5°C/min to 270°C; helium at 16 psi 16
4 GC/EC chromatograms of the Florisil Fractions I, II, and
III of liquid waste No. 1 extract, spiked with the
organochlorine pesticides and PCBs, and analyzed on the
30 m x 0.25 mm ID DB-5 fused-silica capillary column.
Temperature program: 100°C (hold 2 min) to 160°C at
15°C/min, then at 5°C/min to 270°C; helium at 16 psi 17
5 GC/EC chromatograms of the silica gel Fractions I, II,
and III of liquid waste No. 1 extract, analyzed on the
30 m x 0.25 mm ID DB-5 fused-silica capillary column.
Temperature program: 100°C (hold 2 min) to 160°C at
15°C/min, then at 5°C/min to 270°C; helium at 16 psi 41
6 GC/EC chromatograms of the silica gel Fractions I, II,
and III of liquid waste No. 1 extract, spiked with the
organochlorine pesticides and PCBs, and analyzed on the
30 m x 0.25 mm ID DB-5 fused-silica capillary column.
Temperature program: 100°C (hold 2 min) to 160°C at
15°C/min, then at 5°C/min to 270°C; helium at 16 psi 42
Vll
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FIGURES (Continued)
Number Page
7 GC/EC chromatogram of the liquid waste No. 1 extract after
alumina cleanup (10 ml hexane) 45
8 GC/EC chromatogram of the liquid waste No. 1 extract after
alumina cleanup (20 ml hexane) 46
9 GC/EC chromatogram of the liquid waste No. 1 extract after
alumina cleanup (50 ml hexane) 47
10 GC/EC chromatogram of the liquid waste No. 1 extract after
cleanup on alumina deactivated with 15 percent water 50
11 GC/EC chromatogram of the liquid waste No. 1 extract after
cleanup on alumina deactivated with 19 percent water 51
12 GC/EC chromatogram of the sulfur standard before reaction
with the TBA-sulfite reagent 58
13 GC/EC chromatogram of the sulfur standard after reaction with
the TBA-sulfite reagent 59
14 GC/EC chromatograms of the organochlorine pesticide standards
A and B and of a composite of A and B, obtained on the 1.5-
percent OV-17/1.95-percent OV-210 column. Concentrations:
standard A, 0.05 to 0.10 ng/u; standard B, 0.05 to 0.50 ng/uL;
standard A and B composite, 0.05 to 0.10 ng/ut; isothermal
at 190°C 64
15 GC/EC chromatograms of a mixture of PCB 1016/1260 at
0.05 ng/uL and of a composite of organochlorine pesticide
standards A and B and PCB-1016/1260 at 0.05 ng/uL, obtained
on the 1.5-percent OV-17/1.95-percent OV-210 column;
isothermal at 190°C 65
16 GC/EC chromatograms of a PCB-1016/1260 mixture at 0.5 ng/uL
and of a composite of organochlorine pesticide standards A and
B and PCB-1016/1260 at concentrations of 0.05 to 0.50 ng/uL
and 0.50 ng/uL, respectively. The analyses were performed on
the 1.5-percent OV-17/1.95-percent OV-210 column; isothermal
at 190°C 66
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FIGURES (Continued)
Number Page
17 GC/EC chromatograms of the toxaphene standard at 0.10 ng/uL
and of a composite of organochlorine pesticide standards A
and B at 0.05 to 0.50 ng/ul and toxaphene at 0.10 ng/nL,
obtained on the 1.5-percent OV-17/1.95-percent OV-210
column; isothermal at 190°C 67
18 GC/EC chromatograms of the toxaphene standard at 1.0 ng/uL
and of a composite of organochlorine pesticide standards A
and B at 0.05 to 0.50 ng/uL and toxaphene at 1.0 ng/uL,
obtained on the 1.5-percent OV-17/1.95-percent OV-210
column; isothermal at 190°C 68
ix
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TABLES
Number Page
1 Compounds Listed in Method 8080 2
2 Results of the Florisil Evaluation Study 12
3 Evaluation of Method 3510 Using Distilled Water
Samples 19
4 Evaluation of Method 3520 Using Distilled Water
Samples 20
5 Evaluation of Method 3540 Using Sandy Loam Samples 23
6 Evaluation of Method 3550 Using Sandy Loam Samples 24
7 Combinations of 7 Parameters Recommended for Testing the
Ruggedness of Method 3550 26
8 Ruggedness Test for Method 3550 — Recovery Data for the
Organochlorine Pesticides 28
9 Ruggedness Test — Group Differences for the
Organochlorine Pesticides 29
10 Ruggedness Test — Percent Recoveries of the
Organochlorine Pesticides as a Function of Solvent
Composition ~". 30
11 Distribution and Percent Recoveries of the Organochlorine
Pesticides and PCBs in the Silica Gel Column Fractions 32
12 Distribution and Percent Recoveries of the Organochlorine
Pesticides and PCBs Spiked into the Distilled Water
Extracts (Spike Level is 500 ng for Pesticides and 5,000
ng for PCBs) 33
13 Distribution and Percent Recoveries of the Organochlorine
Pesticides and PCBs Spiked into the Liquid Waste No. 1
Extract (Spike Level is 500 ng for Pesticides and 5,000 ng
for PCBs) 34
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TABLES (Continued)
Number
14 Distribution and Percent Recoveries of the Organochlorine
Pesticides and PCBs Spiked into the Liquid Waste No. 1
Extract (Spike Level is 5,000 ng for Pesticides and
50,000 ng for PCBs) 35
15 Distribution and Percent Recoveries of the Organochlorine
Pesticides and PCBs Spiked into the Liquid Waste No. 2
Extract (Spike Level is 500 ng for Pesticides and 5,000 ng
for PCBs) 36
16 Distribution and Percent Recoveries of the Organochlorine
Pesticides and PCBs Spiked into the Liquid Waste No. 2
Extract (Spike Level is 500 ng for Pesticides and
50,000 ng for PCBs) 37
17 Distribution and Percent Recoveries of the Organochlorine
Pesticides and PCBs Spiked into the Sandy Loam
Extract (Spike Level is 500 ng for Pesticides and 5,000 ng
for PCBs) 38
18 Distribution and Percent Recoveries of the Organochlorine
Pesticides and PCBs Spiked into the NBS Sediment
Extract (Spike Level is 500 ng for Pesticides and 5,000 ng
for PCBs) 39
19 Distribution and Percent Recoveries of the Organochlorine
Pesticides and PCBs Spiked into the Solid Waste
Extract (Spike Level is 500 ng for Pesticides and 5,000 ng
for PCBs) 40
20 Recovery of Selected Organochlorine Pesticides After
Chromatography on Neutral Super I Woelm Alumina 43
21 Recovery of Organochlorine Pesticides Spiked into Liquid
Waste No. 1 Extract and Subjected to Alumina Cleanup 43
22 Recovery of the Organochlorine Pesticides Spiked into
Liquid Waste No. 2 and Subjected to Alumina Cleanup 48
23 Alumina Cleanup Recoveries 49
XI
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TABLES (Continued)
Number Page
24 Elution Patterns and Percent Recoveries from the Silica
Gel/Celite Column Cleanup 52
25 Elution Patterns and Percent Recoveries from the Florisil/
Charcoal Cleanup 53
26 Recoveries of the Organochlorine Pesticides and PCBs
Subjected to the TBA-Sulfite Procedure 56
27 Recoveries of the Organochlorine Pesticides Spiked into
Various Matrices and Subjected to the TBA-Sulfite
Procedure 57
28 Summary of Organochlorine Pesticide Retention Times 60
29 Gas Chromatographic Conditions for the Determination of
Pesticides and PCBs by GC/EC Using a 1.5-Percent OV-17/
1.95-Percent OV-210 Column 61
30 Gas Chromatographic Conditions for the Determination of
Pesticides and PCBs by GC/EC Using a 3-Percent OV-1
Column 61
31 Gas Chromatographic Conditions for the Determination of
Pesticides and PCBs by GC/EC Using a DB-5 Fused-Silica
Capillary Column 62
32 Gas Chromatographic Conditions for the Determination of
Pesticides and PCBs by GC/EC Using an SPB-608 Fused-
Silica Capillary Column 63
33 Organochlorine Pesticides and PCBs Average Recoveries from
Silica Gel Chromatography, Determined on an OV-17/OV-210
Column 69
34 Organochlorine Pesticides and PCBs Average Recoveries from
Silica Gel Chromatography, Determined on a 3-percent OV-1
Column 70
XI1
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TABLES (Continued)
Number Page
35 Average Recoveries from Silica Gel Chromatography, Determined
on a 30 m x 0.25 mm ID DB-5 Fused-Silica Capillary Column ... 71
36 Average Recoveries from Silica Gel Chromatography, Determined
on an SPB-608 Fused-Silica Capillary Column .......... 72
37 Reproducibility of the Response Factors of the Organochlorine
Pesticides for 10 Consecutive Replicate Injections ....... 75
38 Reproducibility of the Retention Times of the Organochlorine
Pesticides for 10 Consecutive Replicate Injections ....... 76
39 Reproducibility of the Response Factors for 10 Consecutive
Injections of PCB-1016/1260 Standards ............. 77
40 Reproducibility of the Area Response for 10 Consecutive
Injections of PCB-1016/1260 Standards (Six Major Components). . 78
41 Reproducibility of the Retention Times of Six Major Components
in a PCB-1016/1260 Mixture for 10 Consecutive Injections. ... 79
42 Elution Patterns and Average Recoveries of the
Organochlorine Pesticides and PCBs after Silica Gel
Chromatography (Standards Only) ................ 82
43 Elution Patterns and Average Recoveries of
Organochlorine Pesticides and PCBs after Silica Gel
Chromatography (Liquid Waste No. 1 Extract) .......... 85
44 Elution Patterns and Average Recoveries of the
Organochlorine Pesticides and PCBs after Silica Gel
Chromatography (NBS Sediment Extract) ............. 88
45 Elution Patterns and Average Recoveries of the
Organochlorine Pesticides and PCBs after Silica Gel
Chromatography (Sandy Loam Extract) .............. 91
xiii
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TABLES (continued)
Number Page
46 Elution Patterns and Average Recoveries of the
Organochlorine Pesticides and PCBs after Silica Gel
Chromatography (Soil Extract Contamination with
Toxaphene) 94
47 Results of the Method Detection Limit Determination for
Organochlorine Pesticides and PCBs in Distilled Water 98
48 Results of the Method Detection Limit Determination for
Organochlorine Pesticides and PCBs in Sandy Loam Soil 99
xiv
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SECTION 1
INTRODUCTION
The Environmental Monitoring Systems Laboratory in Las Vegas, Nevada,
Environmental Protection Agency (EPA), has been responsible for evaluating some
of the methods published in "Test Methods for Evaluating Solid Waste —
Physical Chemical Methods" (SW-846) for use with materials characteristic of
the wastes regulated under the Resource Conservation and Recovery Act (RCRA).
One of these methods is Method 8080, entitled "Organochlorine Pesticides and
PCBs," which is used in the determination of chlorinated pesticides and
polychlorinated biphenyls (PCBs) (Table 1) in ground water and in both liquid
and solid matrices.
Acurex, under contract to the EMSL-LV, has been involved in the evaluation
of Method 8080. The first phase of the study addressed the fractionation of
the organochlorine pesticides and PCBs by Florisil chromatography and the
feasibility of substituting the Florisil fractionation procedure with a more
efficient method because the organochlorine pesticides are poorly separated
from the PCBs and toxaphene. Also, the gas chromatographic determination of
the organochlorine pesticides and PCBs using packed and capillary columns was
evaluated, and a literature review of the analytical methodologies for the
determination of the compounds listed in Method 8080 was conducted. Subse-
quent experiments, performed in the second phase, addressed the sample
extraction procedures that are recommended in Method 8080: separatory-funnel
extraction and continuous liquid-liquid extraction for aqueous samples, and
Soxhlet extraction and sonication for solids (Methods 3510, 3520, 3540, and
3550, respectively). In parallel, experiments were conducted to develop a
fractionation scheme using either silica gel or alumina chromatography or a
combination of either silica gel/Celite or Florisi 1/charcoal chromatography.
Upon completion of this phase, a revised protocol was prepared which was then
evaluated in the third phase.
The development of the revised method focused on improving both the
separation of the organochlorine pesticides from the PCBs prior to the gas
chromatographic analysis and their identification by using the resolving power
of a capillary column instead of that of a packed column. The analytical
scheme given in the revised Method 8080 protocol employs silica gel fractiona-
tion (silica gel deactivated with 3.3 percent water). Four fractions are
collected: Fraction I eluted with 80 mL hexane, Fraction II eluted with 50 mL
hexane, Fraction III eluted with 15 mL methylene chloride, and Fraction IV
eluted with 50 mL ethyl acetate. The determination of the organochlorine
pesticides and PCBs which are recovered in these four fractions is performed by
gas chromatography on a fused-silica capillary column and with electron capture
detection.
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TABLE 1. COMPOUNDS LISTED IN METHOD 8080
Parameter
alpha-BHC
beta-BHC
gamma-BHC (Lindane)
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
Storet No.
39337
39338
39340
34259
39410
39330
39420
39350
34361
39320
39380
39390
34356
39310
34366
39018
34351
39300
NA
39400
34671
39488
39492
39496
39500
39504
39508
CAS No.
319-84-6
319-85-7
58-89-9
319-86-8
76-44-8
309-00-2
1024-57-3
57-74-9
959-98-8
72-55-9
60-57-1
72-20-8
33212-65-9
72-54-8
7421-93-4
143-50-0
1031-07-8
50-29-3
72-43-5
8001-35-2
12674-11-2
1104-28-2
11141-16-5
55469-21-9
12672-29-6
11097-69-1
11096-82-5
NA — Storet number not available at the
time of report preparation.
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The revised protocol was evaluated in Phase III by using extracts of
environmental samples spiked with the substances of interest at known concen-
trations. The evaluation studies were conducted at three concentrations, each
in triplicate. The precision and accuracy results indicated that the revised
Method 8080 could be reliably applied to the determination of the organochlorine
pesticides and PCBs in liquid and solid matrices. A method detection limit
determination was performed for both the aqueous and the solid matrices.
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SECTION 2
CONCLUSIONS
Based on the results presented In this report, the following conclusions
can be drawn concerning the determination of the organochlorine pesticides and
PCBs:
° A revision of Method 8080 for the determination of the
organochlorine pesticides and PCBs was necessary because the
Florisil procedure does not allow separation of the organochlorine
pesticides from the multi-component PCB mixture (except for
endosulfans and endrin aldehyde) and because the packed column does
not have enough resolving power to handle complex environmental
samples.
° The revised Method 8080 presented in Appendix B has been evaluated
in a single laboratory with a few relevant liquid and solid
wastes. The utilization of silica gel fractionation and capillary
column analysis was found to be appropriate. When silica gel
fractionation was used, four fractions were collected. The silica
gel procedure is tedious and does account for a major part of the
analysis time. However, we have demonstrated that the method
precision is better than ±20 percent for all compounds, and the
accuracy is greater than 60 percent when standards are processed
through the silica gel procedure. Fraction IV may be combined with
Fraction III in those cases when Kepone is not expected to be present
and when the matrix is not very complex, and thus may reduce the
number of analyses per sample. Furthermore, the capillary column gas
chromatographic analysis allows complete separation of the five
organochlorine pesticides that elute with the PCBs in Fraction I.
The use of a second capillary column as a confirmatory column is
recommended although the presence of Kepone cannot be confirmed with
this column.
0 The detection limits of the revised method for water samples range
from 0.023 to 0.086 pg/L for the organochlorine pesticides and from
0.54 to 0.90 pg/L for the PCBs. The method detection limits for
solid samples is higher, ranging from 1.1 to 5.7 ug/Kg for the
organochlorine pesticides and from 57 to 70 pg/Kg for the PBCs.
These values are representative of clean samples. The actual method
detection limits should be determined for each matrix.
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Toxaphene, if present in the sample at concentrations 10 times as
high as the organochlorine pesticides, is likely to cause problems
in the determination of the organochlorine pesticides and PCBs since
it does not elute from the silica gel column in a narrow band.
Other analytical techniques (e.g., chemical ionization mass
spectrometry) should be investigated for the determination of
toxaphene.
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SECTION 3
RECOMMENDATIONS
The revised Method 8080 presented in this report has been evaluated in a
single laboratory by using a few relevant liquid and solid wastes. However,
the revised method should be evaluated in a number of different laboratories
on additional sample types. This would help identify the applicability range
of the method and define its inter!aboratory method performance.
When the revised Method 8080 is applied to samples which have not been
previously characterized, confirmation of compounds identified in the sample
may be required. Although the revised Method 8080 specifies a second
capillary column for confirmation, additional work should be performed to
determine how reliable the identification obtained by the multi-column
technique really is. Chemical derivatization techniques may prove helpful in
those cases where fewer compounds need to be confirmed. Furthermore, deter-
mination of the organochlorine pesticides and PCBs by gas chromatography/mass
spectrometry should be considered for future investigation since it would
certainly reduce the amount of work required to fractionate a sample extract,
to analyze fractions, and to confirm compound identifications.
The use of internal standards and surrogate spiking compounds needs to
be investigated. Much of the variability inherent to trace analysis in
difficult samples can be reduced by the use of internal standards. Likewise,
surrogate spiking compounds can provide recovery information for each sample.
These surrogate spiking compounds should be selected such that they will
elute into each of the four fractions collected from the silica gel column.
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SECTION 4
LITERATURE REVIEW
In the initial phase of this study, a literature review covering
analytical methods for the determination of the organochlorine pesticides and
polychlorinated biphenyls (PCBs) in water, soil, sediment, and sludge samples
was conducted. This literature review was performed by using the
Computerized Chemical Abstracts search as well as several review books
dealing specifically with the analysis of organochlorine pesticides and PCBs.
Furthermore, recent issues of Analytical Chemistry, the Journal of
Chromatography, the Journal of Agricultural and Food Chemistry, the Journal
of the Association of Official Analytical Chemists, and the Environmental
Science and Technology were searched to gather recent references that were
not yet in the computer data bases.
The computer searches were done by using DIALOG. Chemical Abstracts
files were searched back to 1972 for all references containing "pesticides,"
"PCBs," "gas Chromatography," "extraction," and "cleanup." These searches
printed out approximately 500 citations of which approximately 70 were judged
relevant to the objectives of this study.
Two books, "Analysis of Pesticides in Water," Volumes I and II, by Alfred
S. Y. Chau and B. K. Afghan, CRC Press, 1982, and "Analysis of Pesticide
Residues," edited by H. Anson Moye, John Wiley & Sons, 1981, have been
thoroughly reviewed for additional information and 'citations.
The literature review summary is included in Appendix A in the following
order:
__- »
8 Sample preservation techniques
8 Extraction techniques for organochlorine pesticides and PCBs from
water and soil (type of extraction technique, solvent, factors that
affect recovery)
8 Cleanup techniques
° Gas chromatographic analysis (column, retention time information,
Chromatography problems, etc.)
8 Compound confirmation (chemical derivatization techniques)
8 Stability of organochlorine pesticide solutions.
From the literature review presented in this report, it is evident that
there is sufficient information in the literature on sample extraction,
cleanup, and analysis for organochlorine pesticides and PCBs to form a data
base upon which to design future experiments. However, none of the information
-------
retrieved discusses specifically how to determine all of organochlorine
pesticides and PCBs listed in Method 8080 in hazardous liquid and solid
samples.
Appendix A was prepared in the summer of 1985; since then a few additional
relevant studies on this subject have been published and are worth mentioning
(1-4). One such study deals with the determination of PCBs in environmentally
contaminated sediments (1). Three sediment samples contaminated with PCBs
were analyzed by ten laboratories using standardized procedures for sample
extraction, extract preparation, and chromatography. Six laboratories used
electron capture detectors, and four laboratories used mass spectrometry.
Despite detailed written standardized procedures, there were large differences
in the results reported by the participating laboratories. Total PCB concen-
tration results were more precise than those of the individual PCB mixtures,
and the measurement precisions were better for the electron capture deter-
minations (relative standard deviation of 30 percent).
The approach of determining PCBs as various PCB formulations has several
disadvantages. One of them is that the gas chromatographic patterns in
environmental samples frequently are different from the corresponding Aroclor
patterns because of possible degradation and irreversible adsorption of some
PCBs in the environmental samples. Usually, PCBs enter the environment as
mixtures, and, thus, their determination is complicated. Most important of all
is that their determination as various Aroclor mixtures does not allow the
determination of individual congeners. A procedure to measure concentrations
of PCBs by using nine selected PCB congeners as concentration calibration
standards was developed (2). Concentrations are measured for each isomer group
with different levels of chlorination, and then the total PCB concentration is
calculated. Software was also developed (3) to perform fully automated identi-
fication and measurement of PCBs in environmental samples. These automated
procedures were capable of detecting more PCB congeners than the analyst could.
For example, 52 congeners were detected and measured by automated procedures
while only 42 were identified and measured by the analyst (3). The most impor-
tant aspect of the automation in this case was the cost savings: for each data
file, 12 to 15 hours were required by an experienced analyst to obtain the PCB
data, whereas 0.5 hour of an analyst's time and less than one hour of computer
file time were needed for automated data retrieval (3)7"
Another study, released recently by the EPA Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio, deals with the determination of organo-
chlorine pesticides and PCBs in water and soil samples by gas chromatography/
mass spectometry (4). Either full-range or selected-ion monitoring is
required; the choice of method depends on the pollutant levels to be deter-
mined. In the case of selected-ion monitoring, two analyses are necessary:
one to detect the organochlorine pesticides and one to detect the PCBs. The
PCBs are identified and measured as isomer groups distinguished by their level
of chlorination. This procedure, identified as EPA Method 680 (4), is still
being tested by EPA Cincinnati with real samples to obtain method precision
and accuracy information.
-------
A study worth mentioning which was not covered in the literature review
summary presented in Appendix A is the validation of the Soxhlet extraction
procedure (Method 3540). The objective of this study published by Michael,
et al. (5), was to conduct a single-laboratory evaluation of the Soxhlet pro-
cedure detailed in Method 3540 for the determination of hazardous waste
components in spent diatomaceous earth filter materials. The validation
consisted of two phases: in Phase I, accuracy and precision data were obtained
with diatomaceous earth samples fortified with various compounds including
4,4'-ODO and 4,4'-DDE; in Phase II, precision was determined by the analysis of
diatomaceous earth samples fortified with a pesticide manufacturing waste. The
results showed that Method 3540 is both accurate (average recovery ~90 percent)
and precise (average percent relative standard deviation ~8 percent) when
tested with diatomaceous earth samples.
-------
SECTION 5
METHOD 8080 EVALUATION
Method 8080, as published in the Office of Solid Waste document entitled
"Test Methods for Evaluating Solid Wastes," (6) provides sample extract
cleanup and gas chromatographic conditions for the determination of
part-per-billion (ppb) levels of organochlorine pesticides and PCBs in a
variety of environmental samples including ground water, liquids, and solids.
Following solvent extraction of the liquid samples using a separatory funnel
(Method 3510) or continuous liquid-liquid extractor (Method 3520) and of the
solid samples in a Soxhlet extractor (Method 3540) or with a sonicator
(Method 3550), the extracts are cleaned up by Florisil chromatography.
Elution of compounds from the Florisil column is performed with 6, 15, and
50 percent ethyl ether in hexane (200 ml each). All compounds listed in
Method 8080, except six organochlorine pesticides, elute in Fraction I. From
those six organochlorine pesticides, four (dieldrin, endosulfan II, endrin,
and endrin aldehyde) elute in Fraction II, and two (endosulfan II and
endosulfan sulfate) elute in Fraction III. Endrin aldehyde was also reported
to appear in Fraction III (26 percent recovery).
There are no indications in the protocol of Method 8080 about possible
overlapping of compounds between fractions and about the reproducibility of the
elution scheme. Moreover, the fact that the organochlorine pesticides are not
separated from the PCBs after Florisil cleanup is obviously an indication
that such a scheme would be of no use for samples contaminated with both
organochlorine pesticides and PCBs. Thus, the preliminary evaluation studies
of Method 8080 described in this section were intended to: (a) determine the
recoveries of the organochlorine pesticides and PCBs in the absence of matrix
interferences, (b) determine the extent of overlapping'of compounds between
fractions, and (c) determine the efficiency of such a cleanup scheme with real
samples.
Because of the complex nature of the PCB formulations, we have selected
PCB-1016 and PCB-1260 for the evaluation studies. PCB-1016 is very similar
to PCB-1242 in composition and contains about 40 percent chlorine. PCB-1260
contains about 60 percent chlorine. Thus, since we were mostly interested in
the elution patterns of the PCBs and their recoveries after sample cleanup, we
postulated that the use of the two PCBs will give us enough information about
the procedure, while allowing us to quantify the PCBs in the samples by visual
comparison of the sample extract chromatograms with PCB-1016 or PCB-1260
chromatograms.
10
-------
5.1 EXPERIMENTAL
Column Chromatography Procedure
Florisil was dried overnight at 130°C and was calibrated by the lauric
acid method (7). The amount of Florisil required per column was calculated
to be 11.6 g. Separate column runs were performed in duplicate for standards
of PCBs (PCB-1016 and PCB-1260), toxaphene, technical chlordane, pesticide
group A (gamma-BHC, heptachlor, aldrin, heptachlor epoxide, endosulfan I,
dieldrin, Kepone, endosulfan II, 4,4'-DDT, and endrin aldehyde) and pesticide
group B (alpha-BHC, beta-BHC, delta-BHC, 4,4'-DDE, endrin, 4,4'-DDD, endosulfan
sulfate, and 4,4'-methoxychlor). The amount of each compound added to the
columns is listed in Table 2.
Hexane extracts of environmental samples and standards in 2 to 3 ml hexane
were applied to the columns and eluted in 3 fractions: 200 ml 6-percent ethyl
ether in hexane (Fraction I), 200 ml 15-percent ethyl ether in hexane (Fraction
II), and 200 ml 50-percent ethyl ether in hexane (Fraction III). The fractions
were collected separately in Kuderna-Danish flasks, and each fraction was
concentrated to a 10-mL final volume in hexane. The fractions collected from
pesticide group A were exchanged to 2-percent methanol in benzene because
methanol has been found to enhance and stabilize the detector response of
Kepone when Kepone is dissolved in nonpolar solvents (8).
Sample Analysis
Sample eluates from the Florisil fractionation were analyzed by gas
Chromatography using a 63^ electron capture detector. The chromatographic
conditions are listed in Section 6.3 for the 1.5-percent OV-17/1.95-percent
OV-210 column. Pesticide and PCB concentrations in the samples were
determined by using external standard calibration.
5.2 RESULTS
The recoveries after Florisil cleanup are presented in Table 2 as percent
recovered for each compound in each fraction, as well as the total percent
recovered in the three fractions. The two sets of numbers for each fraction
represent duplicate determinations. The spiking level is given as total M9
loaded onto the Florisil column.
Overall, the recoveries are quantitative (except for Kepone), and the
agreement between the duplicate experiments is excellent. However, the
following discrepancies have been noted between this set of results and the
recovery data given in Method 8080:
o Dieldrin did not elute in Fraction II only but was recovered in both
Fractions I and II
° More Endosulfan I eluted in Fraction I than in Fraction II
11
-------
TABLE 2. RESULTS OF THE FLORISIL EVALUATION STUDY
Compound
Spike
level
(ug)
Fraction I Fraction II Fraction III
Total
0.5
0.5
0.5
0.5
0.5
0.5
0.5
5.0
pa
-------
° A higher percentage of Endosulfan II eluted in Fraction II and not
in Fraction III
» Endrin overlapped in Fractions I and II
« Endosulfan sulfate overlapped in Fractions II and III.
A possible explanation for the discrepancies might be the fact that the
Florisil calcination temperature influences its adsorption properties, as has
been reported (9). Difficulties in obtaining reproducible recoveries and
overlapping of pesticides between fractions have also been reported (7,9,10).
Regardless of the reproducibility of the fractionation scheme, it appears
that the Florisil fractionation is not suitable for those samples that are
contaminated with both organochlorine pesticides and PCBs. Because PCBs are
extracted and isolated along with the organochlorine pesticides, these two
types of analytes need to be separated from each other to a larger extent to
avoid cross-interference. To exemplify this on a real sample, we have extracted
a liquid waste, identified as liquid waste No. 1, and spiked the extract with
known amounts of the organochlorine pesticides and PCBs. Following fractiona-
tion of the extract on Florisil, the three fractions were analyzed by both
packed and capillary column chromatography. Figures 1 through 4 show GC/EC
chromatograms of the three fractions obtained from the unspiked and spiked
liquid waste No. 1 extract. Because of the complexity of this matrix we
were not able to determine any of the spiked compounds when the fractions
were analyzed on the OV-17/OV-210 packed column. Compound resolution was
significantly improved when the DB-5 capillary column was used; however, even
then we were not able to find all of the spiking compounds. In view of these
results, it does not appear that the Florisil fractionation should be
considered for further evaluation. Subsequent sections of this report address
the improvement of the methodology for the determination of the organochlorine
pesticides and PCBs and the selection of a new cleanup procedure that will be
further evaluated for incorporation in the Method 8080 protocol.
13
-------
52 !? 5 •
Figure 1. GE/EC chromatograms of the Florisil Fractions I (top),
II (middle), and III (bottom) of liquid waste No. 1
extract, analyzed on the 1.8 m x 2 mm ID 1.5-percent
OV-17/1.95-percent OY-210 column; isothermal at 190°C.
14
-------
::!•= ii- - *
I
if
1
f:
£25 H 55 f J? ! * ; : ! !
Figure 2. GE/EC chromatograms of the Florisil Fractions I (top),
II (middle), and III (bottom) of liquid waste No. 1
extract, spiked with the organochlorine pesticides
and PCBs, and analyzed on the 1.8 m x 2 mm ID 1.5-percent
OV-17/1.95-percent OY-210 column; Isothermal at 190eC.
15
-------
Figure 3. GE/EC chromatograms of the Florisil Fractions I (top),
II (middle), and III (bottom) of liquid waste No. 1
extract, analyzed on the 30 m x 0.25 mm ID DB-5 fused-
silica capillary column. Temperature program: 100°C
(hold 2 min) to 160°C at 15°C/min, then at 5°C/min to
270°C; helium at 16 psi.
16
-------
Figure 4. GC/EC chromatograms of the Florisil Fractions I (top),
II (middle), and III (bottom) of liquid waste No. 1
extract, spiked with the organochlorine pesticides and
PCBs, and analyzed on the 30 m x 0.25 mm ID DB-5 fused-
silica capillary column. Temperature program: 100°C
(hold 2 min) to 160°C at 15°C/min, then at 5°C/min to
270°C; helium at 16 psi.
17
-------
SECTION 6
METHOD IMPROVEMENT
Improvement of the method for the determination of the organochlorine
pesticides and PCBs in liquid and solid wastes focused on developing a
fractionation procedure to separate the PCBs from the organochlorine
pesticides and, at the same time, to remove interfering coextractants.
Several extract cleanup procedures (e.g., silica, alumina, silica gel/Celite,
Florisi 1/charcoal) were investigated, and a capillary gas chromatographic
method was developed. Furthermore, we have investigated to a limited extent
the extraction techniques recommended in the Method 8080 protocol. For
example, we have used the four extraction techniques (Methods 3510, 3520,
3540, and 3550) to prepare the extracts that were used to evaluate cleanup
techniques, and we have performed a ruggedness test for Method 3550 to
determine how sensitive the method is to changes in solvent composition,
sonicator output setting, volume of the extraction solvent, etc. The results
of these experiments led to the development of a revised protocol for the
analysis of the organochlorine pesticides and PCBs.
This section presents the results of the method improvement studies.
Extraction will be addressed first and will be followed by cleanup techniques
and gas chromatographic analysis.
6.1 EXTRACTION
The purpose of the extraction is to isolate quantitatively the compound(s)
of interest from the matrix. Standard methodologies include extraction of
liquid samples with an organic solvent using a separatory funnel or some sort
of stirring device, whereas solid samples are extracted"either in a Soxhlet
extractor or with a homogenizer, tumbler, or sonicator. Different coextract-
ants derived from the sample matrix may affect the determination of compounds
of interest; therefore, sample extracts must usually be subjected to cleanup
prior to analysis to reduce the amount of coextractants in the matrix. This
section addresses the evaluation of four standard methodologies for sample
extraction. These methodologies are identified as Methods 3510, 3520, 3540,
and 3550 in SW-846.
6.1.1 Extraction of Aqueous Samples
Experimental
One-liter, distilled-water samples were spiked in triplicate at two
concentrations (spike levels are listed in Tables 3 and 4), were extracted by
18
-------
TABLE 3. EVALUATION OF METHOD 3510 USING DISTILLED WATER SAMPLES
Spike level
(M9/D
Average Recovery ± SD (RSD)a
Compound
Cone. Cone.
1 2
Cone. 1
Cone. 2
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4, 4 '-DDE + DieldrinC
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
Toxaphene
PCB-1016
PCB-1260
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1.0
0.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.5
10.0
5.0
5.0
5
5
5
5
5
5
5
10
5
10
10
10
10
10
10
10
10
5
100
50
50
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
94.2
161°
90.4
90.0
70.8
72.0
78.6
73.5
86.9
64.0
98.0
63.0
72.9
72.2
58.5
70.6
84.8
76.7
93.6
84.8
91.9
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
9.1
17.5
8.9
11.0
7.9
12.9
9.5
7.1
6.8
7.4
14.9
6.9
8.3
8.4
43.7
8.5
7.0
10.1
6.8
6.8
7.1
(9.7)
(10.9)
(9.9)
(12.2)
(11.1)
(17.9)
(12.1)
(9.7)
(7.9)
(11.6)
(15.2)
(10.9)
(11.4)
(11.7)
(75.0)
(12.0)
(8.2)
(13.2)
(7.3)
(8.t)}
(7.7)
101
187°
98.8
98.0
85.5
88.9
85.2
80.8
99.8
74.6
77.8
69.6
77.6
86.6
71.5
77.3
76.9
74.1
98.2
87.5
87.0
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
2.8
19.6
3.1
5.3
4.4
3.2
3.8
4.1
6.6
5.2
7.6
7.0
9.3
8.7
27.3
7.9
9.7
9.8
11.9
18.0
15.3
(2.8)
(10.5)
(3.2)
(5.4)
(5.2)
(3.5)
(4.5)
(5.1)
(6.6)
(7.0)
(9.7)
(10.1)
(12.0)
(10.0)
(38.0)
(10.3)
(12.6)
(13.2)
(12.1)
(20.6)
(17.6)
SD ~ Standard deviation.
RSD — Relative standard deviation.
aNumber of determinations is 3.
DHigh recovery possibly because of contamination of unknown origin.
cSpike level of 4,4'-DDE and dieldrin is 0.5 ug/L for cone. 1 and 5.0 ug/L
for cone. 2 for each compound.
19
-------
TABLE 4. EVALUATION OF METHOD 3520 USING DISTILLED WATER SAMPLES
Spike level
(ug/L)
Average Recovery ± SD (RSD)a
Compound
Cone. Cone.
1 2
Cone. 1
Cone. 2
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Al dri n
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE + Dieldrinc
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4, 4 '-DDT
4,4'-Methoxychlor
Toxaphene
PCB-1016
PCB-1260
0
0
0
0
0
0
0
1
0
1
1
1
1
1
1
1
1
0
10
5
5
.5
.5
.5
.5
.5
.5
.5
.0
.5
.0
.0
.0
.0
.0
.0
.0
.0
.5
.0
.0
.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
10.0
5.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
5.0
100.0
50.0
50.0
102
183b
98.7
101
89.6
93.8
87.6
82.9
96.6
72.4
118
72.8
83.7
79.8
69.8
82.3
102
89.7
74.4
99.7
91.0
+
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
+
±
±
1.0
3.2
1.2
2.9
3.7
1.8
3.5
2.8
1.5
1.4
13.3
3.2
5.4
6.0
18.0
6.9
8.8
14.4
23.0
4.8
4.7
(1.0)
(1.8)
(1.2)
(2.9)
(4.1)
(1.9)
(4.0)
(3.3)
(1.6)
(2.0)
(11.3)
(4.4)
(6.4)
(7.6)
(25.9)
(8.4)
(8.6)
(16.1)
(31.0)
(4.8)
(5^2)
101
173b
98.6
96.1
85.2
90.5
84.0
80.1
98.6
71.4
80.5
66.9
74.4
84.6
66.2
76.0
75.6
72.9
80.8
97.9
92.9
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
2.5
6.0
2.3
2.4
2.4
2.1
2.1
1.9
2.5
2.7
4.7
1.3
1.9
2.6
8.3
2.0
2.0
1.8
14.8
10.7
5.1
(2.5)
(3.5)
(2.3)
(2.5)
(2.9)
(2.4)
(2.5)
(2.4)
(2.5)
(3.7)
(5.8)
(1.9)
(2.6)
(3.1)
(12.5)
(2.6)
(2.6)
(2.4)
(18.0)
(10.9)
(5.5)
SD ~ Standard deviation.
RSD — Relative standard deviation.
aNumber of determinations is 3.
bHigh recovery possibly because of contamination of unknown origin.
cSpike level of 4,4'-DDE and dieldrin is 0.5 ug/L for cone. 1 and 5.0 ug/L
for cone. 2 for each compound.
20
-------
using the procedures given in Methods 3510 and 3520 (Appendix C), and the
extracts were analyzed on a 30m x 0.25 mm ID DB-5 fused-silica capillary column
(J&W Scientific, Rancho Cordova, CA).
Experiments were also performed with two liquid wastes identified as
liquid wastes Nos. 1 and 2. Liquid waste No. 1 was collected from a
pesticide waste storage facility at a California agricultural field station.
This waste was found to contain high concentrations of organics (total
organic carbon 520 mg/L; total organic halogen 30 mg/L). The following
organochlorine pesticides were found in the sample (value given in
parentheses is concentration in ug/L): alpha-BHC (0.39), beta-BHC (0.52),
gamma-BHC (0.32), aldrin (0.05), heptachlor epoxide (0.11), gamma-chlordane
(0.11), endosulfan I (0.51), 4,4'-DDE (0.53), endosulfan II (29), 4,4'-DDD
(98), endrin aldehyde (220), and PCB-1260 (0.89). Liquid waste No. 2 was
obtained from a pesticide manufacturing plant. The manufacturer reported the
following: pH 7, NH3 as nitrogen 2200 mg/L, chemical oxygen demand 110,000
mg/L, chloride 21,000 mg/L, cyanide 8.6 mg/L, fluoride 330 mg/L, oil and grease
2400 mg/L, sulfate 12,000 mg/L, total dissolved solids 50,000 mg/L, potassium
1000 mg/L, sodium 16,000 mg/L, sodium acetate 1 percent, methanol 4.98 percent,
acetone 0.67 percent, isopropyl alcohol 0.8 percent, tetrahydrofuran 1.98
percent, dimethylformamide 0.2 percent, methoprene 312 mg/L, and hydroprene
40 mg/L.
Results
The results of the Methods 3510 and 3520 evaluation studies are given in
Tables 3 and 4. Only the results for the distilled water samples are presented
since the extracts were not subjected to any cleanup procedure prior to the gas
chromatographic analysis. The liquid wastes extracts were so complex that they
had to be subjected to alumina cleanup prior to analysis. Consequently, any
recovery data for spiked compounds in liquid wastes Nos. 1 and 2 had to be
lower than those for the distilled water because of losses associated with
alumina cleanup; they cannot be compared with those obtained for the distilled
water samples.
The following conclusions can be drawn from the data in Tables 3 and 4:
Methods 3510 and 3520 gave comparable accuracies when evaluated with
spiked distilled water samples. Except for Kepone and endosulfan II,
all compounds had recoveries >70 percent with either method.
8 The precision was better for Method 3520. Thirty-five out of 42 RSD
values for Method 3520 were below 8.6 percent, with most of these
values below 5 percent. In the case of Method 3510, only 14 out of
42 RSD values were below 8.6 percent.
21
-------
° In the case of liquid waste samples, it is difficult to conclude
from the recovery data whether one method is better than the other,
since cleanup was a must prior to the gas chromatographic analysis.
In the case of liquid waste samples, the volume of sample to be
extracted may be 100 ml, or less if the sample is highly contaminated.
In that case, Method 3520 is not desirable since additional organics-
free water has to be added to the sample for the continuous liquid-
liquid extraction, and, subsequently, there is the possibility of
introducing contamination.
6.1.2 Extraction of Soil Samples
Experimental
Sandy loam used in this study was obtained from Soils Incorporated,
Puyallup, Washington. The following physico-chemical characteristics were
determined: pH 5.9 to 6.0, 89 percent sand, 7 percent silt, 4 percent clay;
cation exchange capacity 7 meq/100 g, and total organic carbon content
1290 ± 185 mg/kg.
The spiked sandy loam samples (spike levels are listed in Tables 5 and 6)
were extracted by following the procedures detailed in Methods 3540 and 3550.
Ten-gram portions of sandy loam soil spiked with the organochlorine pesticides
(RGBs and toxaphene were spiked separately) were mixed with 10 g anhydrous
Na2S04, and the mixtures were then transferred to a Soxhlet extractor; 300 ml
of hexane-acetone (41:59) was used for the 16-hour extraction. Sonication was
also performed on 10-g portions of sandy loam soil. Water (3 ml) was added to
each sample to wet the soil. The spike was added to the soil slurry and was
allowed to equilibrate with the soil for 1 hour. Sonication was performed 3
times for 3 min with 60-mL portions of acetone-hexane (1:1). A sonicator Model
W-375 from Heat Systems-Ultrasonics, Inc., in pulsed mode at 50 percent duty
cycle, was employed. Sandy loam extracts were not subjected to any cleanup
prior to gas chromatographic analysis. All analyses were performed on a
30 m x 0.25 mm ID DB-5 fused-silica capillary column (J&W Scientific, Rancho
Cordova, CA).
Two other solid matrices, a National Bureau of Standards (NBS) reference
sediment material (SRM 1645) and a solid matrix consisting of activated
charcoal mixed with polymeric material, were also used in the evaluation
studies. The NBS SRM 1645 has the following physico-chemical properties:
Kjeldahl nitrogen, 0.0797 ± 0.26 percent; total phosphorus, 0.51 ± 0.0014
percent; loss on ignition (800°C), 10.72 ± 0.28 percent; oil and grease,
1.71 ± 0.26 percent; chemical oxygen demand, 149,400 ± 9,000 mg/Kg; SiOg,
51 percent; MgO, 4 percent; A1203, 4 percent; CaO, 4 percent; total organic
carbon, 30,000 mg/Kg. A physico-chemical characterization of the
charcoal/polymeric material matrix was not performed. Extraction of the
NBS SRM 1645 and of the solid matrix were performed as described above for
the sandy loam.
22
-------
TABLE 5. EVALUATION OF METHOD 3540 USING SANDY LOAM SAMPLES
Spike level
(ing/Kg)
Average Recovery ± SD (RSD)a
Compound
Cone. Cone.
1 2
Cone. 1
Cone. 2
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4 ,4 '-DDE + DieldrinC
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
Toxaphene
PCB-1016
PCB-1260
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.10
0.05
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.50
1.0
0.50
0.50
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1.0
0.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
5.0
1.0
5.0
5.0
50.4
84.8
52.4
34.0
46.0
47.5
61.5
52.8
51.8
67.9
92.1
45.5
76.2
16.2
17.1
46.9
66.7
75.9
35.0
70.9
71.8
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
29.2
6.2
27.7
35.7
23.4
22.9
28.5
13.5
14.6
10.0
37.5
22.1
12.5
7.6
16.2
31.6
31.4
11.3
10.5
26.5
16.7
(59)
(7.3)
(53)
(105)
(51)
(48)
(46)
(26)
(28)
(30)
(41)
(49)
(17)
(47)
(95)
(68)
(47)
(15)
(30)
(37)
(23)
53
115
55
43
46
49
59
56
64
60
77
50
74
22
50
63
70
66
59
86
.0
.8
.6
.5
.4
.5
.6
.2
.7
.2
.8
.8
.5
26,
.6
.3
.3
.1
.4
.9
±
±
±
±
±
±
±
±
±
±
±
±
±
±
,2t
±
±
±
±
±
±
33.8
71.4
33.6
43.7
23.8
23.1
21.2
20.2
10.5
12.9
13.6
6.2
5.7
6.1
>
15.1
7.8
6.9
19.7
23.2
14.9
(64)
(62)
(60)
(100)
(51)
(47)
(36)
(36)
(16)
(21)
(18)
(12)
(7.7)
(27)
(30)
(12)
(9.9)
(30)
(39)
(17)
SD ~ Standard deviation.
RSD — Relative standard deviation.
aNumber of determinations is 3.
^Kepone recovered only in one replicate.
cSpike level of 4,4'-DDE and dieldrin is 0.05 mg/Kg for cone. 1 and 0.5 mg/Kg
for cone. 2 for each compound.
23
-------
TABLE 6. EVALUATION OF METHOD 3550 USING SANDY LOAM SAMPLES
Spike level
(mg/Kg)
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Al dri n
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4, 4 '-DDE + Die! dri nc
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
Toxaphene
PCB-1016
PCB-1260
=====================
Cone.
1
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.10
0.05
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.50
1.0
0.50
0.50
Cone.
2
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1.0
0.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
5.0
1.0
5.0
5.0
Average
Recovery ± SD
Cone. 1
73 4
139b
72.1
59.1
71.2
73.2
72.8
66.4
58.7
75.4
95.3
48.8
71.2
89.4
18.3
61.5
66.4
73.0
48.1
72.1
83.3
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
6.8
5.6
5.0
34.1
2.4
2.2
3.1
1.2
0.6
1.3
5.3
2.2
3.3
58.8
23.2
4.9
4.7
4.9
16.8
6.8
4.3
(9.
(4.
(6.
(58)
(3.
(3.
(4.
(1.
(1.
(1.
(5.
(4.
(4.
(66)
(12.
(7.
(7.
(6.
(35)
(9.
(5.
2)
0)
9)
4)
0)
2)
7)
0)
7)
6)
5)
6)
6)
9)
1)
7)
_
4)
2)
(RSD)a
Cone. 2
80.4
150°
82.1
82.3
68.4
73.2
69.7
66.1
69.5
63.7
69.0
51.1
63.8
29.7
23.9
60.2
58.1
58.6
77.3
89.4
80.6
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
5.5
9.2
2.1
2.6
4.7
5.1
4.7
4.1
4.6
3.6
4.3
3.0
3.7
4.2
4.4
4.0
4.4
3.2
18.5
11.9
7.9
(6.8)
(6.2)
(2.5)
(3.2)
(6.9)
(7.0)
(6.8)
(6.3)
(6.6)
(5.6)
(6.2)
(5.9)
(5.8)
(14.2)
(18.3)
(6.6)
(7.5)
(5.5)
(24)
(13.3)
(9.8)
========
SD — Standard deviation.
RSD — Relative standard deviation.
aNumber of determinations is 3.
bHigh recovery possibly because of interference from matrix.
GSpike level of 4,4'-DDE and dieldrin is 0.05 mg/Kg for cone. 1 and
0.5 mg/Kg for cone. 2 for each compound.
24
-------
Spiking the NBS sediment and the solid matrix with the substances of
interest was performed by adding 3 and 5 ml methanol, respectively, to wet
the solids and to allow for better mixing of the spike with the solid. In
each case, the solid material was allowed to dry at room temperature for
approximately 30 min prior to extraction. All extracts except those from sandy
loam were cleaned up by using alumina deactivated by 15 percent water.
Results
The results of the Methods 3540 and 3550 evaluation studies are given in
Tables 5 and 6. Only the results for the sandy loam samples are presented
since the extracts were not subjected to any cleanup prior to the gas chromato-
graphic analysis. The NBS sediment and solid material extracts were subjected
to alumina cleanup prior to analysis. Consequently, any recovery data for the
spiked compounds in the NBS sediment and the solid wastes were lower than those
obtained for the sandy loam because of losses associated with alumina cleanup,
and they cannot be compared with the recovery data obtained for the sandy loam
samples.
The following conclusions can be drawn from the data presented in
Tables 5 and 6:
° Method 3550 appears to give better precision than Method 3540.
Thirty-four out of 42 RSD values for Method 3550 were below
9.8 percent. Most of the RSD values for Method 3540 were in the
30- to 40-percent range.
° The accuracies of both methods were similar. Low recoveries
(~20 percent) were found for Kepone by either method.
° Spiking of the matrix with the test compounds may have contributed
to the variability of the extraction recoveries. In the case of
Method 3540, separate sample portions were spiked in Petri dishes and
then were transferred to the Soxhlet extractor. Water was added to
the sandy loam samples to slurry and to distribute the spike.
Methanol was added to the sediment reference material since
water did not seem to wet the sediment. Spike volatilization from the
open Petri dish and some loss during the transfer of the solid material
to the Soxhlet extractor cannot be ruled out.-" In the case of Method
3550, the solid material portions were spiked when they were already
in the extraction jar; thus, losses associated with sample transfer
were probably small.
° The determination of the compounds is affected by interferences from
the matrix. In the case of sandy loam which gave relatively clean
extracts, all compounds could be determined by either method; Method
3550 gave better precision than Method 3540. With the standard refer-
ence material and the solid matrix, matrix interferences precluded
the identification of 4,4'-DDT, toxaphene, endosulfan I, dieldrin,
4,4'-ODE, endrin, endosulfan II, and gamma-chlordane even after
alumina cleanup.
25
-------
6.1.3 Ruggedness Test for Method 3550
A ruggedness test was performed for Method 3550 to determine how sensitive
the method is to changes of the parameters listed in Table 7: extraction sol-
vent composition, sonicator output setting, solvent volume, moisture content,
analyte concentration, extract filtration, and the number of times the extrac-
tion was repeated. The seven parameters are assigned the letters A through G,
and the changed values are assigned the same letters in the lower case. For
example, the extraction solvent is hexane-acetone (1:1) in experiments 1
through 4 and hexane-acetone (3:1) in experiments 5 through 8.
TABLE 7. COMBINATIONS OF 7 PARAMETERS RECOMMENDED FOR
TESTING THE RUGGEDNESS OF METHOD 3550
Experiment no.
Condition
Extraction solvent composition
(hexane-acetone 1:1; 3:1)
Sonicator output setting
(8; 9)
Solvent volume
(60 ml, 120 ml)
Volume of water added to soil
(0; 3 ml)
Analyte concentration
(10 ppb; 250 ppb)
Extract filtration
(Celite; Whatman filter)
Number of times the extraction
was repeated (1; 3)
Result (percent recovery of each
test compound)
1
A
B
C
D
E
F
G
s
2
A
B
c
D
e
f
g
t
3
A
b
C
d
E
f
g
u
4
A
b
c
d
e
F
G
V
5
a
B
C
d
e
F
g
w
6
a
B
C
d
E
f
G
X
7
a
b
C
D
e
f
G
y
8
a
b
c
D
E
F
g
z
===========================
26
-------
YA =
YB =
VC =
YD =
YE =
YF =
YG =
l/4(s
l/4(s
l/4(s
l/4(s
l/4(s
l/4(s
l/4(s
+ t
+ t
+ u
+ t
+ u
+ V
+ V
+
+
+
+
+
+
+
u ^
w ^
w H
y H
X H
W H
X H
k V)
> x)
>y)
•• z)
>• z)
1- 2)
>y)
-l/4(w H
-l/4(u -i
-l/4(t H
-l/4(u H
-l/4(t H
-l/4(t H
-l/4(t H
H X
1- V
I- V
H V
1- V
I- U
1- U
+
+
+
+
+
+
+
y +
y +
X +
w +
w +
X +
w +
z)
z)
z)
x)
y)
y)
z)
= A-a
= B-b
= C-c
= D-d
= E-e
= F-f
= G-g
The analytical results are presented as percent recovery for each
organochlorine pesticide in each of the eight experiments (Table 8).
Table 9 shows the group differences VA through VG which were calculated
from equations 1 through 7:
(1)
(2)
(3)
(4)
(5)
(6)
(7)
For example, VA for alpha-BHC represents the average for the A
determinations minus the average for the a determinations [e.g., VA = 1/4(82
+80+86 +68) -1/4(85 + 77 + 82 + 93) = -5.25], which means that
determinations when hexane-acetone (1:1) was used as extraction solvent gave
lower average recoveries than the determinations when hexane-acetone (3:1) was
used as the extraction solvent. Furthermore, for alpha-BHC, parameters such as
the number of times the extraction was repeated, the analyte concentration, the
hexane-to-acetone ratio, and the moisture content of the soil, stand out in
Table 9 as having greater effects on the analytical results than the other
three parameters. No attempt was made to determine the significance of these
four large differences, only those parameters were identified that may be
considered for further investigation.
The following is a brief summary of the parameters which seem to affect
the performance of the method:
° The change in the composition of the solvent was found to affect the
performance of the method for almost all compounds; it is
interesting to note that the algebraic signs of the differences in
Table 9, Column 1, are mostly negative. This indicates that for
most compounds extraction with hexane-acetone (3:1) gave higher
recoveries.
• Sonicator output setting, solvent volume, and_extract filtration did
not appear to affect the performance of the method.
° As expected, improved recoveries were obtained when the extraction
was repeated three times.
8 Analyte concentration appeared to have a strong effect upon compound
recovery; this was quite evident for dieldrin and 4,4'-DDE for which
YE was found to be 50.75, and for endrin aldehyde for which YE was
found to be -10.75. Gas chromatographic analysis probably plays a
major role in both cases since in the case of dieldrin and 4,4'-DDE
the detector response was slightly saturated when the extract from the
250 ppb soil was analyzed, whereas for endrin aldehyde the response
was not linear at lower concentrations because of the possible
degradation of endrin which was also present in the sample extract.
27
-------
TABLE 8. RUGGEDNESS TEST FOR METHOD 3550 — RECOVERY
DATA FOR THE ORGANOCHLORINE PESTICIDES
Percent recovery
Experiment
1
(s)
2
(t)
3
(u)
4
(v)
5
(w)
6
(x)
7
(y)
8
(z)
Compound
alpha-BHC 82 80 86 68 85 77 82 93
beta-BHC 73 80 72 64 82 62 81 81
gamma-BHC NA 91 NA 103 95 NA 89 NA
delta-BHC 79 75 82 66 86 83 84 101
Heptachlor 132 82 97 72 88 86 84 NA
Aldrin 88 82 86 67 85 73 84 101
Heptachlor epoxide 77 80 81 63 84 73 84 90
gamma-Chlordane 95 81 79 64 85 72 83 92
Endosulfan I 112 77 76 65 75 83 76 88
4,4'-DDE + Dieldrin 110 47 106 37 49 95 95 120
Endrin 105 86 100 73 112 110 101 145
Endosulfan II 99 79 78 66 87 77 86 93
4,4'-ODD 99 81 89 66 89 87 86 106
Endrin aldehyde 53 65 61 67 71 58 76 64
Endosulfan sulfate NA 80 70 67 87 74 85 98
4,4'-DDT NA 87 112 67 93 137 88 158
4,4'-Methoxychlor 121 83 107 74 92 112 88 139
NA — Not able to determine recovery because of interference.
Based on the results of the ruggedness test, it seemed that additional
work would be required to determine how the extraction recoveries vary with the
change in the composition of the extraction solvent. Therefore, other experi-
ments were performed in which sandy loam soil spiked with the organochlorine
pesticides was extracted with hexane/acetone (60 ml) mixtures using sonication
(sonicator output setting: 8, sonicator time: 3 min). The extraction was
performed with hexane/acetone 1:1, 2:1, 3:1, and hexane only, and was repeated
three times in each instance. The extracts were filtered through a Whatman
filter. The analyte concentration was kept constant at 250 ppb for each
compound.
28
-------
TABLE 9. RUGGEDNESS TEST -- GROUP DIFFERENCES FOR THE ORGANOCHLORINE
PESTICIDES
Compound
VA
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma- Chlordane
Endosulfan I
4, 4 '-DDE + Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Endosulfan sulfate
4, 4 '-DDT
4,4'-Methoxychlor
-5.25
-4.25
a
-13.0
a
-5.00
-7.50
-3.25
2.00
-14.75
-26
-5.25
-8.25
-5.75
a
a
-11.50
-1.25
0
a
-2.50
a
-2.50
-1.00
3.75
10.50
-14.25
-1.50
4.75
2.25
-5.25
a
a
0
4.25
5.25
a
1.50
a
5.00
5.00
8.25
6.50
15.25
1.00
8.75
5.75
1.75
a
a
0
5.25
8.75
a
5.50
a
11.00
7.50
12.75
13.50
21.25
10.50
12.25
10.25
0.25
a
a
11.50
5.75
-4.75
a
8.50
a
7.50
2.50
6.25
16.50
50.75
22.00
7.25
14.75
-10.75
a
a
35.50
0.75
1.25
a
2.00
a
4.00
-1.00
5.25
7.00
-6.75
9.50
6.25
4.25
-1.25
a
a
9.00
-8.75
-8.75
a
-8.00
a
-10.5
-9.50
-5.75
5.00
3.75
-13.5
-2.25
-6.75
-1.75
a
a
-6.50
aNot able to calculate because some recovery values are missing.
The results are presented in Table 10. It can be concluded that overall
the recoveries were slightly higher when hexane was used as the extraction
solvent, however, the increase was insignificant and does not warrant a change
in the Method 3550 protocol.
6.2 EXTRACT CLEANUP
6.2.1 Silica Gel Cleanup
The silica gel fractionation was performed as specified in a procedure
developed by Biddleman, et al. (11), with slight modifications. The silica
gel (Davidson 923, 100/200 mesh, Supelco, Inc.) was dried for at least
16 hours at 130 to 140°C. Three-g portions were weighed into vials, were
redried for 1 hour, and were deactivated with 0.10 mL of water. The contents
of each vial were mixed thoroughly and were equilibrated for an additional
hour; afterwards, they were emptied into 11-mm-ID glass columns and were
topped with 2 to 3 cm of sodium sulfate. Twenty mL dichloromethane followed
by 20 mL hexane were used to wash the column.
29
-------
TABLE 10. RUGGEDNESS TEST — PERCENT RECOVERIES OF THE ORGANOCHLORINE
PESTICIDES AS A FUNCTION OF SOLVENT COMPOSITION
00
o
Percent Recovery
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE + Dieldrin
Endrin
Endosulfan II
4, 4 '-ODD
Endrin aldehyde
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
Average ± SD (%RSD)
Hexane-Acetone
1:1
75.2
74.2
75.3
73.0
76.8
77.2
74.0
73.8
73.1
82.8
94.5
73.3
75.2
66.6
73.2
79.5
89.6
1
t
76.9 ± 6.7 (8.7)
===================
Hexane-Acetone
2:1
64.3
69.2
70.4
67.2
53.6
53.5
63.9
62.1
60.1
70.0
72.7
60.1
61.9
43.5
56.5
53.1
60.9
61.4 ± 7.6 (12.4)
Hexane-Acetone
3:1
79.7
75.8
78.2
76.9
79.2
75.0
75.0
75.2
72.0
81.2
90.3
73.3
74.2
56.1
71.0
73.0
78.9
75.6 ± 6.7 (8.9)
Hexane
Only
87.1
76.6
85.0
78.5
86.7
84.7
80.8
82.5
80.2
82.8
88.4
76.4
77.1
65.9
71.9
75.0
80.3
80.0 ± 5.9 (7.3)
SD — Standard deviation.
RSD ~ Relative standard deviation.
-------
Separate experiments were run in triplicate and at two concentrations for
each set of organochlorine pesticides and PCBs mixed standards. Technical
chlordane and toxaphene standards were run separately. Standards of the
individual pesticides (alpha-BHC, beta-BHC, gamma-BHC, delta-BHC, heptachlor,
aldrin, heptachlor epoxide, endosulfan I, dieldrin, 4,4'-DDE, Kepone, endrin,
endosulfan 11, 4,4'-DDD, 4,4'-DDT, endrin aldehyde, endosulfan sulfate, and
4,4'-methoxychlor) and PCB-1016 and PCB-1260 were run together. The amount of
each compound added to the columns is given in Table 11.
The spikes were added to the column in 3 to 4 mL of hexane and were eluted
in four fractions with 80 ml hexane (Fraction I), an additional 50 ml hexane
(Fraction II), 15 ml methylene chloride (Fraction III), and 50 mL ethyl acetate
(Fraction IV). The fractions were collected separately in Kuderna-Danish
flasks and were concentrated to a 10-mL final volume in hexane. Because
methanol has been found to enhance and stabilize the response of Kepone, the
ethyl acetate (Fraction IV) was exchanged to 2 percent methanol in benzene.
Six extracts from distilled water, liquid waste No. 1, liquid waste No. 2,
sandy loam, NBS sediment, and solid waste were also subjected to the silica gel
cleanup. Two-milliliter aliquots from each extract were spiked with 500 or
5,000 ng of each organochlorine pesticide and with 5,000 or 50,000 ng of
PCB-1016 and PCB-1260. Technical chlordane was replaced by gamma-chlordane; it
was part of the organochlorine pesticides spike. Kepone was not spiked at all;
however, we did collect and analyze Fraction IV of each extract to verify that
none of the organochlorine pesticides eluted in Fraction IV. Toxaphene was not
used since toxaphene does not elute from the silica gel column in a narrow band
as do the other compounds, and it may preclude the determination of the organo-
chlorine pesticides and PCBs.
Tables 12 through 19 present the data for the 6 matrix extracts spiked
with the organochlorine pesticides and PCB-1016 and PCB-1260. For each matrix,
unspiked extracts were also processed through the silica gel column to
determine which of the organochlorine pesticides and PCBs listed in Method 8080
was present in the unspiked sample extracts.
The following conclusions can be drawn from these data:
° The distribution patterns of the organochlorine pesticides and PCBs
in the four fractions were quite reproducible, with a few exceptions
(i.e., liquid waste No. 2). Compounds found to elute in Fraction I
include heptachlor, aldrin, 4,4'-DDE, 4,4'-DDT, and PCBs. Almost
all the other organochlorine pesticides elute in Fraction III. Only
a few compounds (4,4'-ODD, alpha-BHC, gamma-chlordane) were found in
Fraction II. The advantage of the four-fraction scheme over the
Florisil scheme is in the separation of the PCBs from most of the
organochlorine pesticides.
° All compounds were quantitatively recovered from the silica gel column.
Total recoveries, listed in Tables 11 through 19, were greater than 70
percent, with most values ranging from 80 to 110 percent. Exceptions
were Kepone and technical chlordane when they were spiked at
concentration 1 (Table 11).
31
-------
TABLE 11. DISTRIBUTION AND PERCENT RECOVERIES OF THE OR6ANOCHLORINE
PESTICIDES AND PCBs IN THE SILICA GEL COLUMN FRACTIONSa»t>,c,d,e
u>
Fraction I
Fraction II
Fraction III
Fraction IV
Total recovery
Compound
Cone. 1 Cone. 2 Cone. 1
alpha-BHC
beta-BHC
gamma-BBC
delta-BHC
Heptachlor 109 (4.1) 118 (8.7)
Aldrin 97 (5.6) 104 (1.6)
Heptachlor epoxide
Endosulfan I
4,4'-DDE 86 (5.4) 94 (2.8)
Dieldrin
Endrin
Endosulfan II
4,4'-ODO
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT 86 (13.4)
4,4'-Methoxychlor
PCB-1016 86 (4.0) 87 (6.1)
PCB-1260
Technical
chlordane
Toxaphene
91 (4.1) 95 (5.0)
14 (5.5) 22 (5.3) 19 (6.8)
115 (2.4)
Cone. 2
73 (9.1)
39 (3.6)
17 (1.4)
Cone. 1 Cone. 2 Cone. 1
82 (1.7) 74
107 (2.1) 98
91 (3.6) 85
92 (3.5) 83
95 (4.7.) 88
95 (5.1) 87
96 (6.0) 87
85 (10.5) 71
97 (4.4) 86
102 (4.6) 92
81 (1.9) 76
93 (4.9) 82
15 (18.7) 8.7
99 (9.9) 82
29 (5.0) 37
73 (9.4) 84
(8.0)
(12.5)
(10.7)
(10.6)
(10.2)
(10.2)
(10.6)
(12.3)
(10.4)
(10.2)
(9.5) 29 (4.2)
41 (7.9)
(9.2)
' (15.0)
(10.7) 9.0 (4.8)
(5.1)
(10.7)
Cone, 2 Cone. 1
82 (1.7)
107 (2.1)
91 (3.6)
92 (3.5)
109 (4.1)
97 (5.6)
95 (4.7)
95 (5.1)
86 (5.4)
96 (6.0)
85 (10.5)
97 (4.4)
102 (4.6)
33 (4.7) 110 (3.0)
73 (12.0) 41 (7.9)
93 (4.9)
101 (5.3)
5.7 (3.0) 108 (10.0)
86 (4.0)
91 (4.1)
62 (3.3)
88 (12.0)
Cone. 2
74 (8.0)
98 (12.5)
85 (10.7)
83 (10.6)
118 (8.7)
104 (1.6)
88 (10.2)
87 (10.2)
94 (2.8)
87 (10.6)
71 (12.3)
86 (10.4)
92 (10.2)
109 (5.8)
73 (12.0)
82 (9.2)
82 (23.7)
88 (5.8)
87 (6.1)
95 (5.0)
98 (1.9)
101 (10.1)
aEluant composition:
Fraction I - 80 mL hexane.
Fraction II - 50 ml hexane.
Fraction III - 15 mL methylene chloride.
Fraction IV - 50 mL ethyl acetate.
Concentration 1 is 0.5 ug per column for BHCs, heptachlor, aldrin, heptachlor epoxide, endosulfan I; 1.0 ug per column for dieldrin,
Kepone, endosulfan II, 4,4'-DDT, endrin aldehyde, 4,4'-ODD, 4,4'-ODE, endrin, and endosulfan sulfate; 5 ug per column for
4,4'-itiethoxychlor and technical chlordane; 10 ug per column for toxaphene, PCB-1016,and PCB-1260.
cFor concentration 2 the amounts spiked are 10 times as high as those for concentration 1.
dThe values given represent the average recoveries of three determinations; the numbers in parentheses are the standard deviations;
recovery cutoff point is 5 percent.
eData obtained with standards, as indicated in footnotes b and c, dissolved in 2 mL hexane.
-------
TABLE 12. DISTRIBUTION AND PERCENT RECOVERIES OF THE
ORGANOCHLORINE PESTICIDES AND PCBs SPIKED INTO
THE DISTILLED WATER EXTRACTS (SPIKE LEVEL IS
500 NG FOR PESTICIDES AND 5,000 NG FOR PCBs)
Percent recovery
Compounds
Fraction Fraction Fraction Fraction
I II III IV Total
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PC8-1016)
PCB-1260J
100
88
60
112
92
97
36 56
79
95
83
85
39
83
97
20
83
17 70
87
82
71
92
79
95
83
100
88
85
99
83
112
97
20
83
87
9.8 97
NS NS
16 98
92
71
97
NS -- Not spiked.
Although total recoveries were quantitative for all compounds spiked
into liquid waste No. 2 followed by fractionation on silica gel, the
distribution patterns of the compounds were affected by the fact that
the extract was applied to the column in benzene (because of solu-
bility problems) and not in hexane.
From the examination of the GC/EC chromatograms it is evident that
even for very complex matrices such as liquid waste No. 1 (Figures 5
and 6), liquid waste No. 2, and the NBS sediment material, the
fractionation of the extract on the silica gel column allows precise
determination of the organochlorine pesticides and PCBs.
33
-------
TABLE 13. DISTRIBUTION AND PERCENT RECOVERIES OF THE
ORGANOCHLORINE PESTICIDES AND PCBs SPIKED INTO
THE LIQUID WASTE NO. 1 EXTRACT (SPIKE LEVEL IS
500 N6 FOR PESTICIDES AND 5,000 NG FOR PCBs)
Percent recovery
Fraction Fraction Fraction Fraction
Compounds I II III IV Total
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-10161
PCB-1260J
====================
121
123
78
NA
NA
131
95
=========
41 76
134
64
218a
135
57
118
160a
NA
121
NA
NS
NA
118
=============================
117
134
64
218a
121
123
135
135
118
NA
160a
NA
121
NA
NA
NS
NA
131
118
95
NA — Not able to quantify because of interference.
NS ~ Not spiked.
aHigh recovery probably because of matrix interference.
Careful control of adsorbent activity (i.e., water content) is
essential for reproducible separations. The silica gel material
should be tested prior to use by using standards at concentrations
equivalent to those expected to be found in the samples.
34
-------
TABLE 14. DISTRIBUTION AND PERCENT RECOVERIES OF THE
ORGANOCHLORINE PESTICIDES AND PCBs SPIKED INTO
THE LIQUID WASTE NO. 1 EXTRACT (SPIKE LEVEL IS
5,000 NG FOR PESTICIDES AND 50,000 NG FOR PCBs)
Percent recovery
Compounds
Fraction Fraction Fraction Fraction
I II III IV Total
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
36 55
89
59
NA
97
91
89
59
NA
97
Aldrin 85
Heptachlor epoxide
gamma-Chlordane 65
Endosulfan I
4,4'-DDE 113
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT 79
4,4'-Methoxychlor
PCB-10161 84
PCB-12601
24
11
86
88
110
76
163a
74
80
156a
140a
NS
85
86
89
88
113
110
76
163a
85
80
NS
156a
79
140a
84
NA — Not able to quantify due to interference.
NS — Not spiked.
aHigh recovery probably due to matrix interference.
Interference from phthalate esters was very briefly assessed using
diethylphthalate, di-ni-butyl phthal ate and di-n-octylphthalate. Five
micrograms of each phthalate ester were spike? onto the silica gel column and
were eluted as described above for the organochlorine pesticides and PCBs.
The di-n-butyl- and di-n-octylphthalates were found in Fraction III
(recoveries were 60 and 84 percent, respectively), and the diethylphthalate
35
-------
TABLE 15. DISTRIBUTION AND PERCENT RECOVERIES OF THE
ORGANOCHLORINE PESTICIDES AND PCBs SPIKED INTO
THE LIQUID WASTE NO. 2 EXTRACT (SPIKE LEVEL IS
500 NG FOR PESTICIDES AND 5,000 NG FOR PCBs)
Percent recovery
Compounds
Fraction Fraction Fraction Fraction
I II III IV Total
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
72
32
47
20
96
21
7.2
20
3.9
6.5
46
41
65
100
85
108
89
96
Aldrin 85
Heptachlor epoxide 7.4
gamma-Chlordane 87 2.5
Endosulfan I 4.9
4,4'-DDE 103
Dieldrin
Endrin
Endosulfan II
4,4'-DDD 81 18
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT 86
4,4'-Methoxychlor
PC3-1016) 99
PCB-1260J
NS — Not spiked.
78
81
106
101
87
9.4
70
80
78
NS
85
85
90
86
103
106
101
87
108
70
NS
80
86
78
99
was recovered in Fraction IV. The remainder of the di-n-butylphthalate
(~50 percent) was also recovered in Fraction IV. Additional experiments will
be necessary to assess the impact of the possible interferences from phthalate
esters.
6.2.2 Alumina Cleanup
Alumina cleanup is currently recommended by EPA in the Contract Laboratory
Program Protocol (12). Hexane/acetone extracts are subjected to neutral alu-
mina cleanup on Super I Woelm or equivalent (Universal Scientific, Atlanta, GA,
36
-------
TABLE 16. DISTRIBUTION AND PERCENT RECOVERIES OF THE
ORGANOCHLORINE PESTICIDES AND PCBs SPIKED INTO
THE LIQUID WASTE NO. 2 EXTRACT (SPIKE LEVEL IS
5,000 NG FOR PESTICIDES AND 50,000 NG FOR PCBs)
Percent recovery
Compounds
Fraction Fraction Fraction Fraction
I II III IV Total
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4, 4 '-ODD
Endrin aldehyde
Kepone
Endosulfan sulfate
4, 4 '-DDT
4,4'-Methoxychlor
PCB-1016)
PCB-1260)
77
50
46
36
76
85
88
117
79
92
81
24
7.0
26
6.2
8.5
3.4
3.7
18
3.2
46
38
63
73
88
131
86
98
5.6
89
98
86
104
103
110
105
76
85
82
91
92
117
131
86
98
103
89
NS NS
98
92
86
81
NS ~ Not spiked.
or equivalent) of activity III (3 g). The organochlorine pesticides and PCBs
are eluted with 9 mL hexane. Several reports published in the literature (13,
14) that described alumina cleanup indicated that alumina is more efficient
than silica gel and Florisil in removing naturally occurring organic a'cids,
pigments, and other interferences.
37
-------
TABLE 17. DISTRIBUTION AND PERCENT RECOVERIES OF THE
ORGANOCHLORINE PESTICIDES AND PCBs SPIKED INTO
THE SANDY LOAM EXTRACT (SPIKE LEVEL IS 500 NG
FOR PESTICIDES AND 5,000 NG FOR PCBs)
Percent recovery
Fraction Fraction Fraction Fraction
Compounds I II III IV Total
alpha-BHC 74
beta-BHC
gamma-BHC
delta-BHC
Heptachlor 114
Al dri n 100
Heptachlor epoxide
gamma-Chlordane 96 8.5
Endosulfan I
4,4'-DDE 125
Dieldrin
Endri n
Endosulfan II
4,4'-DDD 63
Endri n aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT 118
4,4'-Methoxychlor
PCB-10161 94
PCB-1260/
14
98
85
97
107
81
138
86
105
43
93
NS
106
97
88
98
85
97
114
100
107
105
81
125
138
86
105
106
93
NS
106
118
97
94
NS — Not spiked.
38
-------
TABLE 18. DISTRIBUTION AND PERCENT RECOVERIES OF THE
ORGANOCHLORINE PESTICIDES AND PCBs SPIKED INTO
THE NBS SEDIMENT EXTRACT (SPIKE LEVEL IS 500 NG
FOR PESTICIDES AND 5,000 NG FOR PCBs}
Percent recovery
Compounds
Fraction Fraction Fraction Fraction
I II III IV Total
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4, 4' -DDT
4,4'-Methoxychlor
PCB-1016J
PCB-1260J
19 69
9.4
90
86
80 16
107
11 53
79
68
26
108
80
97
97
94
120
90
95
43
86
-•
92
108
114
108
89
97
90
86
97
96
94
107
120
90
95
107
86
NS NS
92
79
108
68
NS -- Not spiked.
39
-------
TABLE 19. DISTRIBUTION AND PERCENT RECOVERIES OF THE
ORGANOCHLORINE PESTICIDES AND PCBs SPIKED INTO
THE SOLID WASTE EXTRACT (SPIKE LEVEL IS 500 NG
FOR PESTICIDES AND 5,000 NG FOR PCBs)
Percent recovery
Compounds
Fraction Fraction Fraction Fraction
I II III IV Total
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016)
PCB-1260J
NS — Not spiked.
51
107
96
89
108
32
98
75
48 15
90
26 58
95
100
11
96
109
121
92
52
82
NS
97
92
--
114
90
84
95
107
96
100
100
96
108
109
121
92
84
82
NS
97
98
92
75
The experiments presented in this section address the applicability of
alumina cleanup to the two liquid matrices investigated in this study. In
the case of liquid waste No. 1, a 1-mL aliquot of sample extract was loaded
to a 3-g neutral Super I Woelm alumina activity III column and was eluted
with 10 mL hexane. Activity III was prepared by deactivating alumina with
7 percent water. Recovery of the organochlorine pesticides using alumina
cleanup was determined by using standards as well as liquid waste No. 1
extract spiked with 0.5 to 5 pg of the compounds listed in Tables 20 and 21.
40
-------
•ir
£.
Figure 5. GC/EC chromatograms of the silica gel Fractions I (top),
II (middle), and III (bottom) of liquid waste Ho. 1 extract,
analyzed on the 30 m x 0.25 mm ID DB-5 fused-silica capillary
column. Temperature program: 100°C (hold at 2 min) to 160°C
at 15°C/min, then at 5'C/min to 270°C; helium at 16 psi.
41
-------
! s
SS f-VT *- *o
„!? - ,
i S 5:
I^JU
'-• 9s
5 5 S
1 II
Figure 6: GC/EC chromatograms of the silica gel Fractions I (top),
II (middle), and III (bottom) of liquid waste No. 1 extract,
spiked with the organochlorine pesticides and PCBs, and
analyzed on the 30 m x 0.25 mm ID DB-5 fused-silica capillary
column. Temperature program: 100°C (hold 2 min) to 1608C
at 15°C/min, then at 5°C/min to 270°C; helium at 16 psi.
42
-------
TABLE 20.
RECOVERY OF SELECTED ORGANOCHLORINE
PESTICIDES AFTER CHROMATOGRAPHY ON
NEUTRAL SUPER I WOELM ALUMINA3
Amount spiked
(ug)
Percent recovery
gamma-BHC
Heptachlor
Aldrin
Dieldrin
Endrin
4, 4 '-DDT
0.5
0.5
0.5
0.5
0.5
0.5
107
101
98
100
107
107
aActivity III (deactivated with 7 percent water).
TABLE 21. RECOVERY OF ORGANOCHLORINE PESTICIDES SPIKED INTO LIQUID
WASTE NO. 1 EXTRACT AND SUBJECTED TO ALUMINA CLEANUP
Hexane elution volume
(mL)
Spi
Compound
alpha-BHC
beta-BHC
gamma-BHC
deUa-BHC
Heptachlor
Aldrin
Heptachlor epoxide
Endosulfan I
Dieldrin + 4, 4 '-DDE
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
ke level
(ug)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
5.0
10
79
a
34
23
79
99
4.6
18
69
0.6
2.3
26
0.7
7.6
2.5
76
0.4
20
86
a
104
0
69
82
8D*
74
58
72
3.6
5.7
5.7
0
0
62
0
50
102
a
a
15
99
a
112
102
126
111
0
110
18
0
a
90
142
aRecovery could not be determined because of high background levels
of electron-capturing compounds.
43
-------
The recovery data in Table 20 were obtained with standards and indicate
excellent recoveries for the 6 compounds tested. When alumina cleanup was
attempted on liquid waste No. 1 extract, 12 of the 17 spiking compounds gave
unacceptable recoveries. The experiment was therefore repeated but the volume
of hexane was increased from 10 ml to 20 ml and then to 50 ml. The results of
these experiments are presented in Table 21. Although increasing the hexane
elution volume improved recoveries for many of the organochlorine pesticides,
the amount of interfering compounds eluted from the alumina column was also
greatly increased (Figures 7 through 9).
When liquid waste No. 2 was subjected to alumina cleanup, recoveries of 16
organochlorine pesticides were found to be >79 percent (Table 22). Kepone was
not recovered at all.
Experimental results reported in Table 21 show that an increase in the
hexane elution volume from 10 ml to 50 ml resulted in improved recoveries for
alpha-BHC, heptachlor, heptachlor epoxide, endosulfan I, dieldrin, 4,4'-DDE,
endrin, 4,4'-DDD, and 4,4'-methoxychlor. However, the amount of interfering
compounds eluted from the alumina column was also greatly increased, and this
precluded the quantification of beta- and gamma-BHC, aldrin, and endosulfan
sulfate. An alternative approach to improving recoveries of the test
compounds would be to change the degree of deactivation of the alumina while
keeping the elution volume of hexane constant.
Alumina (Super I Woelm, neutral alumina, 3 g) deactivated with 7, 10,
15, and 19 percent water was tested with a pesticide standard. Recoveries
are presented in Table 23. The results indicate that in order to obtain
quantitative recoveries for all of the 18 organochlorine pesticides, alumina
deactivated with 19 percent water is required. Only Kepone (27 percent
recovery) and endrin aldehyde (50 percent recovery) gave still low recoveries.
Liquid waste No. 1 was also tested by using alumina deactivated with 15 percent
and 19 percent water. The pertaining GC/EC chromatograms are shown in Figures
10 and 11. Although the alumina deactivated with 19 percent water gives higher
recoveries for the organochlorine pesticides of interest, we were not able to
quantify many of the spiked compounds because of material coextracted from
the matrix. When a similar experiment was performed with liquid waste No. 2,
the same conclusion was reached. However, in that case", the extract was
applied to the alumina column in benzene, and the compound elution patterns
were somewhat different.
No experiments were performed to determine recoveries of PCBs by using
the alumina cleanup procedure described above.
44
-------
N
-O
r-
o
Ol
1(1 (DM • MM (MTM
at r-» IMS
•o • — ® • (w>
in
o
n*
n*
Figure 7. GC/EC chromatogram of the liquid waste No.
cleanup (10 ml hexane).
1 extract after alumina
45
-------
Figure 8. GC/EC chromatogram of the liquid waste No.
cleanup (20 ml hexane).
1 extract after alumina
46
-------
o-
(XI
IM
V
01
Figure 9. 6C/EC chromatogram of the liquid waste No.
cleanup (50 ml hexane).
1 extract after alumina
47
-------
TABLE 22. RECOVERY OF THE ORGANOCHLORINE PESTICIDES SPIKED INTO
LIQUID WASTE NO. 2 AND SUBJECTED TO ALUMINA CLEANUP
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
Endosulfan I
Dieldrin + 4,4'-DDE
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
Spike level
(M9)
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
50.0
Percent recovery
100
103
87
94
82
89
90
99
93
88
80
94
79
3
90
88
94
From the data presented in this section it can be concluded that alumina
cleanup may be useful, although this depends on the type of matrix and on
interfering coextracted materials. The usefulness of the alumina cleanup
results primarily from the fact that by controlling the activity of the alumina
adsorbent, one could recover all the organochlorine pesticides in one fraction
while polar compounds such as phthalate esters that may interfere with the gas
chromatographic analysis would be left behind. However, to the best knowledge
of the authors, there are no reports that indicate that the organochlorine
pesticides and PCBs can be fractionated and recovered quantitatively by using
alumina chromatography.
6.2.3 Silica Gel/Celite
The silica gel/Celite cleanup procedure of Armour and Burke (15) was
tested by using a mixture of 5 g acid-washed Celite and 20 g of silica gel
deactivated with 3 percent water. A mixed pesticide standard (0.5 to 1.0 ug)
and 5 M9 of PCB-1016 and PCB-1260 were added to the top of the column in 4 mL
hexane. Fractions 1, 2, and 3 were eluted from the column with 250 mL
hexane, 200 mL acetonitrile:hexane:methylene chloride (1:19:80), 'and 200 mL
methylene chloride, respectively. The fractions were concentrated in a
Kuderna-Danish apparatus, and the solvent was exchanged to hexane and brought
up to 10 mL. Recoveries are given in Table 24.
48
-------
TABLE 23. ALUMINA CLEANUP RECOVERIES
Percent deactivation
Compound
Amount spiked
(M9)
10
15
19
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Al dri n
Heptachlor epoxide
Endosulfan I
Dieldrin + 4,4'-DDE
Kepone
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
5.0
86.0
0
70.6
0
77.1
84.5
8.8
37.7
74.3
10.5
5.2
0
64.4
1.1
0
57.9
0
92.9
0
88.0
0
94.4
99.7
59.2
84.9
70.3
17.5
49.8
1.4
96.4
22.2
2.8
86.6
0
107
101
105
7.8
109
106
107
102
103
8.7
106
0
105
30.4
0
89.6
77.7
99.8
100
101
99.0
98.3
102
101
99.5
99.0
26.7
99.8
96.1
102
50.2
92.3
96.0
94.0
1 mL pesticide standard in hexane added to 3-g alumina column;
compounds eluted with 10 mL hexane.
This fractionation procedure was evaluated because it was reported to
allow separation of the PCBs from the organochlorine pesticides listed in
Method 8080. Specifically, the PCBs are eluted from the silica gel/Celite
column with petroleum ether prior to the elution of the pesticides with
acetonitrile, hexane, and methylene chloride. The data presented in Table 24
do indicate some degree of separation between the PCBs and the organochlorine
pesticides; however, the recoveries are low for gamma-BHC, 4,4'-DDE, endrin
aldehyde, and the PCBs, and beta-BHC, Kepone, and 4,4'-ODD could not be
recovered at all.
6.2.4 Florisi 1/Charcoal
Florisil and charcoal column chromatography were combined by using the
method of Berg et al. (16). Twenty-one grams of activated Florisil were
packed into an 11-mm ID column and were topped with 2 to 3 cm sodium sulfate
(8g). A mixed pesticide standard (0.5 to 1.0 pg each) and 5 ug of PCB-1016 and
5 ug PCB-1260 in 4 mL hexane were added to the top of the column. Two hundred
mL hexane were passed through the column, were concentrated, exchanged with
acetone, and set aside for the charcoal cleanup. Next, Fractions 1, 2, and 3
were eluted from the Florisil column with 200 mL each of 6 percent ethyl
ether/hexane, 15 percent ethyl ether/hexane, and 50 percent ethyl
ether/hexane, respectively.
49
-------
K1
10
m
KI
ro
o-
a—.
fio
Figure 10. 6C/EC chromatogram of the liquid waste No. 1 extract after
cleanup on alumina deactivated with 15 percent water.
50
-------
f-
o>
OD
IM
0
OJ
Kl
(M
IS
(MO1
r- <
(M
• rv
(Mr
Figure 11. GC/EC chromatogram of liquid waste No. 1 extract after cleanup
on alumina deactivated with 19 percent water.
51
-------
TABLE 24. ELUTION PATTERNS AND PERCENT RECOVERIES FROM THE
SILICA GEL/CELITE COLUMN CLEANUP
Fraction*
Amount added
Compound
Total
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
Endosulfan I
Dieldrin
4,4'-DDE
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
0.5
0.5
0.5
0.5
0.5 26.2
0.5 64.4
0.5
0.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
5.0
5.0 26.9
5.0 42.1
53.9
37.3
54.0
63.6
58.1
NI
19.8
65.8
45.5
67.8
83.9
58.3
12.7
0.7
53.9
0
37.3
b
80.2
64.4
2.5 66.1
58.1
3.0 —
19.8
0.9 66.7
7.1 7.1
0
45.5
0
67.8
83.9
58.3
1.9 41.5
42.8
================
NI — Peak not integrated.
aFraction 1 eluted with 250 mL hexane; Fraction 2 eluted
with 200 mL acetonitrile:hexane:methylene chloride
(1:19:80); Fraction 3 eluted with 200 mL methylene chloride.
bNot able to quantify because of interference.
Activated charcoal (1.1 g) was packed into a 6-mm ID column and was
washed with acetone. The first eluate from the Florisil cleanup was added to
the top of the charcoal column and was eluted with 90 mL 25-percent acetone
in benzene (Fraction 4) and then with 60 mL benzene (Fraction .5). Elution
patterns and percent recoveries are given in Table 25.
52
-------
TABLE 25. ELUTION PATTERNS AND PERCENT RECOVERIES FROM THE FLORISIL/CHARCOAL
CLEANUP
Fraction3
Compound
Amount added
(ug) 1
Total
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
Endosulfan I
Dieldrin0!
4,4'-DDEb|
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-ODT
4,4'-Methoxychlor
PCB-1016
PCB-1260
===================
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
5.0
5.0
5.0
46.9
68.7
82.2
43.6
30.6
29.7
73.2
38.3
23.6
3.1
61.7
1.1
37.1
35.3
40.9
47.9
3.8
3.0
18.0
41.6
50.1
42.3
53.6 21.0
58.4
97.0
3.9
15.0 18.3
8.9
88.0
22.9
27.9
50.7
68.7
85.2
61.6
72.2
79.8
73.2
80.6
98.2
61.5
97.0
65.6
34.4
8.9
88.0
60.0
35.3
60.2 100
90.9 167
fractions 1, 2, and 3 eluted from the Florisil column with 200 mL each of
6, 15, and 50 percent ethyl ether in hexane, respectively.
Fractions 4 and 5 were eluted from the charcoal column with 90 mL
25-percent acetone in benzene and with 60 mL benzene, respectively. They
both represent the first eluate from the Florisil column (see text).
bDieldrin and 4,4'-DDE are coeluting.
The fractionalon scheme with Florisil/charcoal resulted in elution
of the organochlorine pesticides and PCBs in five fractions. The first
compounds to elute from the Florisil column are heptachlor, aldrin, dieldrin,
and the PCBs, and they are further fractionated on the charcoal column. The
remainder of the organochlorine pesticides elutes in Fractions 1, 2, and 3.
The more polar compounds such as endosulfan II and endosulfan sulfate are
recovered quantitatively whereas endrin aldehyde and Kepone are only partially
recovered. No further attempts were made to evaluate this fractionation scheme
since the Florisil/charcoal did not seem to be superior to the (simpler) silica
gel cleanup.
53
-------
6.2.5 Sulfur Cleanup
Elemental sulfur, if present, will give large peaks near the solvent area
which often mask the region from the solvent peak to aldrin when analysis of
the organochlorine pesticides is performed on the 1.8-m glass column packed
with 1.5 percent OV-17/1.95 percent OV-210 on Chromosorb WHP. The methods
available for the removal of elemental sulfur have been reviewed in Appendix
A. The procedure selected for evaluation in this study consists of the non-
destructive treatment of the extract with tetrabutylammonium sulfite (17).
This lipophilic ion pair rapidly converts sulfur to thiosulfate.
Sulfur is soluble to some extent in hexane, but not in water, and sodium
sulfite is soluble in water, but not in hexane; therefore, the reaction between
these compounds that results in thiosulfate formation occurs very slowly. When
the tetrabutylammonium ion is present, a lipophilic ion pair is formed with the
sulfite, and added 2-propanol favors the solvation of this ion pair in the
nonpolar phase which allows a momentary reaction with the sulfur. When excess
sodium sulfite is present the sulfur removal is quantitative.
To investigate the removal of sulfur from sample extracts, five matrix
extracts, identified below, were split as follows: 2-mL aliquots of each
matrix extract (liquid waste No. 1, liquid waste No. 2, sandy loam,
NBS sediment, and solid waste) were subjected to a sulfur cleanup with
the tetrabutylammonium (TBA)-sulfite reagent; other 2-mL aliquots from the
five matrix extracts were first spiked with 100 ng or 1000 ng of the
organochlorine pesticides and then were subjected to a sulfur cleanup by the
same procedure; finally, other 2-mL aliquots of the five matrix extracts were
spiked with the organochlorine pesticides and sulfur (300 ug) and were then
subjected to the sulfur cleanup procedure.
The TBA-sulfite reagent was prepared from 3.39 g tetrabutyl ammonium
hydrogen sulfate and 100 mL water. To remove any organic impurities, this
solution was extracted 3 times with 20 mL hexane. One-milliliter aliquots of
the TBA sulfate solution were then transferred to individual vials, and
250 mg of crystalline sodium sulfite were added to each vial. It is very
important that excess sulfite is always present; otherwise, the removal of
sulfur is not complete, and the reaction with the TBA-sulfite reagent has to
be repeated.
The matrix extracts (in hexane) were shaken with 2-propanol (1 mL) and
the TBA-sulfite reagent (1 mL) for at least 1 min. Water (5 mL) was added,
and the vial containing the extract and reagent was shaken for another min
on a Vortex.
The rationale for performing this experiment was two-fold: to determine
if the removal of sulfur is affected by matrix interferences and to determine
if the organochlorine pesticides are recovered quantitatively. In addition to
the 5 matrix extracts, 3 pesticides standards (one of them containing 300 pg
sulfur) were also reacted with the TBA-sulfite reagent to determine how well
the pesticides are recovered in the absence of matrix interferences.
54
-------
The results are presented in Tables 26 and 27. It is clear from the
data in Table 26 that all the organochlorine pesticides are recovered
quantitatively. Because of matrix interferences, quantification of many
organochlorine pesticides was not possible (Table 27). However, from examining
the GC/EC chromatograms (Figures 12 and 13) it appears that sulfur was removed
quantitatively, regardless of the matrix; thus, the procedure is recommended
for incorporation into the Method 8080 protocol. The observations which follow
were made during this study and are considered worthy of note:
° All extracts should be examined visually for the presence of sulfur;
when sulfur crystals are present (yellow color), the extract should be
removed with a syringe or disposable pi pet, transferred to another
vial, and subjected to the TBA-sulfite procedure.
° Even if sulfur crystals are not visible in the extract but sulfur
appears to be present according to the GC/EC analysis (see chromato-
grams in Figures 12 and 13), then the extract should be reacted
with the TBA-sulfite reagent and reanalyzed.
6.3 GAS CHROMATOGRAPHY
The analysis of the organochlorine pesticides and PCBs listed in
Method 8080 by gas chromatography with electron capture detection has been
reviewed in Appendix A, Section 2.4. Basically, the compounds are separated
on a packed or a capillary column, either under isothermal or temperature-
programmed conditions, and are identified by matching the retention times of
the peaks in the sample with those derived from the calibration standard, which
was analyzed under the same conditions as the sample. A second or even a third
column is required to confirm compounds by gas chromatography to minimize or to
avoid misinterpretation of compound identity.
The packed-column chromatographic determination of the organochlorine pes-
ticides, although widely used, does not allow complete separation of the pairs
beta- and gamma-BHC, 4,4'-DDE and dieldrin, and 4,4'-DDD and endosulfan II.
Furthermore, multicomponent mixtures such as toxaphene and PCBs are resolved
into much fewer components with a packed column than with a capillary
column.
This section summarizes the results of a series of experiments performed
with the intent of identifying a set of conditions that will allow adequate
separation of the 19 organochlorine pesticides from the PCB components. Four
columns, two packed (1.5 percent OV-17/1.95 percent OY-210 and 3 percent
OV-1) and two capillary (DB-5 and SPB-608), were evaluated. Standards
containing all 19 organochlorine pesticides were analyzed separately from the
PCBs and toxaphene mixtures. Mixtures containing the organochlorine
pesticides at 0.05 to 0.50 ng/uL and PCBs or toxaphene at 0.05, 0.10, 0.50,
and 1.0 ng/pL were also analyzed to determine a threshold value at which
interferences from the multicomponent mixture can be ignored.
55
-------
TABLE 26. RECOVERIES OF THE ORGANOCHLORINE PESTICIDES
AND PCBs SUBJECTED TO THE TBA-SULFITE
PROCEDURE (STANDARDS ONLY, IN HEXANE)
Percent recovery
Without sulfur With sulfur
Compound added3 added0
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4, 4 '-DDE and Dieldrin
Endrin
Endosulfan II
4, 4 '-ODD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
Toxaphene
PCB-1016
PCB-1260
=========================
110
106
112
100
103
104
107
98
108
91
87
99
103
10
107
94
86
85
93
108
87
================
103
99
108
86
97
99
101
100
106
103
89
99
106
8
63
93
90
68
83
88
68
===========—
aValue given is the average of two determinations.
DSingle determination.
A summary of the organochlorine pesticides retention times is given in
Table 28. Chromatograms of individual as well as composite mixtures of
organochlorine pesticides and PCBs are given in Figures 14 to 18.
Additional Chromatograms from the capillary column analysis are given in
Appendix B.
It can be seen that the separation of the organochlorine pesticides and
PCBs cannot be achieved by using the packed column (1.5 percent OV-17/195-
percent OV-210) recommended by the EPA in Method 8080. Likewise, toxaphene
causes a shift in the baseline when it is present at concentrations twice as
high as some of the organochlorine pesticides.
56
-------
TABLE 27. RECOVERIES OF THE ORGANOCHLORINE PESTICIDES SPIKED INTO VARIOUS
MATRICES AND SUBJECTED TO THE TBA-SULFITE PROCEDURE
Liquid Waste 1 Liquid Waste 2
Sandy loam
NBS sediment
Solid Waste 1
in
Compound
Without With Without With Without With Without With Without With
sulfur sulfur sulfur sulfur sulfur sulfur sulfur sulfur sulfur sulfur
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4, 4 '-DDE + Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Endosulfan sulfate
4, 4 '-DDT
4,4'-Methoxychlor
61
23
a
a
70
a
a
83
92
77
a
a
a
45
a
67
b
56
22
a
a
80
a
a
93
79
67
a
a
a
68
a
102
b
49
7.5
45
0
94
109
a
26
a
75
115
49
41
89
42
94
b
13
0
0
0
104
0
a
0
a
71
79
4.2
0
8.9
0
94
b
95
96
72
67
113
103
103
105
105
81
128
110
102
33
39
118
b
75
97
41
39
114
106
98
104
98
80
132
103
85
83
a
155
b
87
83
90
71
99
66
91
121
96
78
92
87
65
0
49
a
b
84
85
120
61
105
76
74
81
82
73
86
65
60
49
a
a
b
76
85
105
0
59
a
40
a
a
a
a
11
0
0
0
a
b
66
0
27
0
57
a
29
a
a
a
a
0
0
0
0
a
b
'unable to quantify oue to interfering compound(s).
^Unable to quantify 4,4'-methoxychlor because of poor chromatography on the DB-5 column at
the time the experiment was performed.
-------
Figure 12. GC/EC chromatogram of the sulfur standard (concentration 300 ug/mL)
before reaction with the TBA-sulfite reagent.
58
-------
Figure 13. GC/EC chromatogram of the sulfur standard after reaction with
the TBA-sulfite reagent.
59
-------
TABLE 28.
SUMMARY OF ORGANOCHLORINE PESTICIDE RETENTION TIMES (MIN)
Capillary columns
Packed columns
DB-5C
SPB-608d
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
gamma-Chlordane
Heptachlor epoxide
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
alpha-Chlordane
1.5 percent OV-17a +
1.95 percent OV-210
1.54
2.29
1.97
2.69
2.41
2.93
5.00
4.49
5.68
6.65
6.99
8.52
10.48
10.37
14.01 -
8.33
17.05
12.51
24.71
5.47
3 percent OV-lb
1.54
1.66
1.90
1.83
3.07
3.35
5.53
4.80
6.06
7.30
7.22
8.05
8.45
9.34
9.45
10.66
11.00
12.44
19.51
6.18
(1)
6.52
7.28
7.49
8.19
9.83
11.02
13.26
12.43
13.72
14.68
14.68
15.36
16.20
15.80
16.51
16.72
17.39
17.59
19.77
13.48
(2)
12.29
13.13
13.37
14.14
15.91
17.16
19.48
18.60
19.94
20.83
20.91
21.71
22.75
22.38
22.75
23.01
23.64
23.79
25.94
NA
9.46
11.33
10.97
12.73
12.46
13.76
16.70
15.98
17.40
18.36
18.60
19.96
20.69
20.53
21.90
D
22.54
21.72
24.90
17.31
D = degraded on column.
aGC operating conditions are given in Table 29.
bQC operating conditions are given in Table 30.
CGC operating conditions are given in Table 31.
^GC operating conditions are given in Table 32.
NA — data not available.
60
-------
TABLE 29. GAS CHROMATOGRAPHIC CONDITIONS FOR THE DETERMINATION
OF PESTICIDES AND PCBs BY GC/EC USING A 1.5-PERCENT
OV-17/1.95-PERCENT OV-210 COLUMN
Chromatograph:
Column:
Temperatures:
Carrier Gas:
Detector:
Varian Vista 6000 Gas Chromatograph with a Varian 402 Data
System and electron capture detector (constant current-pulsed
frequency)
1.8 m x 2 mm ID glass column
1.5 percent OV-17 + 1.95 percent OV-210 on Chromsorb
W HP 100/120 mesh
Oven 190°C, isothermal
Injector 225°C
Detector 350°C
Nitrogen at 30 mL/min
Range 10
Attenuation 16
Autosampler: Varian Series 8000; 2 uL injection volume
TABLE 30. GAS CHROMATOGRAPHIC CONDITIONS FOR THE DETERMINATION OF
PESTICIDES AND PCBs BY GC/EC USING A 3-PERCENT OV-1 COLUMN
===========================================================
Chromatograph: Varian Vista 6000 Gas Chromatograph with a Yarian 402 Data
System and electron capture detector (constant current-pulsed
frequency)
Column:
Temperatures:
Carrier Gas:
Detector:
1.8 m x 2 mm ID glass column
3 percent OV-1 on Supelcoport 100/12T)~mesh
Oven 182°C, isothermal
Injector 225°C
Detector 3508C
Nitrogen at 30 mL/min
Range 10
Attenuation 16
Autosampler: Varian Series 8000; 2 uL injection volume
61
-------
TABLE 31. GAS CHROMATOGRAPHIC CONDITIONS FOR THE DETERMINATION OF PESTICIDES
AND PCBs BY GC/EC USING A DB-5 FUSED-SILICA CAPILLARY COLUMN
Chromatograph:
Column:
Temperatures:
Carrier Gas:
Detector:
Van'an Vista 6000 Gas Chromatograph with a Varian 402
Data System and electron capture detector (constant
current-pulsed frequency)
Fused-silica capillary column
30 m x 0.25 mm ID DB-5, 0.25 urn coating thickness
Oven (1) 160°C (hold 2 min) to 270°C (hold 1 min) at
5°C/min
(2) 100°C (hold 2 min) to 160°C at 15°C/min, then
at 5°C/min to 270°C
Injector 225°C
Detector 350°C
(1) Nitrogen at 20 psi
(2) Helium at 16 psi
Range 10
Attenuation 16
Splitless Injection: Split flow -- 35 mL/min
Sweep flow -- off
Splitless time: 30 sec
Autosampler:
Varian Series 8000; 2 uL injection volume
In contrast to the packed column, the DB-5 capillary column gives
excellent resolution of the organochlorine pesticides and of the
multicomponent mixtures such as toxaphene, PCBs, and the technical chlordane.
We have investigated two capillary columns and, based on the retention time
data given in Table 28, have concluded that, except for Kepone, both columns
can be used for the analysis of the organochlorine pesticides including
toxaphene and chlordane, and for the PCBs. Because the PCBs are extracted and
isolated along with the organochlorine pesticides, these two types of contami-
nants need to be separated from each other to avoid cross-interference. We
found that the resolving power of the capillary column, although significantly
greater than that of the packed column, is not sufficient to distinguish
between these two groups of compounds. Therefore, we have evaluated the
separation of the organochlorine pesticides and PCBs by column chromatography
prior to the gas chromatographic analysis.
62
-------
TABLE 32. GAS CHROMATOGRAPHIC CONDITIONS FOR THE DETERMINATION
OF PESTICIDES AND PCBs BY GC/EC USING AN SPB-608
FUSED-SILICA CAPILLARY COLUMN
Chromatograph:
Column:
Temperatures:
Carrier Gas:
Detector:
Splitless Injection:
Autosampler:
Varian Vista 6000 Gas Chromatograph with a Varian 402
Data System and electron capture detector (constant
current-pulsed frequency)
Fused-silica capillary column
30 m x 0.25 mm ID SPB-608, 0.25 urn coating thickness
Oven 160°C (2 min hold), then temperature
programmed at 5°C/min to 290°C (1 min hold)
Injector 225°C
Detector 350°C
Nitrogen at 20 psi
Range 10
Attenuation 16
Split flow — 35 mL/min
Sweep flow ~ off
Splitless time: 30 sec
Varian Series 8000; 2 uL injection volume
Tables 33 through 36 present a comparison of four gas chromatographic
analyses of the same "sample" (in this case, there are four fractions collected
from a silica gel column) that was analyzed on four different gas chromato-
graphic columns. The "sample" was obtained by fractionating a standard of
known concentration of organochlorine pesticides (concentration 1 was 0.5 ug
for the BHC isomers, heptachlor, aldrin, heptachlor epoxide, and endosulfan I,
and 1.0 M9 for the remainder of the organochlorine pesticides, except for 4,4'-
methoxychlor which was 5.0 ug; concentration 2 was 10 times concentration 1)
and PCB-1016 and PCB-1260 (10 times concentration 1 of the pesticides).
Technical chlordane and toxaphene were fractionated separately. The values
given in Tables 33 through 36 are the average recoveries and the standard
deviations (numbers in parentheses, from three replicate determinations).
These data indicate the accuracy and precision of a method involving silica
gel fractionation (procedure given in Section 6.2.1) and analysis by gas
chromatography with electron capture detection.
63
-------
!- U
if, Jg ,.,.,i ,1 i
??« ? • ?
::s : s
VJ
r i i M M i "i i i i i i i i i si i i i
Figure 14. GC/EC chromatograms of the organochlorine pesticide standards A
(bottom) and B (middle) and of a composite of A and B (top),
obtained on the 1.5-percent OV-17/1.95-percent OV-210 column.
Concentrations: standard A, 0.05 to 0.10 ng/uL; standard B, 0.05
to 0.50 ng/uL; standard A and B composite, 0.05 to 0.10 ng/uL;
isothermal at 190°C.
64
-------
—*N v irt «r-
1
"\J
5-
! 5 ^ | S,
I I I I'
Figure 15. GC/EC chromatograms of a mixture of PCB-1016/1260 at 0.05 ng/uL
(bottom) and of a composite of the organochlorine pesticide
standards A and B and PCB-1016/1260 at 0.05 ng/pL (top),
obtained on the 1.5-percent OV-17/1.95-percent OV-210 column;
isothermal at 1908C.
65
-------
XXJ
TTSz 1 i ! r
Jfi; -S3;
r
Figure 16. GC/EC chromatograms of a PCB-1016/1260 mixture at 0.5 ng/uL
(bottom) and of a composite of organochlorine pesticide
standards A and B and PCB-1016/1260 (top) at concentrations
of 0.05 to 0.50 ng/uL, respectively. The analyses were
performed on the 1.5-percent OV-1 71/1.95-percent OV-210
column; isothermal at 190°C.
66
-------
TTiz S * ; i
!K« E i J §
s
I
iiir
-J *~
i i l i l 5 I is I I
i
WO
F l ^ l l l l
Figure 17. GC/EC chromatograms of the toxaphene standard at 0.10 ng/pL
(bottom) and of a composite of organochlorine pesticide
standards A and B at 0.05 to 0.50 ng/pL and toxaphene at
0.10 ng/pL (top), obtained on the 1.5-percent OV-17/1.95-
percent OV-210 column; isothermal at 190°C.
67
-------
f
SS
;. m .
ii - i j i
,
I I t I I I I I" I F I I
M X O Z _i *
s 7 s ; i c s
.5 I t 2 i i §
5SI I 'I I I Sl I F I 5I I I I I Is! I I Is
i i i
Figure 18. GC/EC chromatograms of the toxaphene standard at 1.0
(bottom) and of a composite of organochlorine pesticide
standards A and B at 0.05 and 0.50 ng/pL and toxaphene
at 1.0 ng/pL (top), obtained on the 1.5-percent OV-17/1.95-
percent OV-210 column; isothermal at 190°C.
68
-------
TABLE 33. OR6ANOCHLORINE PESTICIDES AND PCBs AVERAGE RECOVERIES FROM SILICA
GEL CHROMATOGRAPHY, DETERMINED ON A OV-17/OV-210 COLUMNa.b»c»d»e
Fraction 1 Fraction II
alpha-BHC
Deta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrln
Heptachlor epoxlde
Endosulfan I
4,4 '-DOE
Oieldrln
Endrln
Endosulfan llf
4.4'-OODf
Kepone
Endrln aldehyde
Endosulfan sulfate
4, 4 '-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Technical chlordane
Toxaphene
aEluant composition:
Fraction I - 80 ml.
Fraction II - 50 mL
Cone. 1 Cone. 2 Cone. 1
169 (10.0) 175 (12.1) 8.1 (6.4)
134 (10.5) 136 (4.1)
82.8 (4.6) 86.9 (5.0) 5.9 (7.6)
93.0 (11.4)
91.5 (10.7) 87.1 (14.1)
90.0 (9.6) 97.8 (4.1)
14.6 (2.3) 16.2 (2.4) 36.4 (1.7)
13.6 (2.1)
hexane.
hexane.
Cone. 2 Cone
77
74
86
90
92
90
91
75
91
91
77
95
80.3 (11.6) 16
109
37.5 (3.4) 42
13.9 (2.4) 80
.8
.2
.3
.4
.1
.3
.4
.4
.3
.3
.6
.2
.4
.2
.0
Fraction III
. 1
(1.4)
(1.1)
(3.1)
(3.3)
(3.2)
(3.2)
(2.5)
(7.3)
(1.2)
(1.2)
(9.8)
(5.2)
(18.4)
(0.8)
(3.3)
(4.1)
Cone. 2
64.5
67.8
76.9
78.5
79.6
82.0
79.2
61.6
77.8
77.8
73.4
79.7
9.0
88.0
38.8
82.7
(7
(8
(8
(9
(8
(7
(8
(10
(9
(9
(8
(10
(14
(13
(6
(2
Fraction IV
Cone. 1 Cone
.7)
.0)
.5)
.6)
.6)
.6)
.6)
.8)
.4)
.4)
46.6 (3.3) 77.4
.7) 27.6 (4.6) 33.5
.7) 7.2 (5.1)
.0)
.9) 6.9 (1.9) 5.3
.3)
.7)
Total
. 2 Cone. 1
77.8
74.2
86.3
90.4
178
134
92.1
90.3
88.7
91.4
75.4
91.3
91.3
(5.1) 46.6
(5.4) 105
102
109
(2.8) 115
91.5
90.0
93.2
93.6
(1.4)
(1.1)
(3.1)
(3.3)
(15.7)
(10.5)
(3.2)
(3.2)
(8.8)
(2.5)
(7.3)
(1.2)
(1.2)
(3.3)
(14.1)
(10.2)
(9.7)
(2.1)
(10.7)
(9.6)
(0.9)
(2.4)
Cone. 2
64.5
67.8
76.9
78.5
175
136
79.6
82.0
86.9
79.2
61.6
77.8
77.8
77.4
107
79.7
89.3
93.3
87.1
97.8
93.3
96.3
(7.7)
(8.0)
(8.5)
(9.6)
(12.1)
(4.1)
(8.6)
(7.6)
(5.0)
(8.6)
(10.8)
(9.4)
(9.4)
(5.1)
(7.2)
(10.7)
(23.4)
(12.0)
(14.1)
(4.1)
(6.6)
(1.2)
Fraction III - IS ml methyl ene chloride.
Fraction IV - 50 nL
bConcentrat1on 1 1s
Kepone, endosulfan
ethyl acetate.
0.5 ug per column for BHCs. hbptachlor,
II, 4,4'-OOT, endrln aldehyde, 4.4'-DOD,
aldrln, heptachlor
4,4'-OOE, endrln,
epoxlde, endosulfan I; 1.0 ug per column for
and endosulfan sulfate; 5 ug per column for
dleldrln,
4,4'-methoxychlor and technical chlordane; 10 wg per column for toxaphene, PCB-1016.and PCB-1260.
cFor concentration 2 the amounts spiked are 10 times as high as those for concentration 1.
dThe values given represent the average recoveries of three determinations; the numbers In parentheses are the standard deviations;
recovery cut-off point Is 5 percent.
eData obtained with standards, as Indicated in footnotes b and c, dissolved In 2 mL hexane.
f4,4'-DDD and endosulfan II are not resolved, and an average recovery is given.
-------
TABLE 34. ORGANOCHLORINE, PESTICIDES AND PCBs AVERAGE RECOVERIES FROM SILICA
GEL CHROMATOGRAPHY, DETERMINED ON A 3-PERCENT OV-1 COLUMN3,b.c.d.e
Fraction I Fraction 11
Cone. 1 Cone. 2 Cone. 1
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor 94.4 (2.5) 102 (7.6)
Aldrin 110 (9.1) 111 (1.6)
Heptachlor epoxide
Endosulfan I
4, 4 '-DDE 93.4 (4.6) 96.6 (4.9) 7.9 (8.0)
Dieldrin
Endrin
Endosulfan II
4,4'-DDDf
Endrin aldehyde'
Kepone
Endosulfan sulfate
4.4'-DDT 84.7 (10.3)
4,4'-Methoxychlor
PCB-1016 87.7 (4.5) 86.4 (9.1)
PCB-1260 94.8 (9.3) 99.7 (6.7)
Technical chlordane 17.8 (1.4) 18.5 (2.8) 37.8 (1.0)
Toxaphene 12.9 (2.4)
Cone. 2 Cone
78
102
79
90
92
93
92
69
97
SO
50
93
73.6 (9.4)
115
37.8 (2.6) 38
12.7 (3.4) 75
.1
.7
.1
.7
.0
.1
.0
.0
.2
.2
.1
.3
.9
Fraction III
. 1
(2.
(2.
(1.
(6.
(9.
(4.
(3.
(9.
(0.
(7.
(7.
(1.
(1.
(2.
(8.
7)
2)
7)
1)
1)
1)
2)
6)
9)
5)
5)
2)
4)
4)
1)
Cone
67.8
89.2
77.0
74.2
80.2
79.0
78.5
58.2
80.1
54.6
54.6
74.8
89.9
34.0
79.6
. 2
(6.
(9.
(7.
(6.
(8.
7)
9)
8)
6)
3)
(9.5)
(7.
(17.
(9.
(9.
(9.
(7.
(9.
(4.
(3.
3)
9)
3)
8)
8)
7)
2)
6)
7)
Fraction IV
Cone. 1 Cone. 2 Cone
78.1
102
79.7
90.1
94.4
110
92.7
93.0
101.1
92.1
69.0
97.0
15.8 (3.2) 21.4 (2.7) 66.1
15.8 (3.2) 21.4 (2.7) 66.1
57.9 (8.4) 94.8 (8.4) 57.9
93.1
84.7
7.4 (1.8) 6.3 (3.4) 122
87.7
94.8
93.9
88.8
Total
. 1
(2.7)
(2.2)
(1.7)
(6.1)
(2.5)
(9.1)
(4.1)
(4.1)
(10.9)
(3.2)
(9.6)
(0.9)
(10.7)
(10.7)
(8.4)
(1.2
(10.3)
(0.8)
4.5)
(9.3)
(0.8)
(7.2)
Cone
67.8
89.2
77.0
74.2
102
111
80.2
79.0
96.6
78.5
58.2
80.1
76.0
76.0
94.8
74.8
73.6
96.2
86.4
99.7
90.4
92.1
. 2
(6.7)
(9.9)
(7.8)
(6.2
(7.6
(1.6]
(8.3;
(9.5]
(4.9]
(7.3]
(17.9]
(9.3]
(15.2]
(15.2]
(8.4
(7.7
(9.4
(7.5
(9.1
(6.7
(3.0)
(0.1)
aEluant composition:
Fraction 1 - 80 ml hexane.
Fraction II - 50 ml hexane.
Fraction III - 15 ml raethylene chloride.
Fraction IV - 50 ml ethyl acetate.
^Concentration 1 is 0.5 ug per column for BHCs,, heptachlor, aldrin. heptachlor epoxide, endosulfan 1; 1.0 ug per column for dleldrin,
Kepone, endosulfan II, 4,4'-DDT, endrln aldehyde, 4,4'-DDD, 4,4'-DDE, endrin, and endosulfan sulfate; 5 pg per column for
4,4'-methoxychlor and technical chlordane; 10 119 per column for toxaphene, PCB-1016,and PCB-1260.
cFor concentration 2 the amounts spiked are 10 times as high as those for concentration 1.
dThe values given represent the average recoveries of three determinations; the numbers in parentheses are the standard deviations;
recovery cut-off point is 5 percent.
eData obtained with standards, as indicated 1n footnotes b and c, dissolved In 2 ml hexane.
f4,4'-DDD and endrin aldehyde are not resolved, and an average recovery 1s given.
-------
TABLE 35. AVERAGE RECOVERIES FROM SILICA GEL CHROMATOGRAPHY, DETERMINED ON A
30 m X 0.25 mm ID DB-5 FUSED-SILICA CAPILLARY COLUMN*,b.c.d.e
Fraction 1
Cone. 1 Cone. 2
alpha-BHC
beta-BHC
gamma -BHC
delta-BHC
Heptachlor 109 (4.1) 118 (8.7)
Aldrin 97.5 (5.6) 104 (1.6)
Heptachlor epoxlde
Endosulfan I
4,4'-DO£ 86.1 (5.4) 94.0 (2.8)
Oieldrtn
Endrin
Endosulfan 11
4, 4 '-000
Endrin aldehyde
Kepone
Endosulfan sulfate
4.4'-DDT
4,4'-Methoxychlor
PCB-1016 86.1 (4.0) 86.8 (6.1)
PCB-1260 90.9 (4.1) 95.3 (5.0)
Technical chlordane 13.9 (5.5) 22.4 (5.3)
Toxaphene
Fraction II
Fraction III
Cone. 1 Cone. 2 Cone
7.4 (5.5) 82.
107
91.
92.
94.
95.
96.
84.
96.
102
80.
93.
86.2 (13.4) 72.7 (9.1) 14.
98.
19.3 (6.8) 38.7 (3.6) 29.
14.9 (2.4) 16.7 (1.4) 73.
1
3
4
9
1
5
7
8
8
0
7
8
2
1
. 1
(1.
(2.
(3.
(3.
(4.
(5.
(6.
(10.
(4.
(4.
(1.
(4.
(18.
(9.
(5.
(9.
7)
1)
6)
5)
7)
1)
0)
5)
4)
6)
9)
9)
7)
9)
0)
4)
Cone
73.6
98.1
85.2
83.5
88.2
87.0
86.8
71.0
85.6
92.2
75.8
82.2
82.3
37.3
84.2
. 2
(8.
(12.
(10.
(10.
(10.
(10.
(10.
(12.
(10.
(10.
(9.
(9.
(10.
(5.
(10.
Fraction IV
Cone. 1
0)
5)
7)
6)
2)
2)
6)
3)
4)
2)
5) 29.4 (4.2)
40.7 (7.9)
2)
7) 9.0 (4.8)
1)
7)
Cone. 2 Cone
82.1
107
91.3
92.4
109
97.5
94.9
95.1
86.1
96.5
84.7
96.8
102
33.2 (4.7) 110
73.0 (12.0) 40.7
93.0
101
5.7 (3.0) 108
86.1
90.9
62.4
88.0
Total
. 1
(1.7)
(2.1)
(3.6)
(3.5)
(4.1)
(5.6)
(4.7)
(5.1)
(5.4)
(6.0)
(10.5)
(4.4)
(4.6)
(3.0)
(7.9)
(4.9)
(5.3)
(10.0)
(4.0)
(4.1)
(3.3)
(12.0)
Cone
81.0
98.1
85.2
83.5
118
104
88.2
87.0
94.0
86.8
71.0
85.6
92.2
109
73.0
82.2
72.7
88.0
86.8
95.3
98.4
101
. 2
(6.8)
(12.5)
(10.1)
(10.6)
(8.7)
(1.6)
(10.2)
(10.2)
(2.8)
(10.6)
(12.3)
(10.4)
(10.2)
(5.8)
(12.0)
(9.2)
(9.1)
(5.8)
(6.1)
(5.0)
(1.9)
(10.1)
aEluant composition:
Fraction I - 80 ml hexane.
Fraction 11 - 50 ml hexane.
Fraction III - 15 mL methylene chloride.
Fraction IV - 50 al ethyl acetate.
bConcentration 1 is 0.5 vg per column for BHCs, heptachlor, aldrin, heptachlor epoxide, endosulfan I; 1.0 ug per column for dleldrin,
Kepone, endosulfan II, 4.4'-DDT, endrin aldehyde, '4,4'-ODD. 4,4'-DDE, endrin, and endosulfan sulfate; 5 ug per column for
4,4'-methoxychlor and technical chlordane; 10 pg per column for toxaphene, PCB-1016,and PCB-1260.
°For concentration 2 the amounts spiked are 10 times as high as those for concentration 1.
dThe values given represent the average recoveries of three determinations; the numbers in parentheses are the standard-deviations;
recovery cut-off point is 5 percent.
eData obtained with standards, as Indicated in footnotes b and c, dissolved in 2 raL hexane.
-------
TABLE 36. AVERAGE RECOVERIES FROM SILICA GEL CHROMATOGRAPHY, DETERMINED ON
AN SPB-608 FUSED-SILICA CAPILLARY COLUMN*»b»c»d»e
ro
Fraction I Fraction 11
Cone. 1
alpna-SHC
oeta-BHC
gamma-BHC
delta-BHC
Heptachlor no (4.1)
Aldrin 97.8 (7.8)
Heptachlor epoxide
Endosulfan I
4,4'-ODE 87.3 (5.1)
Dieldrln
Endrin
Endosulfan II
4,4'-DDO
Endrin aldehyde
Endosulfan sulfate
4.4'-DDT
4,4'-Hethoxychlor
PCB-1016 86.6 (3.2)
PCB-1260 87.9 (5.3)
Technical chlordane 24.8 (2.1)
Toxaphene
Cone. 2 Cone. 1
116 (6.0) 9.1 (6.9)
102 (2.8)
92.5 (4.2) 9.4 (10.1)
76.2 (5.4)
76.7 (7.0)
93.1 (7.4)
24.1 (3.1) 47.9 (10.2)
14.5 (1.8)
Cone. 2 Cone
85.
109
86.
93.
86.
86.
83.
80.
84.
95.
70.
95.
64.4 (9.8)
83.
42.8 (9.3) 40.
16.6 (0.6) 77.
7
6
1
0
3
9
8
5
8
1
4
9
2
9
Fraction III
. 1
(1
(1
(4
(1
(2
(2
(1
(6
(0
(2
(11
(1
(1
(1
(2
.7)
.0)
.0)
.3)
.8)
.5)
.7)
.1)
.2)
.8)
.2)
.9)
.8)
.7)
.4)
Cone
70.8
93.7
72.9
78.7
75.4
76.0
73.4
64.7
73.2
81.6
66.6
80.6
71.0
31.7
83.7
. 2
(7.4)
(11.4)
(8.1)
(8.9)
(7.6)
(8.0)
(6.8)
(10.2)
(7.6)
(7.0)
(7.1)
(8.5)
(6.8)
(3.6)
(0.7)
Fraction IV
Cone. 1 Cone. 2 Cone
85.7
109
86.6
93.1
119
97.8
86.0
86.3
96.7
83.9
80.8
84.5
95.8
22.0 (4.8) 28.6 (4.7) 92.1
95.4
76.2
6.8 (0.8) 6.1 (2.8) 90.7
86.6
87.9
113
92.4
Total
. 1
(1.7)
(1.0)
(4.0)
(1.3)
(7.1)
(7.8)
(2.8)
(2.S)
(9.5)
(1.7)
(6.1)
(0.2)
(2.8)
(14.6)
(1.9)
(5.4)
(1.0)
(3.2)
(5.3)
(14.0)
(4.1)
Cone
70.8
93.7
72.9
78.7
116
102
75.4
76.0
92.5
73.4
64.7
73.2
81.6
95.2
80.6
64.4
77.1
76.7
93.1
93.6
100
. 2
(7.4)
(11.4)
(8.1)
(8.9)
(6.0)
(2.8)
(7.6)
(8.0)
(4.2)
(6.8)
(10.2)
(7.6)
(7.0)
(4.7)
(8.5)
(9.8)
(5.0)
(7.0)
(7.4)
(9.2)
(0.7)
acluant composition:
Fraction 1 - 80 ml hexane.
Fraction II - 50 ml hexane.
Fraction III - 15 raL methylene chloride.
Fraction IV - 50 mL ethyl acetate.
"Concentration 1 is 0.5 vg per column for BHCs, heptachlor, aldrtn, heptachlor epoxlde, endosulfan I; 1.0 vg per column for dieldrin,
Kepone, endosulfan II, 4,4'-OOT, endrin aldehyde/4,4'-ODD, 4,4'-DDE, endrin, and endosulfan sulfate; 5 vg per column for
4,4'-methoxychlor and technical chlordane; 10 vg p'er column for toxaphene, PCB-1016,and PCB-1260.
cFor concentration 2 the amounts spiked are 10 times as high as those for concentration 1.
dThe values given represent the average recoveries of three determinations; the numbers in parentheses are the standard deviations;
recovery cut-off point is 5 percent.
eData obtained with standards, as Indicated in footnotes b and c, dissolved in 2 ml hexane.
-------
6.4 RECOMMENDATIONS FOR METHOD REVISION
Upon completion of the various method improvement studies described in
Sections 6.1, 6.2, and 6.3, the following recommendations were made for
consideration in revising Method 8080:
° Methods 3510 and 3520 give comparable accuracies when evaluated with
spiked distilled water samples. Except for Kepone and endosulfan II,
all compounds yielded recoveries >70 percent with either method.
Method precision was better for Method 3520. However, when the liquid
samples are highly contaminated and sample portions of less than 100
ml are extracted, Method 3520 is not desirable with the equipment
specified in the current method since additional organics-free water
has to be added which increases the possibility of introducing
contamination.
8 Methods 3540 and 3550, recommended for the extraction of solid
matrices, give comparable accuracies. Method 3550 appears to give
better precision than Method 3540. The change in the composition of
the extraction solvent was found to affect the performance of
Method 3550 slightly, however, it did not justify a change in the
Method 3550 protocol.
° The silica gel fractional!"on procedure separates the PCBs from most
of the organochlorine pesticides. The distribution patterns of the
organochlorine pesticides in the four fractions were
reproducible. Components found to elute in Fraction I include
heptachlor, aldrin, 4,4'-DDE, 4,4'-DDT, and the PCBs. Almost all
other organochlorine pesticides elute in Fraction III. A few
compounds, 4,4'-DDD, beta-BHC, and gamma-chlordane, were distributed
between two or three fractions. This fractionation procedure should
be further evaluated with additional real samples to obtain method
performance information.
° Packed column analysis should be replaced with the capillary column
analysis. Two capillary columns have been selected for further
evaluation.
73
-------
SECTION 7
REVISED METHOD 8080 — EVALUATION STUDIES
The performance of the revised Method 8080 was evaluated to determine the
reproducibility and efficiency of the silica gel fractionation, the precision
of the identification and the measurement of the gas chromatographic technique,
and the minimum detectable levels for the organochlorine pesticides and
PCBs. The subsections address the reproducibility of the gas chromatographic
analysis, the method precision and accuracy, and the method detection limits.
7.1 REPRODUCIBILITY STUDY OF THE GAS CHROMATOGRAPHIC ANALYSIS
To establish the precision of the gas chromatographic method, ten
replicate injections of a standard containing the organochlorine pesticides
were performed by using an autosampler. Separately, another set of ten
replicate injections were performed of a mixture of PCB-1016 and PCB-1260
and of toxaphene. The results are presented as the average response factors
of the ten replicates and the relative standard deviations (RSDs), and as the
average retention times of the ten replicates and the RSDs (Tables 37
and 38).
The reproducibility of the injection technique was better than
10 percent for the early eluting compounds, and was between 10 and 16 percent
for 4,4'-DDE, 4,4'-DDD, 4,4'-DDT, endosulfan sulfate, endrin aldehyde, and
4,4'-methoxychlor. The reproducibility of the retention times was excellent
(<0.15 percent) for the organochlorine pesticides.
In the case of the PCBs, the peak areas were summed for all peaks, and a
response factor was determined from the total response" and from the amount
injected (Table 39). Six major components that represent the mixture were
selected and were used to calculate the relative standard deviation of the
ten replicate measurements (Table 40). The reproducibility of the injection,
when the sum of all peaks was used in the calculations, was better than
15 percent. As expected, the reproducibility was better for the early
eluting peaks but degraded for the late-eluting components of the PCB
mixture. The reproducibility of the retention time was also excellent for
the two PCB mixtures tested (Table 41). This indicates that the identification
of the organochlorine pesticides and PCBs by retention time match is reliable
for screening purposes.
74
-------
in
TABLE 37. REPRODUCIBILITY OF THE RESPONSE FACTORS OF THE OR6ANOCHLORINE
PESTICIDES FOR 10 CONSECUTIVE REPLICATE INJECTIONSa
Response' factor x
Compounds
alpha-BHC
beta-BHC
gamma -BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE + Oieldrin
Endrin
Endosulfan II
4,4'-ODD
Endrin aldehyde
Endosulfan sulfate
4, 4' -DDT
4,4'-Methoxychlor
No. 1
2.76
0.94
2.54
2.40
2.19
2.22
2.03
2.05
1.90
3.29
1.28
1.75
1.50
1.35
1.51
1.47
0.57
No. 2
2.87
0.86
2.54
2.29
2.11
2.10
1.88
1.85
1.67
2.92
1.01
1.50
1.23
1.10
1.20
1.15
0.42
No. 3
2.97
0.99
2.70
2.56
2.33
2.36
2.23
2.20
2.07
3.50
1.32
1.86
1.60
1.46
1.59
1.50
0.61
No. 4
2.92
0.90
2.60
2.41
2.20
2.23
2.07
2.01
1.82
3.12
1.08
1.63
1.38
1.28
1.35
1.29
0.49
No. 5
2.99
0.99
2.67
2.57
2.34
2.39
2.23
2.21
2.01
3.50
1.26
1.87
1.60
1.42
1.58
1.50
0.57
No. 6
2.83
0.84
2.50
2.27
2.07
2.08
1.90
1.86
1.67
2.88
9.10
1.49
1.23
1.15
1.20
1.13
0.41
10-6
No. 7
2.88
0.92
2.58
2.45
2.24
2.28
2.17
2.13
1.93
3.34
1.06
1.78
1.47
1.36
1.46
1.37
0.47
No. 8
2.79
0.81
2.47
2.23
2.04
2.04
1.85
1.82
1.63
2.81
8.76
1.45
1.17
1.05
1.16
1.09
0.39
No. 9
2.97
0.99
2.65
2.57
2.34
2.39
2.27
2.24
2.05
3.55
1.25
1.91
1.64
1.53
1.63
1.53
0.59
No. 10
2.89
0.92
2.58
2.44
2.25
2.29
2.13
2.13
1.93
3.36
1.16
1.79
1.52
1.42
1.52
1.43
0.55
Average
2.88
0.92
2.58
2.42
2.21
2.23
2.08
2.05
1.87
3.23
1.12
1.70
1.43
1.32
1.42
1.35
0.51
RSD
(percent)
2.5
6.9
2.9
5.2
5.0
5.8
7.5
8.0
8.7
8.6
14.1
10.1
12.0
12.4
12.6
12.5
16.1
»
aThe amount injected is 100 pg per component; the response factor is defined as peak area divided by the
amount injected.
bThe analyses were performed on a 30 m x 0.25 mm ID DB-5 fused-silica capillary column; temperature program:
160°C (2 rain hold) to 270°C (1 min hold) at 5°C/min; carrier gas: nitrogen at 20 psi.
-------
TABLE 38. REPRODUCIBILITY OF THE RETENTION TIMES OF THE OR6ANOCHLORINE
PESTICIDES FOR 10 CONSECUTIVE REPLICATE INJECTIONSa
Retention
Compounds
alpha-BHC
beta-BHC
gamma -BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma -Chi ordane
Endosulfan I
4,4'-DDE ••• Dieldrln
Endrin
Endosulfan II
4,4'-DOD
Endrin aldehyde
Endosulfan sulfate
4, 4 '-DDT
4,4'-Methoxychlor
No. 1
6.515
7.274
7.499
8.206
9.893
11.099
12.531
13.399
13.849
14.799
15.590
15.933
16.341
16.634
17.528
17.729
19.924
No. 2
6.511
7.261
7.475
8.200
9.880
11.093
12.521
13.398
13.831
14.794
15.582
15.923
16.329
16.638
17.523
17.728
19.917
No. 3
6.506
7.261
7.476
8.197
9.875
11.085
12.517
13.381
13.833
14.790
15.581
15.922
16.326
16.631
17.513
17.722
19.918
No. 4
6.500
7.260
7.482
8.195
9.882
11.087
12.516
13.381
13.829
14.792
15.580
15.920
16.333
16.632
17.523
17.726
19.918
No. 5
6.487
7.243
7.464
8.173
9.865
11.071
12.503
13.367
13.812
14.776
15.571
15.908
16.313
16.618
17.503
17.711
19.900
No. 6
6.520
7.267
7.495
8.208
9.889
11.095
12.525
13.390
13.843
14.801
15.586
15.922
16.333
16.633
17.522
17.730
19.916
time (rain.)
No. 7
6.512
7.265
7.496
8.208
9.886
11.102
12.529
13.398
13.834
14.801
15.589
15.924
16.338
16.642
17.523
17.739
19.922
No. 8
6.501
7.267
7.486
8.195
9.880
11.095
12.524
13.393
13.834
14.800
15.583
15.923
16.333
16.636
17.524
17.735
19.917
No. 9
6.501
7.254
7.473
8.193
9.870
11.083
12.507
13.369
13.820
14.784
15.569
15.917
16.320
16.623
17.508
17.721
19.907
No. 10
6.498
7.253
7.481
8.185
9.877
11.082
12.521
13.382
13.832
14.787
15.579
15.916
16.323
16.628
17.513
17.722
19.913
Average
6.505
7.260
7.482
8.196
9.880
11.089
12.519
13.386
13.832
14.792
15.581
15.921
16.329
16.631
17.518
17.726
19.915
RSD
(percent)
0.149
0.120
0.151
0.133
0.086
0.084
0.071
0.087
0.075
0.056
0.044
0.040
0.052
0.042
0.047
0.045
0.035
aThe analyses were performed on a 30 m x 0.25 mm ID DB-5 fused-silica capillary column; temperature program:
(2 min hold) to 270°C (1 min hold) at 5°C/min; carrier gas: nitrogen at 20 psi.
160°C
-------
TABLE 39. REPRODUCIBILITY OF THE RESPONSE FACTORS FOR 10 CONSECUTIVE
INJECTIONS OF PCB-1016/1260 STANDARDS3-15
Response factor
Compounds No. 1 No. 2 No. 3 No. 4 No. 5 No. 6
PCB-1016/1260 1.868 x 106 1.514 x 106 1.310 x 106 1.630 x 106 1.283 x 10$ 1.858 x 106
Response factor
RSD
Compounds No. 7 No. 8 No. 9 No. 10 Average (percent)
PCB-1016/1260 1.525 x 106 1.711 x 106 1.805 x 106 1.284 x 106 1.579 x 106 14.7
===========a==============================3======================================================:
aThe amount injected is 100 pg iper component; the response factor is defined as peak area divided
by the amount injected.
bThe analyses were performed on a 30 m x 0.25 mm ID DB-5 fused-silica capillary column;
temperature program: 160°C (2 min hold) to 270°C (1 min hold) at 5°C/min; carrier gas:
nitrogen at 20 psi.
-------
TABLE 40. REPRODUCIBILITY OF AREA RESPONSE FOR 10 CONSECUTIVE
INJECTIONS OF PCB-1016/1260 STANDARDS (SIX MAJOR
COMPONENTS)3
Peak area
Retention time
(mln.) No. 1
RSD
No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10 Average (percent)
00
9.327 107856 100454 112331
15.936 64705 53292 69306
18.520 52137 42329 56945
19.371 44206 36759 50050
20.402 106234 90652 126169
21.467 48269 41379 57653
99213 121480 108640 117956 115803
52933 80229 65305 72680 75767
41930 67907 54236 60270 63450
35770 59016 46733 53064 56815
86008 149645 114682 133804 142659
38885 68117 51297 60409 65657
93180 115883 109280 8.4
54488 75623 66433 15.1
44894 63106 54720 17.0
38747 56027 47719 18.1
95441 144975 119027 19.9
42936 68507 54310 20.8
aAnalyses were performed on a 30 m x 0.25 mm ID DB-5 fused-silica capillary column; temperature program:
(2 min hold) to 270°C (1 m1n hold) at 5°C/m1n; carrier gas: nitrogen at 20 psi.
160°C
-------
TABLE 41. REPROnUCIBILITY OF RETENTION TIMES OF SIX MAJOR COMPONENTS
IN A PCB-1016/1260 MIXTURE FOR 10 CONSECUTIVE INJECTIONS3
Retention time (m1n)
RSD
Peak No. No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10 Average (percent)
to
1
2
3
4
5
6
9.333
15.963
18.523
19.377
20.402
21.474
9.327
15.962
18.520
19.371
20.393
21.467
9.340
15.966
18.528
19.380
20.405
21.472
9.35
15.968
18.529
19.381
20.407
21.482
9.335
15.969
18.528
19.379
20.403
21.477
9.335
15.963
18.525
19.378
20.407
21.481
9.312
15.948
18.504
19.356
20.382
21.458
9.321
15.958
18.522
19.378
20.397
21.471
9.346
15.967
18.518
19.372
20.392
21.466
9.335
15.956
18.514
19.367
20.390
21.465
9.32
15.962
18.521
19.374
20.398
21.471
0.104
0.040
0.042
0.040
0.041
0.035
aAnalyses were performed on a 30 m x 0.25 mm ID OB-5 fused-siUca capillary column; temperature program:
160°C (2 mln hold) to 270°C (1 mln hold) at 5°C/m1n; carrier gas: nitrogen at 20 ps1.
-------
7.2 METHOD PRECISION AND ACCURACY
Experimental Section
Sample Collection: Liquid waste Ho. 1 was collected from a pesticide
waste storage facility at a California agricultural field station. This
waste contains high concentrations of organics (total organic carbon 520 mg/L;
total organic halogen 30 mg/L) and was briefly described in Section 5. The
standard reference material was NBS SRM 1645. According to the NBS, the
material was dredged from the bottom of the Indiana Harbor Canal near Gary,
Indiana. The material was screened to remove foreign objects, freeze-dried,
and sieved (particle size <180 urn). The material was sterilized by radiation
to minimize biological activity. A gross characterization done by NBS was also
given in Section 5.
Sandy loam was obtained from Soils Incorporated, Puyallup, Washington;
the following physicochemical characteristics were determined: pH 5.9 to 6.0;
89 percent sand; 7 percent silt; 4 percent clay; cation exchange capacity
7.5 meq/lOOg; total organic carbon content 1290 ± 185 mg/Kg.
Reagents. Stock solutions of 4,4'-methoxychlor (5000 ng/uL in methylene
chloride), gamma-chlordane (5000 ng/uL in hexane) and a composite solution of
aldrin, alpha-BHC, beta-BHC, gamma-BHC, delta-BHC, 4,4'-DDD, 4,4'-DDE,
4,4'-DDT, dieldrin, endosulfan I, endosulfan II, endosulfan sulfate, endrin,
endrin aldehyde, heptachlor, and heptachlor epoxide, at 2,000 ng/uL in
toluene-hexane (1:1), were obtained from the U.S. EPA Quality Assurance
Materials Bank, Research Triangle Park, North Carolina. Neat standards of
Kepone, PCB-1016, and PCB-1260 were obtained from the U.S. EPA Pesticides and
Industrial Chemicals Repository, Research Triangle Park, North Carolina, and
were used to prepare stock solutions of Kepone at 1000 ng/uL each in benzene
and of PCB-1016 and PCB-1260 together at 2,500 ng/uL in hexane. These individ-
ual stock solutions and the composite solutions were further diluted with
hexane to obtain the spiking solutions and the analytical standards for
instrument calibration.
Sample Extract Preparation. The liquid waste No._l (300 mL) was
extracted in a separatory funnel at neutral pH with 60 mL methylene
chloride. The extraction was repeated two times; fresh aliquots of methylene
chloride were used each time. The extracts were combined, were dried through
a column of anhydrous sodium sulfate, and were then concentrated in a
Kuderna-Dam'sh evaporator. The solvent was exchanged to hexane and
concentrated to 30 mL. The 30-mL concentrated extract was split as follows:
a 2-mL aliquot was used for background analysis; three aliquots of 6 mL each
were spiked with 1,500 ng (concentration 1), 3,000 ng (concentration 2), and
15,000 ng (concentration 3) of the organochlorine pesticides, and 15,000 ng
(concentration 1), 30,000 ng (concentration 2), and 150,000 ng
(concentration 3) of PCB-1016 and of PCB-1260, respectively.
A 30-g portion of the NBS SRM-1645 or of the sandy loam was extracted
with 60-mL portions of hexane-acetone (1:1) by using a sonicator model W-375
from Heat Systems-Ultrasonics, Inc. The extraction was performed three times
80
-------
for 3 min each time with fresh portions of hexane-acetone. The extracts were
combined, were concentrated to 30 ml in a Kuderna-Dam'sh evaporator, and were
split and spiked as described above for liquid waste No. 1.
Silica Gel Fractionation. All extracts were subjected to silica gel
chromatography on silica deactivated with 3.3 percent water (Davidson grade
923, 100/200 mesh, Supelco Inc.). The elution patterns and the recoveries of
the 19 organochlorine pesticides and of PCB-1016 and PCB-1260 were determined
in triplicate at three concentrations (1,500 ng, 3,000 ng, 15,000 ng for each
compound per column) corresponding to the spiking levels in the liquid waste
and in the solid samples. PCBs were spiked at concentrations ten times as high
as those of the pesticides. Four fractions were collected: Fraction I eluted
with 80 ml hexane; Fraction II eluted with 50 mL hexane; Fraction III eluted
with 15 ml methylene chloride; and Fraction IV eluted with 50 ml ethyl acetate.
The Fraction III solvent was exchanged to hexane, and the Fraction IV solvent
was exchanged to 2 percent methanol in benzene prior to analysis. The final
volume of each fraction was 10 ml.
Gas Chrpmatography/Electron Capture Detection. A Varian Vista 6000 gas
chromatograph equipped with a split/splitless injector and fused-silica
capillary column (30 m x 0.25 mm ID; film thickness 0.25 urn) DB-5 (J&W
Scientific, Rancho Cordova, California) was used for analyzing all sample
extracts and silica gel fractions. The oven was held at 100°C for 2 min
during sample injection, the temperature was then programmed to Increase
at 15°C/min to 160°C and at 5'C/min to 270°C. The injector and detector
temperatures were set at 225°C and at 300°C, respectively. Quantification
was performed by external standard calibration. The PCBs were identified by
visual comparison of the sample extract chromatograms with the PCB standard
chromatograms. Five to six gas chromatographic peaks were selected to quantify
each PCB mixture, and the response factors were determined by the analysis of
standards containing known concentrations of each PCB mixture.
RESULTS AND DISCUSSION
The accuracy and the precision data are given in Tables 42 through 46 as the
average percent recovery ± one standard deviation and as the relative standard
deviation of the average recovery (number of determinations is three). The
recovery was calculated as the ratio of the amount of a given compound detected
to the amount added to the sample extract prior to silica gel fractionation.
The amount of compound detected is computed from the detector response relative
to the standard. Precision is expressed as percent relative standard deviation.
From these data we conclude that the four-fraction silica gel
fractionation allows separation of the organochlorine pesticides from PCBs,
with the exception of heptachlor, aldrin, gamma-chlordane, 4,4'-DDE, and
4,4'-DDT. The silica gel procedure is tedious and does account for a major
part of the analysis time. However, we have demonstrated that the method
precision is better than ±20 percent for all compounds, and the accuracy is
greater than 60 percent when standards are processed through the silica gel
procedure. Furthermore, the use of a capillary column for gas chromatographic
81
-------
TABLE 42. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE
PESTICIDES AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (STANDARDS ONLY)
Average ± SD (RSD) at Concentration la
00
PO
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4, 4 '-DDE
Dieldrin
Endrin
Endosulfan II
4, 4 '-ODD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Fraction I
hexane
(80 mL)
79 ± 11 (13)
88 ± 11 (13)
78 ± 13 (17)
92 ± 12 (14)
i
74 ± 19 (26)
94 ± 14 (15)
92 ± 12 (13)
Fraction III
Fraction II methyl ene Fraction IV
hexane chloride ethyl acetate
(50 mL) (15 mL) (50 mL) Total recovery
59 ± 15 (25) 25 ± 9.2
86 ± 15
85 ± 15
87 ± 12
94 ± 6.7
15 ± 4.7 (31)
89 ± 12
89 ± 12
66 ± 11
86 ± 7.9
39 ± 15 (38) 50 ± 11
83 ± 8.3
91 ± 5.2
98 ± 2.6
(37)
(17)
(18)
(14)
(7.1)
(14)
(14)
(17)
(9.2)
(22)
(10)
0
(5.7)
(2.7)
83 ± 16
86 ± 15
85 ± 15
87 ± 12
79 ± 11
88 ± 11
94 ± 6.7
94 ± 13
89 ± 12
92 ± 13
89 ± 12
66 ± 11
86 ± 7.9
89 ± 12
83 ± 8.3
0
91 ± 5.2
74 ± 19
98 ± 2.6
94 ± 14
92 ± 12
(19)
(17)
(18)
(14)
(13)
(13)
(7.1)
(14)
(14)
(14)
(14)
(17)
(9.2)
(14)
(10)
(5.7)
(26)
(2.7)
(15)
(13)
aThe values given represent the average percent recoveries of the organochlonne pesticides and PCBs from
five replicate determinations ± one standard deviation; the numbers in parentheses are the relative
standard deviations.
-------
TABLE 42. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE
PESTICIDES AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (STANDARDS ONLY)
(CONTINUED)
Ave ± SD (RSD) at Concentration
00
CJ
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxlde
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4, 4 '-ODD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Fraction I Fraction II
hexane hexane
(80 mL) (50 mL)
64 ± 6.0 (9.5)
94 ± 9.5 (10)
107 ± 9.5 (8.9)
86 ± 8.4 (9.4) 23 ± 3.2 (14)
107 ± 15 (14)
38 ± 3.5 (9.2)
i
88 ± 13 (15)
93 ± 6.5 (7.0)
87 ± 15 (17)
Fraction III
methyl ene Fraction IV
chloride ethyl acetate
(15 mL) (50 mL) Total recovery
41 ± 5.9
110 ± 10
108 ± 11
109 ± 12
109 ± 14
108 ± 13
112 ± 13
65 ± 10
111 ± 14
71 ± 13
102 ± 19
112 ± 21
104 ± 18
(14)
(9.5)
(10)
(11)
(13)
(12)
(12)
(16)
(13)
(18)
(19)
0
(19)
(17)
106 ± 6.8
110 ± 10
108 ± 11
109 ± 12
94 ± 9.5
107 ± 9.5
109 ± 14
110 ± 11
108 ± 13
107 ± 15
112 ± 13
65 ± 10
111 ± 14
110 ± 9.8
102 ± 19
0
112 ± 21
88 ± 13
104 ± 18
93 ± 6.5
87 ± 15
(6.4)
(9.5)
(10)
(11)
(10)
(8.9)
(13)
(10)
(12)
(14)
(12)
(16)
(13)
(8.9)
(19)
(19)
(15)
(17)
(7.0)
(17)
aThe values given represent the average percent recoveries from three replicate determinations ± one
standard deviation; the numbers in parentheses are the relative standard deviations.
-------
TABLE 42. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE
PESTICIDES AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (STANDARDS ONLY)
(CONCLUDED)
Average ± SD (RSD) at Concentration
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor
epoxide
gamma-Chlordane
Endosulfan
4,4'-DDE
Dieldrin
Endrin
Endosulfan
4,4'-DDD
I
II
Fraction I Fraction II
hexane hexane
(80 mL) (50 mL)
83
89
70
89
45 ± 12 (26)
± 6.6 (7.9)
± 4.2 (4.7)
± 7.7 (11) 20 ± 6.8 (34)
± 4.5 (5.0)
24 ± 10 (42)
Endrin aldehyde ',
Kepone
Endosulfan
4,4'-DDT
sulfate
73
± 5.8 (8.0)
4,4'-Methoxychlor
PCB-1016
PCB-1260
93
78
± 2.0 (2.2)
± 4.0 (5.1)
Fraction III
methylene Fraction IV
chloride ethyl acetate
(15 mL) (50 mL)
47
98
99
97
100
99
102
64
101
73
95
104
104
± 10
± 2.0
± 2.3
± 1.6
± 23
± 2.3
± 1.5
± 8.3
± 0.6
± 8.8
± 3.0
± 2.5
± 3.2
(22)
(2.1)
(2.3)
(1.6)
(23)
(2.3)
(1.5)
(13)
(0.6)
(12)
(3.2)
58 ± 25 (44)
(2.4)
(3.1)
Total
91
98
99
97
83
89
100
91
99
89
102
64
101
97
95
58
104
73
104
93
78
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
recovery
4.
2.
2.
1.
6.
4.
23
1.
2.
4.
1.
8.
0.
1.
3.
25
2.
5.
3.
2.
4.
6
0
3
6
6
2
2
3
5
5
3
6
7
0
5
8
2
0
0
(5.1)
(2.1)
(2.3)
(1.6)
(7.9)
(4.7)
(23)
(1.3)
(23)
(5.0)
(1.5)
(13)
(0.6)
(1.8)
(3.2)
(44)
(2.4)
(8.0)
(3.1)
(2.2)
(5.1)
values given represent the average percent recoveries from three replicate determinations ± one
standard deviation; the numbers in parentheses are the relative standard deviations.
-------
00
in
TABLE 43. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES
AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (LIQUID WASTE NO. 1 EXTRACT)
Average ± SD (RSD) at Concentration la
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrln
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrln
Endosulfan II
4, 4 '-ODD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Fraction I Fraction II
hexane hexane
(80 ml) (50 ml)
58 ± 13 (22)
87 ± 13 (15)
96 ± 8.9 (9.3)
83 ± 12 (14) 17 ± 6.3 (37)
119 ± 11 (8.9)
32 ± 13 (40)
i
101 ± 23 (23)
114 ± 6.0 (5.3)
99 ± 4.6 (4.6)
Fraction III
methyl ene Fraction IV
chloride ethyl acetate
(15 ml) (50 mL) Total recovery
39 ±
92 ±
91 ±
111 ±
95 ±
88 ±
101 ±
i
1
i
132 ±
49 ±
5.5
10
10
17
6.5
3.2
5.9
17
14
(14)
(11)
(11)
(15)
(6.8)
(3.6)
(5.8)
0
(13)
(29)
96 ± 7.0
92 ± 10
91 ± 10
i
87 ± 13
96 ± 8.9
111 ± 17
100 ± 8.5
95 ± 6.5
119 ± 11
88 ± 3.2
101 ±. 5.9
i
i
i
0
132 ± 17
101 ± 23
49 ± 14
114 ± 6.0
99 ± 4.6
(7.3)
(11)
(11)
(15)
(9.3)
(15)
(8.5)
(6.8)
(8.9)
(3.6)
(5.8)
(13)
(23)
(29)
(5.3)
(4.6)
aThe values given represent the average percent recoveries from three replicate determinations ±
standard deviation; the numbers in parentheses are the relative standard deviations.
i — Unable to determine the recovery because of interference.
one
-------
TABLE 43.
ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES
AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (LIQUID WASTE NO. 1 EXTRACT)
(CONTINUED)
Average ± SD (RSD) at Concentration 2a
CO
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dleldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Fraction I
hexane
(80 mL)
94 ± 10
98 ± 9.4
89 ± 6.5
113 ± 2.5
83 ± 11
122 ± 10
102 ± 4.7
Fraction II
hexane
(50 mL)
60 ± 5.7 (9.5)
(11)
(9.6)
(7.3) 13 ± 4.2 (32)
(2.2)
37 ± 19 (50)
i
i
(13)
(8.3)
(4.6)
Fraction III
methyl ene
chloride
(15 mL)
37 ± 7.4 (20)
100 ± 4.0 (4.0)
100 ± 5.5 (5.5)
i
109 ± 14 (13)
100 ± 12 (12)
86 ± 9.5 (11)
90 ± 9.9 (11)
i
i
i
127 ±22 (17)
58 ± 9.3 (16)
Fraction IV
ethyl acetate
(50 mL) Total recovery
97 ± 3.5 (3.6)
100 ± 4.0 (4.0)
100 ± 5.5 (5.5)
i
94 ± 1 (11)
98 ± 9.4 (9.6)
109 ± 14 (13)
103 ± 2.5 (2.4)
100 ± 12 (12)
113 ± 2.5 (2.2)
86 ± 9.5 (11)
90 ± 9.9 (11)
i
i
i
0 0
127 ±22 (17)
83 ± 11 (13)
58 ± 9.3 (16)
122 ± 10 (8.3)
102 ± 4.7 (4.6)
aThe values given represent the average percent recoveries from three replicate determinations ±
standard deviation; the numbers in parentheses are the relative standard deviations.
i — Unable to determine the recovery because of interference.
one
-------
TABLE 43. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES
AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (STANDARDS ONLY) (CONCLUDED)
Average ± SD (RSD) at Concentration 3a
00
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4, 4 '-DDE
Dieldrin
Endrin
Endosulfan II
4, 4 '-ODD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Fraction I
hexane
(80 mL)
90 ± 11
92 ± 9.2
85 ± 7.2
95 ± 16
88 ± 18
118 ± 9.8
100 ± 18
Fraction II
hexane
(50 mL)
57 ± 2.5 (4.4)
(12)
(10)
(8.5) 10 ± 9.2 (92)
(17)
33 ± 4.0 (15)
i
(21)
(8.3)
(18)
Fraction III
methyl ene Fraction IV
chloride ethyl acetate
(15 mL) (50 mL)
22 ± 9.2 (42)
90 ± 3.1 (3.4)
90 ± 4.0 (4.4)
90 ± 11 (8.8)
89 ± 4.1 (4.6)
88 ± 3.8 (4.3)
82 ± 4.3 (5.3)
65 ± 3.1 (4.7)
79 ± 7.1 (9.0)
43 ± 16 (37)
i
113; 223°
83 ± 4.0 (4.8)
75 ± 4.6 (6.1)
Total recovery
79 ± 10 (13)
90 ± 3.1 (3.4)
90 ± 4.0 (4.4)
90 ± 11 (8.8)
90 ± 11 (12)
92 ± 9.2 (10)
89 ± 4.1 (4.6)
95 ± 8.0 (8.4)
88 ± 3.8 (4.3)
95 ± 16 (17)
82 ± 4.3 (5.3)
65 ± 3.1 (4.7)
79 ± 7.1 (9.0)
76 ± 16 (21)
i'b
113; 223°
83 ± 4.0 (4.8)
88 ± 18 (21)
75 ± 4.6 (6.1)
118 ± 9.8 (8.3)
100 ± 18 (18)
aThe values given represent the average percent recoveries from three replicate determinations ± one
standard deviation; the numbers in parentheses are the relative standard deviations.
DDuplicate determinations.
i -- Unable to determine the recovery because of interference.
-------
TABLE 44. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES
AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (NBS SEDIMENT EXTRACT)
Average ± SD (RSD) at Concentration la
00
CO
Fraction I Fraction II
hexane hexane
Compound (80 mL) (50 mL)
alpha-BHC 52 ± 6.2 (12)
beta-BHC
gamma-BHC
delta-BHC
Heptachlor 53 ± 10 (19)
Aldrin 69 ± 3.6 (5.2)
Heptachlor epoxide
gamma-Chlordane 67 ± 7.4 (11) 10 ± 2.6 (26)
Endosulfan I
4,4'-DDE 71 ± 7.1 (10)
Dieldrin
Endrin
Endosulfan II
4,4'-DDD 37 ± 8.9 (37)
Endrin aldehyde
Kepone '.
Endosulfan sulfate
4,4'-DDT 54 ± 13 (24)
4,4'-Methoxychlor
PCB-1016 104 ± 9.1 (8.7)
PCB-1260 92 ± 9.2 (10)
Fraction III
methyl ene
chloride
(15 mL)
21
88
83
85
91
85
92
100
80
69
70
75
±
±
±
±
±
±
±
±
±
±
±
±
6
4
3
4
4
5
8
9
7
12
5
9
.1
.1
.0
.6
.9
.5
.6
.5
.4
Fraction IV
ethyl acetate
(50 mL) Total recovery
(29)
(4
(3
(5
(5
(6
(9
(9
(9
.7)
.6)
.4)
.4)
.5)
.4)
.5)
.2)
(18)
.7
.0
(8
.2)
0
(12)
73
88
83
85
53
69
91
77
85
71
92
100
80
106
70
0
75
54
i
104
92
+
±
±
±
±
±
±
±
±
±
±
±
±
±
+
±
±
±
±
2
4
3
4
10
3
4
5
5
7
8
9
7
6
5
9
13
9
9
.1
.1
.0
.6
.6
.9
.3
.5
.1
.6
.5
.4
.4
.7
.0
.1
.2
(2.9)
(4.7)
(3.6)
(5.4)
(19)
(5.2)
(5.4)
(6.9)
(6.5)
(10)
(9.4)
(9.5)
(9.2)
(6.0)
(8.2)
(12)
(24)
(8.7)
(10)
aThe values given represent the average percent recoveries from three replicate determinations ±
standard deviation; the numbers in parentheses are the relative standard deviations.
i — Unable to determine the recovery because of interference.
one
-------
TABLE 44. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES
AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (NBS SEDIMENT EXTRACT)
(CONTINUED)
Average ± SD (RSD) at Concentration 2a
00
10
Fraction I Fraction II
hexane hexane
Compound (80 mL) (50 mL)
alpha-BHC 55 ± 6.1 (11)
beta-BHC
gamma-BHC
delta-BHC
Heptachlor 70 ± 7.7 (11)
Aldrin 65 ± 4.6 (7.1)
Heptachlor epoxide
gamma-Chlordane 71 ± 3.2 (4.5) 10 ± 2.0 (20)
Endosulfan I
4,4'-DDE 76 ± 7.1 (9.3)
Dieldrin
Endrin
Endosulfan II
4,4'-DDD 36 ± 2.0 (5.6)
Endrin aldehyde '.
Kepone
Endosulfan sulfate
4,4'-DDT 61 ± 7.9 (13)
4,4'-Methoxychlor
PCB-1016 104 ± 2.5 (2.4)
PCB-1260 95 ± 7.5 (7.9)
Fraction III
methyl ene
chloride
(15 mL)
20
94
89
92
91
88
85
87
81
49
71
86
99
±
±
±
±
±
±
±
±
±
±
±
±
±
1
3
4
5
5
5
9
6
4
1
9
5
17
.7
.0
.1
.2
.7
.1
.4
.4
.5
.2
.2
.0
(8.
(3.
(4.
(5.
(6.
(5.
(11
(7.
(5.
(2.
Fraction IV
ethyl acetate
(50 mL) Total recovery
7)
2)
6)
6)
3)
8)
)
3)
5)
4)
(13)
(5.
(17
0
8)
)
75
94
89
92
70
65
91
81
88
76
85
87
81
85
71
0
86
61
99
104
95
±
±
±
±
+
±
±
+
±
±
±
±
±
±
±
±
±
±
+
±
6.
3.
4.
5.
7.
4.
5.
4.
5.
7.
9.
6.
4.
3.
9.
5.
7.
17
2.
7.
0
0
1
2
7
6
7
9
1
1
4
4
5
1
2
0
9
5
5
(8.0)
(3.2)
(4.6)
(5.6)
(11)
(7.1)
(6.3)
(6.1)
(5.8)
(9.3)
(11)
(7.3)
(5.5)
(3.6)
(13)
(5.8)
(13)
(17)
(2.4)
(7.9)
aThe values given represent the average percent recoveries from three replicate determinations ± one
standard deviation; the numbers in parentheses are the relative standard deviations.
-------
TABLE 44.
ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES
AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (NBS SEDIMENT EXTRACT)
(CONCLUDED)
Average ± SD (RSD) at Concentration 3a
10
o
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4, 4 '-ODD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
aThe values given
Fraction I
hexane
(80 mL)
88 ±
72 ±
75 ±
84 ±
76 ±
102 ±
91 ±
represent
4.1
1.0
1.5
1.0
2.5
4.6
4.0
the
Fraction 11
hexane
(50 mL)
55 ± 3.6
(4.7)
(1.4)
(2.0) 10 ± 2.9
(1.2)
42 ± 2.3
i
(3.3)
(4.5)
(4.4)
average percent
Fraction III
[ methyl ene Fraction IV
chloride ethyl acetate
(15 mL) (50 mL) Total
(6.6) 21
92
93
94
93
(29)
91
94
76
91
(5.5) 47
88
72
92
recoveries
±
±
±
±
±
±
±
±
±
±
±
±
±
2
7
8
8
8
9
10
9
12
5
12
11
17
Trom
.7 (13)
.1 (7.7)
.1 (8.7)
.7 (9.3)
.6 (9.2)
.1 (10)
(11)
.9 (13)
(13)
.6 (12)
(14)
225 ±
(15)
(19)
three replicate
76
92
93
94
88
72
93
85
91
84
94
76
91
90
88
99 (44) 225
72
76
92
102
91
determinati
±
±
±
±
±
±
±
±
±
±
±
+
+
+
±
±
±
±
±
±
±
ons
recovery
5.6
7.1
8.1
8.7
4.1
1.0
8.6
1.0
9.1
1.0
10
9.9
12
7.2
12
99
11
2.5
17
4.6
4.0
(7.3)
(7.7)
(8.7)
(9.3)
(4.7)
(1.4)
(9.2)
(1.2)
(10)
(1.2)
(11)
(13)
(13)
(8.0)
(14)
(44)
(15)
(3.3)
(19)
(4.5)
(4.4)
± one
standard deviation; the numbers in parentheses are the relative standard deviations.
-------
TABLE 45. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES
AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (SANDY LOAM EXTRACT)
Average ± SD (RSD) at Concentration la
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4, 4 '-ODD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Fraction I Fraction II
hexane hexane
(80 mL) (50 mL)
61 ± 13 (21)
89 ± 9.8 (11)
99 ± 4.4 (4.4)
87 ± 9.6 (11) 13 ± 3.5 (27)
105 ± 14 (13)
42 ± 5.5 (13)
i
i
107 ± 25 (23)
90 ± 15 (17)
99 ± 6.8 (6.9)
Fraction III
methyl ene Fraction IV
chloride ethyl acetate
(15 mL) (50 mL) Total recovery
25 ± 3.5
94 ± 8.4
92 ± 11
94 ± 12
96 ± 11
95 ± 10
113 ± 12
74 ± 5.6
97 ± 14
61 ± 4.1
86 ± 11
112 ± 19
91 ± 14
(14)
(8.9)
(12)
(13)
(11)
(11)
(11)
(7.5)
(14)
(6.7)
(13)
0
(17)
(15)
86 ± 9.5
94 ± 8.4
92 ± 11
94 ± 12
89 ± 9.8
99 ± 4.4
96 ± 11
100 ± 8.3
95 ± 10
105 ± 14
113 ± 12
74 ± 5.6
97 ± 14
103 ± 9.6
86 ± 11
0
112 ± 19
107 ± 25
91 ± 14
90 ± 15
99 ± 6.8
(11)
(8.9)
(12)
(13)
(11)
(4.4)
(11)
(8.3)
(11)
(13)
(11)
(7.5)
(14)
(9.3)
(13)
(17)
(23)
(15)
(17)
(6.9)
«The values given represent the average percent recoveries from three replicate determinations ±
standard deviation; the numbers in parentheses are the relative standard deviations.
one
-------
TABLE 45. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES
AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (SANDY LOAM EXTRACT) (CONTINUED)
Average ± SD (RSD) at Concentration 2a
ro
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Fraction I Fraction II
hexane hexane
(80 mL) (50 mL)
49 ± 3.5 (7.1)
83 ± 9.1 (11)
88 ± 2.0 (2.3)
70 ± 6.0 (8.6) 23 ± 5.1 (22)
93 ± 5.6 (6.0)
21 ± 1.2 (5.6)
i
83 ± 5.3 (6.4)
92 ± 4.5 (4.9)
89 ± 8.9 (10)
Fraction III
methyl ene Fraction IV
chloride ethyl acetate
(15 mL) (50 mL) Total recovery
38
90
91
89
90
89
99
60
86
67
82
91
89
±
+
±
±
±
±
±
±
±
±
±
±
±
4
4
3
3
4
5
4
7
6
7
7
13
9
.2
.1
.0
.6
.6
.3
.6
.8
.7
.4
.6
.8
(11)
(4.5)
(3.3)
(4.1)
(5.1)
(5.9)
(4.6)
(13)
(7.8)
(11)
(9.3)
0
(14)
(11)
87
90
91
89
83
88
90
93
89
93
99
60
86
88
82
0
91
83
89
92
89
±
±
±
±
±
±
±
±
±
±
±
±
±
+
±
±
±
~+
±
±
4.
4.
3.
3.
9.
2.
4.
3.
5.
5.
4.
7.
6.
7.
7.
13
5.
9.
4.
8.
9
1
0
6
1
0
6
0
3
6
6
8
7
8
6
3
8
5
9
(5.7)
(4.5)
(3.3)
(4.1)
(11)
(2.3)
(5.1)
(3.2)
(5.9)
(6.0)
(4.6)
(13)
(7.8)
(8.9)
(9.3)
(14)
(6.4)
(11)
(4.9)
(10)
aThe values given represent the average percent recoveries from three replicate determinations ±
standard deviation; the numbers in parentheses are the relative standard deviations.
one
-------
TABLE 45. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES
AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (SANDY LOAM EXTRACT) (CONCLUDED)
Average ± SD (RSD) at Concentration 3a
vo
CO
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Fraction I
hexane
(80 mL)
79 ± 17
82 ± 12
63 ± 8.8
82 ± 11
64 ± 13
85 ± 10
85 ± 13
Fraction II
hexane
(50 mL)
46 ± 4.6 (10)
(21)
(15)
(14) 24 ± 2.4 (10)
(13)
18 ± 2.7 (15)
i
(21)
(12)
(15)
Fraction III
methylene
chloride
(15 mL)
43 ± 2.0 (4.8)
93 ± 5.0 (5.4)
95 ± 4.6 (4.8)
93 ± 6.1 (6.5)
94 ± 6.6 (7.0)
93 ± 7.5 (8.1)
93 ± 8.2 (8.8)
47 ± 11 (24)
89 ± 11 (12)
72 ± 5.2 (7.2)
73 ± 20 (28)
88 ± 11 (13)
86 ± 14 (16)
Fraction IV
ethyl acetate
(50 mL) Total recovery
89 ± 2.5 (2.8)
93 ± 5.0 (5.4)
95 ± 4.6 (4.8)
93 ± 6.1 (6.5)
79 ± 17 (21)
82 ± 12 (15)
94 ± 6.6 (7.0)
87 ± 11 (13)
93 ± 7.5 (8.1)
82 ± 11 (13)
93 ± 8.2 (8.8)
47 ± 11 (24)
89 ± 11 (12)
90 ± 7.8 (8.6)
73 ± 20 (28)
260 ± 55 (21) 260 ± 55 (21)
88 ± 11 (13)
64 ± 12 (21)
86 ± 14 (16)
85 ± 10 (12)
85 ± 13 (15)
aThe values given represent the average percent recoveries from three replicate determinations ±
standard deviation; the numbers in parentheses are the relative standard deviations.
one
-------
TABLE 46. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES AND
PCBs AFTER SILICA GEL CHROMATOGRAPHY (SOIL EXTRACT CONTAMINATED WITH TOXAPHENE)
Average ± SD (RSD) at Concentration la
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Fraction I Fraction II
hexane hexane
(80 mL) (50 mL)
67 ± 15 (22)
82 ± 9.0 (11)
84 ± 7.6 (9.1)
83 ± 7.1 (8.5) 10 ± 2.0 (20)
70 ± 0.6 (0.8)
66 ± 11 (17)
i
66 ± 11 (17)
89 ± 20 (22)
89 ± 23 (26)
Fraction III
methyl ene Fraction IV
chloride ethyl acetate
(15 mL) (50 mL) Total recovery
12 ± 3.7
97 ± 6.5
86 ± 12
95 ± 11
103 ± 15
100 ± 14
103 ± 16
93 ± 10
100 ± 13
30 ± 6.6
91 ± 14
127 ± 26
99 ± 12
(31)
(6.7)
(14)
(12)
(15)
(14)
(15)
(11)
(13)
(22)
(15)
NA
(20)
(12)
79 ± 18
97 ± 6.5
86 ± 12
95 ± 11
82 ± 9.0
84 ± 7.6
103 ± 15
93 ± 8.5
100 ± 14
70 ± 0.6
103 ± 16
93 ± 10
100 ± 13
96 ± 18
91 ± 14
NA
127 ± 26
66 ± 11
99 ± 12
89 ± 20
89 ± 23
(22)
(6.7)
(14)
(12)
(11)
(9.1)
(15)
(9.1)
(14)
(0.8)
(15)
(11)
(13)
(18)
(15)
(20)
(17)
(12)
(22)
(26)
NA — not analyzed.
aThe values given represent the average percent recoveries from three replicate determinations ± one
standard deviation; the numbers in parentheses are the relative standard deviations.
-------
TABLE 46. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES AND
PCBs AFTER SILICA GEL CHROMATOGRAPHY (SOIL EXTRACT CONTAMINATED WITH TOXAPHENE)
(CONTINUED)
Average ± SD (RSD) at Concentration 2a
en
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4, 4 '-ODD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Fraction I Fraction II
hexane hexane
(80 mL) (50 mL)
48 ± 4.7 (10)
74 ± 12 (16)
82 ± 5.6 (6.8)
83 ± 6.8 (8.2) 4.9 ± 0.7 (14)
71 ± 12 (17)
50 ± 5.9 (12)
i
56 ± 23 (41)
98 ± 23 (23)
77 ± 18 (23)
Fraction III
methyl ene Fraction IV
chloride^ ethyl acetate
(15 mL) (50 mL) Total recovery
4.0
73
53
68
74
68
71
63
70
11
59
88
70
; 6.7
; 74
; 63
; 74
; 74
; 70
72
67
70
12
68
NA
; 73
; 70
47 ; 58
73 ; 74
53 ; 63
68 ; 74
74 ± 12
82 ± 5.6
74 ; 74
87 ± 6.4
68 ; 70
71 ± 12
71 ; 72
63 ; 67
70 ; 70
57 ; 69
59 ; 68
NA
88 ; 73
56 ± 23
70 ; 70
98 ± 23
77 ± 18
(16)
(6.8)
(7.4)
(17)
(41)
(23)
(23)
NA — not analyzed.
aThe values given represent the average percent recoveries from three replicate determinations ± one
standard deviation; the numbers in parentheses are the relative standard deviations.
t>The values given represent duplicate determinations.
-------
TABLE 46.
10
ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE PESTICIDES AND
PCBs AFTER SILICA GEL CHROMATOGRAPHY (SOIL EXTRACT CONTAMINATED WITH TOXAPHENE)
(CONCLUDED)
Average ± SD (RSD) at Concentration 3a
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Fraction I
hexane
(80 mL)
63 ± 8.7 (14)
83 ± 6.2 (7.5)
80 ± 7.6 (9.5)
81 ± 9.7 (12)
i
i
72 ± 8.9 (12)
53 ± 3.5 (6.6)
49 ± 5.3 (11)
Fraction III
Fraction II methyl ene Fraction IV
hexane chloride ethyl acetate
(50 mL) (15 mL) (50 mL) Total
61 ± 5.5 (9.0) 11
87
78
89
93
7.0 ± 1.1 (16)
84
88
77
85
58 ± 4.5 (7.8) 24
84
87
85
±
±
±
±
±
±
±
±
+
±
+
±
+
2
6
12
7
2
7
8
7
8
4
8
9
11
.3
.5
.5
.6
.2
.5
.0
.1
.0
.7
.1
(21)
(7.5)
(15)
(8.4)
(2.8)
(8.6)
(9.7)
(9.1)
(9.6)
(17)
(10)
NA
(10)
(12)
73
87
78
89
63
83
93
87
84
81
88
77
85
82
84
NA
87
72
85
53
49
±
±
±
+
±
+
±
±
±
±
±
±
±
+
±
±
±
±
±
±
±
recovery
5.
6.
12
7.
8.
6.
2.
6.
7.
9.
8.
7.
8.
7.
8.
9.
8.
11
3.
5.
9
5
5
7
2
6
8
2
7
5
0
1
2
7
1
9
5
3
(8.0)
(7.5)
(15)
(8.4)
(14)
(7.5)
(2.8)
(7.8)
(8.6)
(12)
(9.7)
(9.1)
(9.6)
(8.8)
(10)
(10)
(12)
(12)
(6.6)
(11)
NA — not analyzed.
aThe values given represent the average percent recoveries from three replicate determinations ± one
standard deviation; the numbers in parentheses are the relative standard deviations.
bThe values given represent duplicate determinations.
-------
analysis allows complete separation of the five organochlorine pesticides that
elute with the PCBs in Fraction 1.
7.3 METHOD DETECTION LIMIT
The determination of the method detection limit was performed according
to the following procedure:
8 Make an estimate of the detection limit from the concentration value
that corresponds to an instrument signal-to-noise ratio in the range
of 5 to 10.
0 Prepare reagent water or soil at a concentration that is in the same
concentration range as the estimated method detection limit; in this
case the concentration is 0.1 ug/L for water (except PCB-1016 and
PCB-1260 at 1.0 M9/U and 10 ug/Kg for soil (except PCB-1016 and
PCB-1260 at 100 ug/Kg).
° Take seven aliquots of the matrix and process each aliquot through
the entire analytical method. Make all computations according to
the protocol written for the revised Method 8080. A blank
measurement is also performed to calculate the concentration of the
compound in the unspiked sample and to subtract it from the
respective spiked sample measurements.
° Calculate the standard deviation (SD) of the seven replicate
measurements and compute the MDL as follows:
MDL - t(n.1§ 0.99) x SD
where t(n«i 0.99) 1S tne student's t value appropriate for a 99%
confidence level and a standard deviation with n-1 degrees of
freedom, and SD is the standard deviation of the seven replicate
measurements. The value of t for 6 degrees of freedom is 3.143; a
t value of 3 was used to compute the MDL values in Tables 47 and 48
for water and soil samples, respectively.
Data are not available for either Kepone or toxaphene. Kepone was not
detected in any of the seven replicates, and toxaphene_was not spiked into any
of the samples since it would interfere with the determination of the
organochlorine pesticides and PCBs.
97
-------
TABLE 47. RESULTS OF METHOD DETECTION LIMIT (MDL) DETERMINATION FOR
ORGANOCHLORINE PESTICIDES AND PCBs IN DISTILLED WATER
oo
Compound
alpha-BHC
beta-BHC
gamma -BHC
delta -BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma -Chi ordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4, 4 '-ODD
Endrin aldehyde
Endosulfan sulfate
4, 4 '-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Amount
spiked
(ug/L)
0.100
0.100
0.100
0.100
0.100
0.100
0.100
0.100
0.100
0.100
0.100
0.100
0.100
0.100
0.100
0.100
0.100
0.100
1.0
1.0
Amount found In a given spiked replicate
(ug/L)
(1)
0.057
0.077
0.083
0.076
0.060
0.052
0.070
0.050
0.075
0.066
0.077
0.083
0.075
0.074
0.054
0.080
0.057
0.125
0.74
0.40
(2)
0.086
0.098
0.102
0.099
0.083
0.058
0.088
0.051
0.092
0.075
0.094
0.108
0.092
0.099
0.072
0.103
0.077
0.180
0.81
0.96
(3)
0.069
0.083
0.089
0.085
0.070
0.046
0.080
0.046
0.080
0.062
0.085
0.094
0.081
0.090
0.066
0.092
0.062
0.155
0.68
0.84
(4)
0.085
0.093
0.102
0.095
0.096
0.073
0.084
0.063
0.086
0.100
0.084
0.078
0.082
0.089
0.066
0.085
0.112
0.134
1.2
1.3
(5)
0.059
0.095
0.091
0.096
0.095
0.074
0.102
0.076
0.103
0.108
0.116
0.096
0.108
0.100
0.092
0.102
0.120
0.115
1.0
1.1
(6)
0.062
0.093
0.090
0.091
0.089
0.074
0.095
0.071
0.095
0.104
0.104
0.069
0.094
0.093
0.079
0.089
0.106
0.090
0.80
1.1
(7)
0.069
0.083
0.081
0.085
0.076
0.062
0.094
0.046
0.098
0.073
0.111
0.085
0.110
0.129
0.103
0.113
0.061
0.137
1.0
0.73
Average
0.070
0.089
0.091
0.090
0.081
0.063
0.088
0.058
0.090
0.084
0.096
0.088
0.092
0.096
0.076
0.095
0.085
0.134
0.89
0.92
SO
0.012
0.008
0.008
0.008
0.013
0.011
0.011
0.012
0.010
0.019
0.015
0.013
0.013
0.017
0.017
0.012
0.027
0.029
0.18
0.30
MDL
0.035
0.023
0.025
0.024
0.040
0.034
0.032
0.037
0.030
0.058
0.044
0.039
0.040
0.050
0.050
0.035
0.081
0.086
0.54
0.90
Average
percent
recovery
70
89
91
90
81
63
88
58
90
84
96
88
92
96
76
95
R5
134
89
92
-------
TABLE 48. RESULTS OF THE METHOD DETECTION LIMIT (MDL) DETERMINATION FOR
ORGANOCHLORINE PESTICIDES AND PCBs IN SANDY LOAM SOIL
Compound
alpha-BHC
beta-BHC
gamma -BHC
delta-BHC
Heptachlor
Aldrln
Heptachlor epoxlde
gamma-Chlordane
Endosulfan I
4,4'-DDE
D1eldr1n
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Endosulfan sulfate
4.4'-DDT
4,4'-Methoxychlor
PCB-1016
PCB-1260
Amount
spiked
(ug/Kg)
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
100
100
Amount found In a given spiked replicate
(wg/kg)
(1)
1.5
3.9
4.1
4.3
4.8
3.7
4.6
3.4
4.5
4.4
NA
3.6
4.7
4.0
2.7
4.5
4.0
4.3
150
130
(2)
0.9
3.1
2.6
4.4
4.5
3.4
3.6
3.2
4.4
4.6
NA
3.5
4.9
4.4
3.1
5.5
4.5
', 4.3
130
100
(3)
1.6
3.5
3.8
3.7
3.9
2.7
3.8
3.2
3.7
3.8
NA
3.0
3.8
3.1
2.6
3.4
3.9
3.1
120
88
(4)
0.8
4.8
3.8
4.1
3.4
2.2
3.8
2.8
3.5
4.1
NA
2.7
3.8
3.0
2.6
4.1
3.6
3.9
110
85
(5)
1.6
6.3
4.8
4.2
2.9
1.8
3.8
2.6
3.7
3.9
NA
3.8
4.2
3.8
2.6
4.4
3.7
5.0
89
73
(6)
1.1
3.6
3.5
3.8
3.4
2.1
3.7
4.2
3.7
6.0
NA
3.6
4.0
2.8
2.9
3.7
6.5
4.0
120
130
(7)
2.7
4.3
3.7
4.8
3.5
2.1
5.5
3.3
5.5
5.5
NA
6.5
6.0
6.9
4.1
7.0
6.0
9.0
110
120
Average
1.5
4.2
3.8
4.2
3.8
2.6
4.1
3.2
4.1
4.6
NA
3.8
4.5
4.0
2.9
4.7
4.6
4.8
120
100
SO
0.64
1.1
0.66
0.37
0.67
0.73
0.69
0.51
0.71
0.84
_.
1.2
0.79
1.4
0.54
1.2
1.2
1.9
19
23
MDL
1.9
3.3
2.0
1.1
2.0
2.2
2.1
1.5
2.1
2.5
—
3.6
2.4
4.2
1.6
3.6
3.6
5.7
57
70
Average
percent
recovery
15
42
38
42
38
26
41
32
41
46
—
38
45
40
29
47
46
48
120
100
NA -- Not able to determine because of matrix interference.
-------
REFERENCES
1. AT ford-Stevens, A. L., W. L. Budde, and T. A. Bellar. Interlaboratory
Study on Determination of Polychlorinated Biphenyls in Environmentally
Contaminated Sediments. Anal. Chem. 57: 2452 (1985).
2. Gebhart, 0. E., T. L. Hayes, A. L. Alford-Stevens, and W. L. Budde.
Mass Spectrometric Determination of Polychlorinated Biphenyls as Isomer
Groups. Anal. Chem. 57: 2458 (1985).
3. Slivon, L. E., J. E. Gebhart, T. L. Hayes, A. L. Alford-Stevens, and
W. L. Budde. Automated Procedures for Mass Spectrometric Determination
of Polychlorinated Biphenyls as Isomer Groups. Anal. Chem. 57: 2464
(1985).
4. Method 680. Determination of Pesticides and PCBs in Water and
Soil/Sediment by Gas Chromatography/Mass Spectrometry.
U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio 45268, November 1985.
5. Michael, L. C., M. A. Moseley, J. H. Hines, and E. D. Pellizzari.
Validation of Soxhlet Extraction Procedure for SW-846. EPA 600/4-85-073,
October 1985.
6. Test Methods for Evaluating Solid Wastes — Physical Chemical Methods.
U.S. Environmental Protection Agency, SW-846, Second Edition,
July 1982.
7. Mills, P. A. Variation of Florisil Activity: Simple Method for
Measuring Adsorbent Capacity and Its Use in Standardizing Florisil
Columns. J. Assoc. Off. Anal. Chem. 51: 29 (1968).
8. Beroza, M., and J. Sherma. Manual of Analytical Methods for the Analysis
of Pesticides in Humans and Environmental Samples. EPA-600/8-80-038,
U.S. Environmental Protection Agency, Research Triangle Park, North
Carolina, 1980. 686 pp.
9. Burke, J. A., and Malone, B. Effects of Calcination Temperature and Time
on Retentive Properties of Florisil Used for Pesticide Residue Analysis.
J. Assoc. Off. Anal. Chem. 49: 1003 (1966).
10. Bevenue, A., and N. J. Ogata. A Note on the Use of Florisil Adsorbent
for the Separation of Polychlorobiphenyls from Chlorinated Pesticides.
J. Chromatogr. 50: 142 (1972).
100
-------
11. Biddleman, T. F., et al. Separation of Polychlorinated Biphenyls,
Chlordane, and p,p'-DDT from Toxaphene by Silicic Acid Column
Chromatography. J. Assoc. Off. Anal. Chem. 61: 820-828 (1978).
12. U.S. Environmental Protection Agency -- Contract Laboratory Program
Protocol for the Analysis of Hazardous Substances List (HSL) Compounds,
revised July 1985.
13. Goerlitz, D. F., and L. M. Law. Determination of Chlorinated
Insecticides in Suspended Sediments and Bottom Material. J. Assoc. Off.
Anal. Chem. 57: 176 (1974).
14. Millar, J. D., R. E. Thomas, and H. J. Schattenberg. Determination of
Organochlorine Pesticides and Polychlorinated Biphenyls in Water by Gas
Chromatography. Anal. Chem. 53: 214 (1981).
15. Armour, J. A., and J. A. Burke. Method for Separating Polychlorinated
Biphenyls from DDT and Its Analog. J. Assoc. Off. Anal. Chem. 53:
761-768 (1970).
16. Berg, 0. W., P. L. Diosady, and G. A. V. Rees. Column Chromatographic
Separation of Polychlorinated Pesticides and Their Subsequent Gas
Chromatographic Quantisation in Terms of Derivatives. Bull. Environ.
Contamin. and Toxicol. 7: 338-345 (1972).
17. Jensen, S., L. Renberg, and L. Reutergardh. Residue Analysis of
Sediment and Sewage Sludge from Organochlorines in the Presence of
Elemental Sulfur. Anal. Chem. 49: 316 (1977).
101
-------
APPENDIX A
LITERATURE REVIEW
102
-------
TABLE OF CONTENTS
Section Page
1 Structures 110
2 Analytical Methodologies for Organochlorine Pesticides
and PCBs 116
2.1 Sample Preservation 116
2.2 Extraction 117
2.2.1 Extraction of Water Samples 120
2.2.2 Extraction of Sediment and Soil Samples 125
2.3 Extract Cleanup 135
2.3.1 Liquid-Liquid Partitioning 135
2.3.2 Gel Permeation Chromatography 137
2.3.3 Sulfur Removal 137
2.3.4 Liquid-Solid Chromatography 140
2.3.5 High-Pressure Liquid Chromatography 149
2.3.6 Selection of the Cleanup Technique 152
2.4 GC Analysis 152
2.4.1 Gas Chromatographic Columns 156
2.4.2 Problems With Chromatography 159
2.5 Confirmation of Compound Identity -." 163
2.6 Stability of Pesticide Solutions 168
104 Preceding page blank
-------
FIGURES
Number Page
A-l Effect of silicic acid water content on elution and
separation of PCB-1260 and DDT analogs 147
A-2 Fractionation scheme employing Woelm neutral alumina,
activity I, and silica gel deactivated with 3 percent water
for the separation of organochlorine pesticides and PCBs. . . . 153
A-3 Fractionation of organochlorine pesticides, PCBs, PCNs,
and phthalate esters using neutral alumina, activity I,
and silica gel deactivated with 0.3 percent water 154
A-4 Florisi 1/charcoal column fractionation of organochlorine
pesticides and PCBs 155
A-5 1.5 percent OV-17 + 1.95 percent OF-1 158
A-6 4 percent SE-30 + 6 percent OV-210 158
A-7 5 percent OV-210 158
A-8 3 percent DEGS 160
A-9 10 percent DC-200 160
A-10 1.6 percent OF-17 + 6.4 percent OV-210. ..._.. 160
A-ll 5 percent DC-200 + 7.5 percent OF-1 160
A-12 GC/EC chromatogram of composite organochlorine pesticide
and PCB-1016/1260 standard (30 m x 0.25 mm ID DB-5 fused-
silica capillary column; 100 to 200 pg pesticides
1,000 PCBs injected) 161
A-13 GC/EC chromatogram of composite organochlorine pesticide
and PCB-1016/1260 standard (1.8 m x 4 mm ID packed 1.5
percent OV-17 + 1.95 percent OV-210, 25 to 50 pg injected
for pesticides and 500 pg injected for PCBs) 162
105
-------
FIGURES (Continued)
Number Page
A-14 Chromatograms illustrating column overloading and
subsequent rejuvenation 164
A-15 Reduction in decomposition of endrin resulting from
column silylation 167
A-16 Comparative evaporation rates (mL/wk) of hexane and
isooctane from different containers at ambient temperature. . . 175
106
-------
TABLES
Number
A-l
A-2
A- 3
A-4
A-5
A-6
A-7
A-8
A-9
A-10
A-ll
A-12
A- 13
A-14
A- 15
Nomenclatures, Structures, Formulae, and Molecular
Weights of the Organochlorine Pesticides
Mean Recoveries from Preservation Tests in Clean Water
after 7 days at 48C
Mean Recoveries from Preservation Tests in Clean Water
after 7 days at 24°C
Extraction of Organochlorine Pesticides from Water
Samples
Organochlorine Pesticides and PCBs Recoveries by
Extraction with Methyl ene Chloride from Water
Recoveries from Wastewaters (Percent of Spiked Amount)
Recoveries of PCBs and Pesticides from Water
Extraction of Organochlorine Pesticides from Soil and
Sediment Samples
Recoveries of Organochlorine Pesticides Added to
Christiana Clay Loam
Comparison of Extraction Efficiencies ....-'.
Comparison of Extraction Methods for Efficiency of
Extraction of Heptachlor Epoxide and Dieldrin from a
Wet and Dry Clay Soil Using Acetone
Comparison of Extraction Efficiencies of Solvents
Extraction of Contaminated Bottom Material
Influence of Moisture Content
Effect of Water on Recovery in ppm of Organochlorine
Pesticides from Two Samples of Silt Loam Soil by Soxhlet
Extraction
Page
111
118
119
121
122
123
124
126
127
129
130
132
133
134
136
107
-------
TABLES (Continued)
Number Page
A-16 Effect of Amount of Water Added to an Air-Dried Clay
Loam Soil on the Recovery of Chlordane Residues by 1:1
Hexane:Acetone (Mechanical Shaker Method) 136
A-17 Solvent Partition of Pesticides and Butter Oil Between 1
Part Hexane and 3 Parts Polar Solvent 137
A-18 Elution Volumes of Organochlorine Pesticides by Gel
Permeation Chromatography 138
A-19 Effect of Exposure of Organochlorine Pesticides to
Various Sulfur Removal Reagents 139
A-20 Results of Recovery Experiments from Spiked Sediments
Using Tetrabutyl Ammonium Sulfite 140
A-21 Column Chromatographic Cleanup of Organochlorine
Pesticides and PCBs Using Florisi 1 141
A-22 Mills 1972 Modified Procedure for Elution of
Organochlorine Pesticides from Florisi 1 143
A-23 Pesticide Recoveries and Fat Elution from Florisil of
Different Calcinations 145
A-24 Elution Patterns of Organochlorine Pesticides"and PCBs
Using Armour/Burke's Procedure with Silica Gel/Celite 545 ... 146
A-25 Elution Patterns of Organochlorine Pesticides and PCBs
Using Leoni's Silica Gel Procedure (5 Percent Deactivated). . . 148
A-26 Elution Patterns of Organochlorine Pesticides and PCBs
Using Biddleman's Silica Gel Procedure (Modified by Acurex) . . 149
A-27 Column Chromatographic Cleanup of Organochlorine
Pesticides and PCBs Using Silica Gel 150
A-28 Column Chromatographic Cleanup of Organochlorine
Pesticides and PCBs Using Alumina 151
108
-------
TABLES (Continued)
Number Page
A-29 Relative Retention Times of Some Common Organochlorine
Pesticides 157
A-30 Summary of Pesticide Retention Times (min) on Two Fused-
Silica Capillary Columns 163
A-31 On-Column Decomposition of 4,4'-DDT 165
A-32 On-Column Decomposition of Endrin 166
A-33 Type of Reactions Used for the Confirmation of
Organochlorine Pesticides by Chemical Derivatization —
GC Techniques 169
A-34 Applications of Alkali Dehydrochlorination for DDT and
Analogs 170
A-35 Summary of Chemical Confirmation Procedures for Endrin
and Dieldrin 171
A-36 Summary of Chemical Confirmation Procedures for Aldrin 171
A-37 Summary of Chemical Confirmation Procedures for the
Chlordane Group 172
A-38 Chemical Confirmation of BHC Isomers 173
A-39 Summary of Chemical Confirmation Procedures for Endosulfans . . 173
A-40 Evaporation Rates from 10-mL Glass-Stopper Volumetric
Flasks and Vapor Pressure/Surface Tension Ratios for
Some Common Organic Solvents 174
A-41 Organochlorine Isooctane Solutions and 2-Year Stability
Test Results 177
109
-------
SECTION 1
STRUCTURES
The chemical structures, common names, formulae, molecular weights, and
nomenclatures of the organochlorine pesticides listed in Method 8080 are given
in Table A-l.
Organochlorine pesticides consist of three different major groups based
on their molecular structures: the cyclopentadiene group, the cyclohexane
group, and the DDT group. Except for toxaphene, the cyclopentadiene pesticides
are the Diels-Alder reaction products of hexachlorocyclopentadiene and suitable
unsaturated compounds. Toxaphene is a mixture of chlorinated camphenes
containing approximately 67 to 69 percent chlorine.
PCBs are complex mixtures of chlorinated biphenyls containing up to 209
isomers. Commercial mixtures of PCBs are known as Aroclors 1016, 1221, 1232,
1242, 1248, 1254, and 1260 (the last two digits indicate the percentage of
chlorine in the mixture, except for Aroclor 1016 which contains 41.5 percent
chlorine). In addition to PCBs, commercial preparations also contain
chlorinated naphthalenes and traces of chlorinated dibenzofurans.
110
-------
TABLE A-l. NOMENCLATURES, STRUCTURES, FORMULAE, AND MOLECULAR WEIGHTS OF
THE ORGANOCHLORINE PESTICIDES
Common name
Structure
Formula and
mol. wt»
Nomenclature
1. alpha-BHC H
H (290.8)
L.CI
H
.
H
Cl
alpha-l,2,3,4,5,6-hexa-
chlorocyclohexane
2. beta-BHC
cr-
Cl..
H
CgHgCU
(290.8)
"Cl
beta-l,2,3,4,5,6-hexa-
chlorocyclohexane
3. gamma-BHC
.(lindane)
(active form)
Cl
gamma-1,2,3,4,5,6-nexa-
chlorocyclohexane
4. delta-BHC
fi delta-l,2,3,4,5,6-hexa
(290.8) chlorocyclohexane
Tc'ontili'uedf
111
-------
TABLE A-l. (continued)
Common name
Structure
Formula and
mol. wt.
Nomenclature
5. alpha-Chlordane
(cis-Chlordane)
09.8
Cl
Cl
1-exo, 2-exo, 4,5,6,7,8,8-
octachloro 2,3,3a,4,7,7a-
hexahydro-4,7-methanoi n-
dene
6. gamma-Chlordane
(trans-Chlordane)
7. Heptachlor
8. Heptachlor cl
epoxide
Cl —
C10H6C18
(409.8)
C10H5C17
(373.3)
(389.3)
1-exo, 2-endo, 4,5,6,7,8,8-
octachloro-2,3,3a,4,7,7a-
hexahydro-4,7-methanoin-
dene
1,4,5,6,7,8,8-heptachlor-
3a,4,7,7a-tetrahydro-4,7-
methanoindene or 1,4,5,6,
7,8,8-heptachloro-4,7-en-
domethylene-3a,4,7,7a-te-
trahydroindene
2,3-epoxy-l,4,5,6,7,8,8-
heptachloro-3a,4,7,7a-
tetrahydro-4,7-methanoin-
dene
fcontinuedf
112
-------
TABLE A-l. (continued)
Common name
Structure
Formula and
mol. wt.
Nomenclature
9. alpha-Endosulfan
(Thiodan-I) Cl
C9H6Clfi03S
(406.9)
6, 7, 8, 9, 10, 10-hexachl oro
l,5,5a,6,9,9a-hexahydro-
6,9-methano-2,4,3-benzo-
dioxathiepin-3-oxide
(o-isomer)
10. beta-Endosulfan
(Thiodan-II)
Cl
(406.9;
S.O
6,7,8,9,10,10-nexachloro-
l,5,5a,6,9,9a-hexahydro-
6,9-rnethano-2,4,3-benzo-
dioxathiepin-3-oxide
(B-isomer)
11. Endosulfan
sulfate
Cl
Cl
Cl
5048
(422.9)
6,9-methano-2,4,3-benzo-
dioxathiepin, 6,7,8,9,10,
10-hexachl oro-1 ,5 ,5a ,6 ,9 ,
9a-hexahydro-3,3-di oxi de
12. Aldrln
Cl
CigHacjs
(364.9)
1,2,3,4,10,10-hexachloro-
l,4,4a,5,8,8a-hexahydro-
endo-1, 4-exo-5,8-di-
methanonaphthalene
"(TonTSlrTued)'
113
-------
TABLE A-l. (continued)
Common name
Structure
Formula and
rnol. wt»
Nomenclature
13. Dieldrin
(HEOD)
C12H8C160
(380.9)
1,2,3,4,10,10-hexachloro-
exo-6,7-epoxy-l,4,4a,5,6,
7,8,8a-octahydro-l,4-
endo, exo-5,8-dimethano-
naphthalene
14. Endrin
C12H8C16° 1,2,3,4,10,10-hexachloro-
(380.9) exo-6,7-epoxy-l,4,4a,5,6,
7,8,8a-octahydro-l,4-
endo, endo-5,8-dimethano-
naphthalene
15. Endrin
aldehyde
C12H8C160
Cl (380.9)
Cl
1,2,4-rnethenocyclopenta
(cdjpentalene-5-carbox-
aldehyde, 2,2a,3,4,7-
hexachlorodecahydro-,
(la,23,2a3,4B,4aB,5B,6a3,
6be,7R*)-
0 H
16. 4,4'-DOT
Cl —
cci
(354.5)
Cl
l,l,l-trichloro-2,2-bis
(p-chlorophenyl)-ethane
"ifcbntinuedf
114
-------
TABLE A-l. (concluded)
17.
Common name
4, 4 '-ODD
(TDE)
CI-/Y"
Structure
^-L/Rw
Formula and
mol. wt.
(320.1)
Cl
Nomenclature
l,l-dichloro-2,2-bis
(p-chlorophenyl )ethane
CHC1,
18. 4,4'-DDE
Cl
C_
ecu
_C1
(318.1)
l,l-dichloro-2,2-bis
(p-chlorophenyl)ethane
19. 4,4'-Methoxychlor
l,l,l-trichloro-2,2-bis
(p-methoxyphenyl )ethane
CC1.
OCH.
20. Kepone
(chlordecone)Cl
21. Toxaphene
io ig
Cl. ^0(490.6)
1,2,3,5,6,7,8,9,10,10-
decachloro {5.2.1.02*6
Q3.9o5.8| decano-4-one or
decachlorooctahydro
l,3,4-methano-2H-cyclo-
buta--{cd}-pentalen-2-one
Toxaphene [mixture of
chlorinated camphenes
(chlorine 67-69 percent)
of uncertain identity]
115
-------
SECTION 2
ANALYTICAL METHODOLOGIES FOR ORGANOCHLORINE PESTICIDES AND PCBs
The discussion of analytical methodologies for organochlorine pesticides
and PCBs addresses the following: sample preservation, extraction, cleanup, GC
analysis, compound confirmation, and stability of organochlorine pesticide
solutions.
2.1 SAMPLE PRESERVATION
The choice of the preservation method depends on the type of sample, the
compounds to be determined, the duration of sample storage prior to analysis,
and the analytical procedure to be used. The method chosen must not impair
the analytical procedure to be used in determining the compound.
The containers in which the samples are collected must be made of suitable
material for the preservation method and for the chemical to be determined.
Narrow-mouth glass bottles with aluminum-foil-lined lids are recommended for
the collection of water samples (1). Also, emptied 1-gal amber solvent bottles
previously used for acetone, hexane, and petroleum ether can be used for sample
collection. The use of Teflon liners instead of aluminum foil is not recom-
mended because of possible adsorption of some pesticides onto Teflon (1).
Weil and Quentin (2) investigated the effect of container, temperature,
and light on the storage of water samples containing DDT, dieldrin, heptachlor
epoxide, lindane, and methoxychlor at 10 ug/L and reported substantial losses
of the test compounds during a 2-week storage period in polyethylene con-
tainers. For aluminum and glass containers, the recoveries were in the range
of 40 to 90 and 50 to 100 percent, respectively. Small losses of DDT,
dieldrin, and heptachlor epoxide were reported from samples which were stored
at room temperature and in sunlight.
In another study, dealing with the persistence of organochlorine
pesticides in river water, Eichelberger and Lichtenberg (3) reported that
heptachlor, aldrin, alpha- and beta-endosulfan, and chlordane degrade totally
or partially during an 8-week storage period in glass containers in sunlight
and fluorescent light.
The effects of pH, temperature, and residual chlorine on the
preservation of spiked water samples in glass bottles sealed with
aluminum-foil-lined caps for a period of 7 days were investigated by Millar
116
-------
et al. (4). The results of Millar's study are given in Tables A-2 and A-3
and are summarized briefly as follows:
° The best overall conditions for the storage of water samples
containing organochlorine pesticides and PCBs are 4°C and pH 2.
« From water samples stored at 4°C, recoveries at pH 7 were similar to
those at pH 2 and pH 10, except for endosulfan I and II.
• From water samples stored at 24°C, alpha-, gamma-, and delta-BHCs
showed only 19, 15, and 36 percent recovery, respectively.
o Heptachlor disappeared when stored at 24°C in the absence of
chlorine.
° Endrin recovery was only 23 percent when water samples were adjusted
to pH 2 and stored at 24°C.
0 Endosulfan sulfate recovery was only 5 to 10 percent under all
storage conditions investigated for spiked clean water samples. In
addition, endosulfan sulfate did not disappear from wastewater after
7 days of storage. (A possible explanation given by the authors was
the fact that the spiking level of endosulfan sulfate was increased
50 times in the case of wastewater.)
The stability of the organochlorine pesticides in soil has not been
systematically investigated. Storage of soil samples at room temperature
should be avoided since degradation of some organochlorine pesticides does
occur (5). Losses of lindane and aldrin, after incubation of spiked soil
samples at 25°C under aerobic conditions, were noticeable 7 days after the
initiation of the experiment, and only 35 to 40 percent of the two pesticides
were found at day 30 of the experiment (5).
Deep-freezing to -20°C appears to be the most suitable method for the
storage of solid matrices since it has the widest range of application, causes
the least changes in the samples, and makes the addition of preservatives
unnecessary.
2.2 EXTRACTION
A number of methods have been used to extract PCBs and organochlorine
pesticides from water, soil, and sediment samples. The extraction techniques
that have been used on water samples include solvent extraction by stirring,
separatory funnel partitioning, and liquid-liquid extraction; adsorption onto
charcoal, polyurethane, and XAD-resins; and steam distillation. The methods
that have been reported for soil and sediment samples include shaking,
Soxhlet extraction, blending, and ultrasonication. Each of these techniques
will be reviewed below.
117
-------
TABLE A-2. MEAN RECOVERIES FROM PRESERVATION TESTS IN CLEAN
WATER AFTER 7 DAYS AT 4°Ca
Percent recovery
Compound
pH 2
pH 7
pH 10
Aldrin
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
4, 4 '-ODD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Chlordane
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
89b (15)c
64
86
100
80
98
94
94
105
96
102
<10
92
88
82
d
93
103
98
98
89
95
93
99
96
118b (12)c
60
98
100
108
100
89
90
107
103
98
<10
102
89
91
d
104
101
95
92
95
88
91 -'
97
91
86
82
81
84
74
95
92
93
102
0
0
<10
99
99b (0)c
77b (401)C
d
101
120
97
93
95
98
93
95
91
aAverage of four replicates unless accompanied by
parenthetical values. Data taken from Ref. 4.
bAverage of two replicates.
cWhen residual chlorine was present; average of two
replicates.
^Interference prevented assay.
118
-------
TABLE A-3. MEAN RECOVERIES FROM PRESERVATION TESTS IN CLEAN
WATER AFTER 7 DAYS AT 24°Ca
Percent recovery
Compound
pH 2
pH 7
pH 10
Al dri n
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
4, 4 '-ODD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Chlordane
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PC8-1242
PCB-1248
PCB-1254
PCB-1260
91b (23)c
65
84
94
100b (64)c
101
97
98
95
104b (78)c
92b (80)c
<10
23
94b (71)c
14
d
97
101
96
89
92
97
90
94
92
94b (15)C
68b (25)c
87
93
101b (77)C
96
89
93
101
90
81
<10
96
85K
15b (84)c
d
98
101
99
52b (94)c
72b (85)c
93
93 -'
97
90
89
19
95
15
36b (26)C
99
117
81
106
0
0
<10
88b (119)c
86b (0)c
10b (433)c
d
103
126
93
86
96
98
92
95
86
aAverage of four replicates unless accompanied by
parenthetical values. Data taken from Ref. 4.
bAverage of two replicates.
°When residual chlorine was present; average of two
replicates.
^Interference prevented assay.
119
-------
2.2.1 Extraction of Water Samples
Table A-4 is a summary of extraction methods published in the
literature. The following describes in detail some of the procedures for
the extraction of organochlorine pesticides and PCBs from water samples.
2.2.1.1 Stirring
This technique calls for stirring the sample with a nonpolar solvent in
the original sample container. After stirring, the sample may then be
transferred to a separatory funnel for further extraction. This method is
particularly suited for aqueous samples with suspended material which can
adhere to the walls of the sample container. Particles have been known to
adsorb organochlorine compounds; their transfer to a separatory funnel is
usually incomplete (1).
2.2.1.2 Separatory Funnel Partitioning
In this technique, up to 1 liter of aqueous sample is poured into a
separatory funnel and is extracted by shaking with an organic solvent. The
layers are allowed to separate, and the organic fraction is drawn off.
Solvents with a specific gravity greater than 1 are preferred since the lower
layer can be removed more easily.
The major drawbacks of this method are the limit of the sample size and
the tendency of many wastewaters to form emulsions during extraction. To
break emulsions, it has been recommended that the extract be passed through a
25-mm-thick glass-wool pad (27, 28).
Using separatory funnel partitioning on distilled water fortified with
pesticides, Millar et al. (4) obtained the recoveries given in Table A-5.
Although 15 percent methylene chloride in hexane gave slightly higher
recoveries overall, methylene chloride alone settled to the bottom of the
funnel and could be drained off more easily. Recoveries were greater than
80 percent under all conditions studied, except for aldrin and heptachlor.
No reasons for the lower recoveries of these two pesticides were given.
Method recoveries from two wastewater samples after cl-e'anup are given in
Table A-6. Lower recoveries in wastewater 2 may have been the result of a
greater degree of emulsion formation or the result of erratic recovery from
sulfur cleanup (4).
The American Society for Testing and Materials recommends that 1-liter
water samples be extracted twice with 60 mL of ethyl ether:hexane (15:85).
After saturating the solution with sodium sulfate, the sample is re-extracted
with hexane. By using this procedure, recoveries ranging from 65 to
118 percent were obtained for aldrin, lindane, dieldrin, and DDT (29).
2.2.1.3 Liquid-Liquid Extraction
In this technique, several liquid-liquid extractors can be used in
series to continuously extract an aqueous sample with an organic solvent.
120
-------
TABLE A-4. EXTRACTION OF ORGANOCHLORINE PESTICIDES FROM WATER SAMPLES
Method
Activated carbon
Continuous
Solvent (vortex)
Solvent (sep. funnel)
RP L-L partition
Solvent
Polyurethane foams
Supported-bonded
s1 11 cones
Continuous
XAD-2
XAD-2
XAO-2, 4, 8, 12
Polyurethane foams
RP L-L partition
XAD-2
XAD-2
Activated carbon
Solvent (sep. funnel)
Solvent (sep. funnel)
Solvent (sep. funnel)
XAD-2, XAD-4, Tenax
Solvent
Chloroform
PE3
Hexane and mixed
solvents
Benzene
PE
Hexane
Hexane
Pentane
Cyclohexane, hexane
Acetonltrile
Di ethyl ether
10 percent ether 1n
hexane
Hexane
PE
Hexane
01 ethyl ether
Chloroform
Hexane
Methyl ene chloride
Benzene
Acetone, methanol ,
acetonltrlle,
n-pentane
Pesticides
Mult1-OCS
Aldrln, Isodrln,
dleldrin, endrln
Dleldrin, endrin
Multl-OCS
Mult1-OCS
Toxaphene
Multi-OCS
Mult1-OCS
Multl-OCS
Multl-OCS
Mult1-OCS
Mult1-OCS
Mult1-OCS
Mult1-OCS
Mult1-OCS
Mult1-OCS
Mult1-OCS
Multl-OCS
Mult1-OCS
Kepone
Chlorinated
hydrocarbons
Recovery
(percent)
75 to 86
95 to 100
Average
61 to 95
90 to 100
50 to 100
75 to 85
87 to 100
—
83 to 96
—
47 to 95
100 except
aldrln 61
100
73 to 107
75 to 100
Average 96
distilled
water
Average 85
natural water
except for
mlrex 52
5 to 90
84 to 114
74 to 104
90 to 96
—
Concentration
2.5 to 5 ppm
7 to 340 ppb
0.2 to 0.5 ppb
0.06 to 1.2 ppb
5 to 60 ppt
0.25 to 1.25 ppm
0.5 to 1 ppb
10 to 20 ppt
0.6 to 7.5 ppb
--
20 ppt
1 to 10 ppb
1 to 10 ppb
1 to 10 ppb
1.5 to 3 ppt
0.01 to 0.1 ppb
2 ppb
0.03 to 1.3 ppb
0.09 to 66.5 ppb
2 to 5 ppb
ppt
Ref.
6
7
8
9
10
11
12
13
30
15
16
17
18
19
20
21
22
23
24
25
26
aPE, petroleum ether.
OCS - organochlorine pesticides.
121
-------
TABLE A-5. ORGANOCHLORINE PESTICIDES AND PCBs
RECOVERIES BY EXTRACTION WITH
METHYLENE CHLORIDE FROM WATER3
Percent recovery
Compound
Aldrin (30)b
alpha-BHC (20)
beta-BHC (40)
gamma-BHC (20)
delta-BHC (40)
4,4'-DDD (120)
4,4'-DDE (60)
4,4'-DDT (160)
Dieldrin (60)
Endosulfan I (50)
Endosulfan II (100)
Endosulfan sulfate (310)
Endrin (90)
Endrin aldehyde (230)
Heptachlor (20)
Heptachlor epoxide (40)
Chlordane (380)
Toxaphene (3800)
PCB-1016 (1290)
PCB-1221 (3000)
PCB-1232 (3000)
PCB-1242 (1500)
PCB-1248 (2250)
PCB-1254 (750)
PCB-1260 (1500)
===== = = = = = s:= = = = 3 = = = = = = = ==:
pH 2
64C
97
94
109
104
97
94
102
96
98
98
98
106
100
59
100
103
93
89
77
92
99
96
91
101
pH 7
46d
95
95
92
92
97
92
104
100
102
99
92
108
92
60
100
99
104
87
96
84"
101
92
101
100
============
pH 10
68C
92
91
101
97
93
81
92
101
101
94
89
108
99
72
89
102
111
88
80
83
104
91
96
100
========
aData taken from Ref. 4.
bAmount in ng added to 1 liter of water.
cAverage of three replicates.
dAverage of four replicates.
122
-------
TABLE A-6. RECOVERIES FROM WASTEWATERS
(PERCENT OF SPIKED AMOUNT)3
Compound Wastewater 1 Wastewater 2
Aldrin
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
4, 4 '-ODD
4,4'^ODE
4,4'-ODT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Chlordane
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
=—========—==—=========
93b
102
93
91
98
99
99
104
97
97
110
105
95
95
95
99
96
103
92
102
100
97
99
95
95
:====—=========
943
82
80
96
79
90
84
87
94
95
84
102
95
86
88
91
86
93
95
98
86
83
87
91
91
________ — _ — _
aData taken from Ref. 4.
bAverage of three replicates.
This method has the advantages of being applicable to very large samples and
of decreasing the likelihood of emulsion formation (26).
Using a 45-min petroleum ether extraction, Kahn and Wayman (7) obtained
95 to 100 percent recoveries of aldrin, isodrin, dieldrin, and endrin from
wastewater. Using a modification of this liquid-liquid extraction and
running at a pump rate of 52 L/hr, Ahnoff and Josefsson (30) obtained 89 to
96 percent extraction efficiencies for several organochlorine pesticides from
water. Cyclohexane was chosen as the extracting solvent because of its
123
-------
greater phase separation from water. The amount of pesticides lost by
adsorption to the glass reservoir walls was significant. For aldrin, which
was the worst case, losses were 3.7 and 15.8 percent at 1.0 and 4.5 hours,
respectively.
2.2.1.4 Steam Distillation
In this method, 2.5-liter water samples are heated to boiling. The
steam is condensed and passed through a low-density solvent, such as
isooctane or toluene, to extract chlorinated pesticides and PCBs.
According to Veith and Kiwus (31), the main advantage of this procedure
is that it also serves as a cleanup method. Sample extracts may be directly
injected onto the GC. Extraction recoveries for several PCBs and
organochlorine pesticides by steam distillation are given in Table A-7.
2.2.1.5 Adsorption
Several techniques have been used to adsorb organochlorine pesticides
and PCBs onto solid supports. The materials used include activated carbon
(5, 22), support-bonded or coated polymers (13), polyurethane foams (12, 14,
and 18), and polystyrene resins (15, 16, 17, 20, 21, 26). Once adsorbed from
TABLE A-7. RECOVERY OF PCBs AND PESTICIDES FROM WATER3
Quantity of Steam-distillation
chemical present recovery
Compound (ug) (percent)
PCB-1016
PCB-1242
PCB-1248
PCB-1254
4,4'-DDE
Heptachlor epoxide
Mi rex
0.04b
0.04
0.04
0.04
0.02
0.01
0.04
97.9
99.4
100.0
-99.1
104.0
89.9
89.5
aData taken from Ref. 31.
^Quantity added to water, individual analyses.
124
-------
water, pesticides and PCBs may be desorbed by either elution or Soxhlet
extraction with an organic solvent such as chloroform (22), petroleum ether
or pentane (26), or hexane (18). While many of these methods work well for
distilled water fortified with pesticides, recoveries are often poor for
wastewaters. Furthermore, adsorption sites on these resins can become
deactivated by impurities in natural waters (1).
2.2.2 Extraction of Sediment and Soil Samples
This section summarizes the extraction techniques reported in the
literature for soil and sediment samples. A detailed discussion of the
extraction of organochlorine pesticides from soil was published by Chiba
(32) and will be summarized below. Examples of extraction techniques,
solvent and solvent mixtures used for extraction, and recovery data are
presented in Table A-8.
Techniques used for the extraction of organochlorine pesticides from soil
can be classified into four major groups: (a) surface rinsing by rotating
or tumbling, (b) blending with solvents, (c) Soxhlet extraction, and
(d) sonication. Each of these techniques will be described in detail below.
After the description of extraction techniques, other factors that affect the
extraction recovery such as solvent(s), contact time between the solvent and
the solid matrix, and soil factors (e.g., moisture) will be addressed.
2.2.2.1 Extraction Equipment
Tumbling and Shaking
Several types of tumblers including end-over-end tumbler, rotary shaker,
Northcott shaker, head-to-end tumbler, mechanical shaker, and wrist-action
shaker have been used for pesticide residue determination.
The tumbling techniques have the advantage of giving a smaller amount of
coextractants than other methods, but the recoveries may vary greatly between
laboratories (1 and 32).
Wool son (48) used a 1:1 solution of hexane and acetone to extract soil
premoistened with 0.2M NH^l. Recoveries from spiked samples averaged
92 percent with a 13.58-percent coefficient of variation (Table A-9).
Wheatly (49) reported recoveries between 51 and 64 percent for dieldrin
when the tumbling technique was used with hexane:2-propanol or
hexane:acetone.
Lichtenstein (50) used the tumbling method and reported recoveries of 92
to 98 percent for DDT, 92 to 97 percent for aldrin, 90 to 94 percent for
dieldrin, and 100 percent for lindane.
125
-------
TABLE A-8. EXTRACTION OF ORGANOCHLORINE PESTICIDES FROM SOIL AND SEDIMENT
SAMPLES
ro
01
Method
Ultrasonic
Blender
Soxhlet
Soxhlet
Blender
Shaker
Soxhlet
Multi
Shaker
Blender
Soxhlet
Shaker
Shaker
Rotation
Soxhlet
Col umn
Column
Soxhlet
============
Sample
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Sediments
Sediments
Sludge
Sediment
Soil
Sediment
Soil
Sediment
Solvent
Acetone
21 solvent systems
41:59 hexane: acetone
41:59 hexane: acetone
Methyl ene chloride
3 solvent systems
5 solvent systems
Multi
1:1 hexane: acetone3
1:1 hexane :acetone
41:59 hexane :acetone
2:1 and 4:1
hexane :acetone
1:2 hexane: acetone
Acetone;
1:3 acetone: hexane
1:9 ace tone :PE
>
1:1 hexane: acetone
4:3:3 hexane:
acetone :methanol
35:65 ether: hexane
Pesticides
Multi -OCS
Aldrin,
Dieldrin
Multi -OCS
Multi -OCS
Multi -OCS
Multi -OCS
Multi -OCS
Dieldrin
Chlordanes and
others
Chlordanes and
others
Toxaphene
Multi -OCS
OCS and PCBs
OCS and PCBs
OCS and PCBs
Multi -OCS
4,4'-DDT
Kepone
Recovery
(percent)
94 to 104
—
—
95 to 108
22 to 155
Average 74.6
Average 87
—
—
—
Average 88
75 to 99
—
OCS = 73 to
OCS = 82 to
—
78 to 87
103
Level
2 ppm
0.1 to 0.9 ppm
0.01 to 0.4 ppm
0.25 to 6.5 ppm
0.4 and 4.0 ppm
0.5 to 6.0 ppm
0.5 to 6 ppm
—
2 to 4 ppm
2 to 4 ppm
0.25 ppm
0.5 to 1 ppb
OCS = 0.005 to
0.24 ppm
97 OCS = 50 ppb
112 OCS = 1.3 to
3.3 ppb
—
10 ppm
4 ppm
Ref.
33, 34
35
36
37
5
38
39
39
40
41
41
11
42
43
44
45
27
46
47
aOther solvents evaluated include: hexane:2-propanol (1:1);
OCS - organochlorine pesticides.
PE - petroleum ether.
methanol; benzene:methanol (1:1).
-------
TABLE A-9. RECOVERIES OF ORGANOCHLORINE PESTICIDES ADDED TO CHRISTIANA
CLAY LOAM3
Average Recovery
Compound
Lindane
Heptachlor
Aldrin
Heptachlor epoxide
4,4'-DDE
Dieldrin
Endri n
4,4'-TDE
2,4'-DDT
4, 4 '-DDT
Added
(ppm)
0.94
1.52
0.98
0.49
0.82
1.56
1.17
1.35
3.64
4.27
(ppm)
0.81
1.32
0.84
0.49
0.79
1.42
1.09
1.25
3.28
3.82
(percent)
86
87
86
100
96
91
93
97
90
89
Standard
deviation
(ppm)
0.08
0.20
0.11
0.07
0.06
0.15
0.13
0.20
0.84
0.48
Coefficient
of
variation
(percent)
9.35
15.25
13.12
13.80
7.62
10.45
11.65
16.03
25.47
12.69
Average of all data
=========================:
aData taken from Ref. 48.
92
0.23
13.58
The sample size used in tumbling extraction was generally larger than that
used in shaking extraction; for example, most workers used 100 to 500 g for
tumbling and 10 to 200 g for shaking.
Blending
In this technique, soil samples are blended with solvent for a relatively
short period of time which is generally 2 to 15 min. The method is very
popular for the extraction of plant materials but is less widely used with
soils. Sample sizes of 25 to 100 g are generally required (1 and 32).
Several types of blenders are available commercially; the most commonly
used are Waring and Omni-Mix blenders. Lichtenstein (50) used a Volu-Mix
Lourdes homogenizer at medium speed for 5 min for 75 g soil and 150 mL hexane:
acetone (1:1). Chiba and Morley (36) used an Omni-Mix high-speed blender and
found that it was necessary to repeat the 5-min extraction with the same
solvent. This was done not only to achieve complete extraction but also to
minimize loss due to pesticide readsorption during filtration.
In a comparison of the efficiencies of tumbling, Soxhlet extraction, and
blending at equal extraction times, blending was the most efficient technique
(32). When acetonitrile was used as the extracting solvent, recoveries of
127
-------
several organochlorine pesticides and PCBs were over 95 percent (51). Another
study (36) compared blender extraction efficiency on aldrin and dieldrin. The
extraction solvents dimethyl formamide, dimethyl sulfoxide, and acetone gave
the best results while the results with methylene chloride, n-hexane, and
propylene carbonate were poor (36).
Soxhlet Extraction
This technique has been commonly used for extracting pesticides from
soil. The main advantage of this technique is the ease and neatness in
handling. Because of the nature of the system, air-dried soil is preferred;
however, wet soil has also been extracted in a Soxhlet by using water-miscible
solvents (28).
Chiba and Morley (36) reported that the Soxhlet extraction method was
very efficient with most solvent systems although the amounts of
coextractants were always higher when compared to the blending technique
(Table A-10). The authors concluded that there is little advantage in using
the Soxhlet procedure since a stringent cleanup method is required to remove
the large amount of coextractants. Furthermore, they reported that it is
difficult to obtain good reproducibility because of the difficulty in keeping
the solvent-recycle time constant. Acetonitrile gave the fastest rate of
extraction while n-hexane:acetone gave the slowest. Despite the fact that
n-hexane:acetone has a slow solvent-recycle time, the use of hexane:acetone
(41:59) which forms an azeotrope with a boiling point of 50°C decreases
the chance of pesticide degradation (1, 37). When acetonitrile (boiling
point 82°C) was used, for example, DDT degraded to DDE (37).
By using Soxhlet extraction with hexane:acetone, it was determined that
the moisture content of the soil could affect extraction efficiency. At least
5 percent water was essential for good recoveries, and up to 40 percent water
had no detrimental effect (37).
Ultrasonication
This technique uses an ultrasonicator to extract pesticides into an
organic solvent. It has the advantage of providing quantitative recoveries
in short periods of time. It may also be particularly useful for the
extraction of "aged" samples because of its ability to break up cells and soil
colloids (34).
With acetone, a 30-sec ultrasonication gave higher recoveries than the
roller or blender methods and was better than or equal to an 8-hour Soxhlet
extraction (Table A-ll) (35).
2.2.2.2 Solvents
Several solvents and solvent mixtures have been used for the extraction
of organochlorine pesticides from soil. These include: petroleum ether (52),
hexane (52), pentane (52), hexane:acetone (5, 37, 41, 42, 43, 44, 27),
acetone (33, 34, 35), acetonitrile (54), propylene carbonate (59), benzene
128
-------
TABLE A-10. COMPARISON OF EXTRACTION EFFICIENCIES3
Extraction
No. Solvent
13
22
23'
24
25
9
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
50 percent acetone :n-hexane
50 percent acetonern-hexane
50 percent acetone:n-hexane
50 percent acetone :n-hexane
50 percent acetone :n-hexane
Dimethyl formamide
Dimethyl formaml de
01 methyl formamlde
01 methyl formamlde
Methanol
Methanol
50 percent methanol : benzene
50 percent methanol .'benzene
AcetonltMle
Acetonitrile
Acetone
Acetone
Benzene
Benzene
50 percent 2-propanol :n-hexane
50 percent acetone: benzene
Methylene chloride
Coextractants
Method (mg/g)
B 5 x 2
19 B 5 x 2
B 1 hr x 1
Soxhlet, 12 hr
B 5 hr x 1
B 5 x 2
B 2 hr x 1
19 B 5 x 2
Soxhlet, 20 hrb
19 B 5 x 2
Soxhlet, 12 hr
19 B 5 x 2
Soxhlet, 12 hr
19 B 5 x 2
Soxhlet, 12 hr
19 B 5 x 2
Soxhlet, 12 hr
19 B 5 x 2
Soxhlet, 12 hr
Soxhlet, 12 hr
19 B 5 x 2
Soxhlet, 12 hr
0.085
0.097
0.150
0.340
0.190
0.022
0.038
0.048
0.470
0.043
0.180
0.320
0.490
0.033
0.200
0.045
0.062
0.077
0.570
0.230
0.150
0.220
Aldrin
(ppm)
0.51
0.83
0.88
0.90
0.92
0.92
0.88
0.88
0.99
0.90
1.01
0.97
0.92
0.72
1.00
0.86
0.89
0.49
0.81
0.62
0.61
0.18
D1eldr1n
(ppm)
0.47
0.87
0.93
0.87
0.89
0.87
0.95
1.02
1.08
1.04
1.06
0.95
1.07
0.66
1.05
0.93
0.90
0.41
0.86
0.77
0.66
0.48
aData taken from Ref. 36.
bTemperature of oil bath adjusted so that 5 min were required to finish one extraction cycle.
Note:
B 5 x 2. Air-dried soil (100 grams) and the extraction solvent (200 mL) were blended for
5 min with external cold-water cooling. The mixture was filtered with suction by using
Whatman No. 1 filter paper with a 1-cm pad of SuperCel. The container was rinsed with 2 x
25 ml of the solvent; the soil, filter paper, and pad were returned to the blender, and the
extraction procedure was repeated.
B 5 x 1. The B 5 x 2 procedure was modified by adding anhydrous N32S04 or by using moist
soil in experiments where comparisons with the standard method were being made. The
extraction was done just once; 4 x 50 ml of the solvent were used for rinsing instead of
2 x 25 mL.
19 B 5 x 2. To check the influence of the contact time between solvent and soil, soil
(100 grams) was kept in contact with solvent in a container for 19 hours (overnight) prior
to carrying out the standard procedure.
Soxhlet. A soil-solvent ratio of 1:5 was used, and the steam bath was adjusted so that it
took 5 m1n to complete one extraction cycle with methanol (boiling point 65°C). In the
case of mixed solvents, soil extraction was actually made by the azeotropes, but the normal
1:1 ratio of solvents was added to the flask Initially so that working-up procedures could
be kept consistent.
B 1 hr x 1, 8 2 hr x 1, and B 5 hr x 1 are identical to B 5 x 2 except that the blending
time was extended to 1 hr, 2 hr, and 5 hr, respectively.
129
-------
TABLE A-ll. COMPARISON OF EXTRACTION METHODS FOR EFFICIENCY
OF EXTRACTION OF HEPTACHLOR EPOXIDE AND DIELDRIN
FROM A NET AND DRY CLAY SOIL USING ACETONEa
Mean percent recovery
and deviation from mean
Extraction
method Soil moisture Heptachlor epoxide
Dieldrin
Ultrasound
Blender
Roller
Soxhlet
Dry
Wet
Dry
Wet
Dry
Wet
Dry
Wet
97.6 ± 0.4
99.3 ± 0.1
84.2 ± 2.7
95.2 ± 0.6
87.8 ± 1.1
93.7 ± 0.1
86.5
99.8 ± 1.9
98.3 ± 0.0
96.0 ± 1.1
79.3 ± 2.7
93.1 ± 0.3
85.8 ± 0.9
89.7 ± 0.1
82.0
97.0 ± 2.4
aData taken from Ref. 35.
(36), pentane:2-propanol:glacial acetic acid (4:1:1) (55), benzene:
2-propanol (56), n-hexane: 2-propanol (56), pentane.-acetone (56); chloroform:
methanol (1:1) (57), and methylene chloride:methanol (1:1) (58).
Observations made by the various authors referenced above include:
° Petroleum ether has been recommended for most organochlorine
compounds because the solvent will give satisfactory clean extracts
(52).
° n-hexane:acetone (4:1) extracted 10 to 20 percent more aldrin than
hexane alone (53).
0 Although mixtures of n-hexane:acetone appear "to be fairly efficient,
the best mixing ratio has not been determined, and ratios varying
from 1:1 to 19:1 have been used (1).
° Acetonitrile was used primarily for vegetables and fruits. Duffy
and Wong (53) evaluated this solvent for a number of soils and found
that it was less efficient than a mixture of n-hexane and 2-propanol
(3:1).
° In general, improved extraction efficiency was achieved when two
different solvent systems were combined (60). However, this was only
advantageous when the solvent was the only factor controlling the
extraction efficiency. It was possible to achieve satisfactory
extraction with one solvent by changing the moisture content of the
sample, the contact time between solvent and soil, and the type of
extractor.
130
-------
Twenty-one solvent systems were investigated by Chiba and Morley (36) for
the extraction of aldrin and dieldrin from different types of soils (Table
A-12). Of the nine single-solvent systems, dimethylformamide (DMF) showed the
best results, followed by dimethyl sulfoxide (DMSO) and acetone. Benzene,
methylene chloride, n-hexane, and propylene carbonate gave poor results, and
methanol gave good recovery for dieldrin only. Furthermore, most polar
solvents tended to give lower amounts of coextractants whereas the nonpolar
solvents did the opposite. No correlation between the concentration of the
pesticides and the amount of coextractants was found.
Williams compared the extraction efficiency of three solvent systems using
mechanical shaking (37). With one soil, hexane nsopropanol gave highest
recoveries while with another one, hexane:acetone was superior. In a measure
of the extraction of technical chlordane from aged soil samples when the
mechanical shaker method was used, recovery decreased in the order
methanol>ethanol>hexane:acetone>hexane:i sopropanol >acetone (38).
Johnsen and Starr (35) studied the extraction efficiency of six different
solvent systems for the extraction of dieldrin and heptachlor epoxide from
sandy loam soil by using three different methods over three time intervals.
The solvent systems investigated were acetonitrile, water-saturated benzene,
benzene:2-propanol (2:1), benzene:methanol (2:1), n-hexane:2-propanol (2:1),
and n-hexane:acetone (4:1). They reported that benzene was a poor solvent, that
benzene:methanol gave the highest average recoveries for all three extraction
times, and that n-hexane:acetone gave the highest recoveries for all three
methods when the sample was extracted one day after spiking with the pesti-
cides, but that the efficiency of this solvent system decreased significantly
when the sample was extracted one month after spiking.
Residues of chlordane were extracted from clay loam soil (38) with
different solvents under various conditions; the results indicate that mixtures
of hexane:acetone gave better recoveries than hexane:2-propanol and that
acetone alone gave poor recoveries. The use of the hexane:acetone mixture
increased the efficiency of extraction, but there was no significant
improvement in efficiency by increasing the proportion of hexane in acetone
from 50 to 90 percent. Furthermore, methanol gave higher recoveries than any
other solvent or mixture of solvents.
2.2.2.3 Time
The time factor was investigated by Chiba and Morley (36). As shown in
Table A-10, the time factor was very important when the blending method was
used. Only 0.51 ppm aldrin and 0.47 ppm dieldrin were extracted when the
standard contact time was used, but all other modified methods showed nearly
double the amount of each pesticide. The 19-hour contact time with the
solvent, which was followed by normal blending, was one of the most promising
methods because the recovery of pesticides was high, and the amount of
coextractants was low. There appeared to be no advantage in extending the
blending time to more than 1 hour.
Goerlitz and Law (42) have performed five separate extractions of fortified
sediment samples with acetone/hexane and have reported 97.9 percent average
131
-------
TABLE A-12. COMPARISON OF EXTRACTION EFFICIENCIES OF SOLVENTS'*
Co-
Extraction extractants, Aldrin
number Solvents (mg/g) (ppm)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Methyl ene chloride
Methanol
Propylene carbonate
n-Hexane
Benzene
Acetonitrile
Di me thy Isulf oxide (DMSO)
Acetone
Di methyl formamide (DMF)
50 percent acetone :methyl ene
chloride
50 percent 2-propanol :n-hexane
10 percent acetone :n-hexane
50 percent acetone :n-hexane
50 percent acetom'trile:acetone
50 percent acetone: benzene
50 percent acetonitrile:n-hexane
50 percent acetonitrile:benzene
50 percent DMSO:benzene
50 percent methanol .-methyl ene
chloride
50 percent methanol : benzene
50 percent DMF:benzene
0.079
0.032
0.016
0.110
0.069
0.025
0.011
0.032
0.022
0.140
0.076
0.071
0.085b
0.061
0.130
0.040
0.086
0.190
0.180
0.240
0.170
0.10
0.10
0.17
0.21
0.33
0.50
0.74
0.81
0.92
0.30
0.33
0.45
0.51b
0.57
0.60
0.60
0.61
0.79
0.82
0.85
0.89
Dieldrin
(ppm)
0.30
0.55
0.09
0.19
0.29
0.45
0.80
0.73
0.87
0.38
0.32
0.39
0.47&
0.51
0.44
0.53
0.55
0.72
0.72
0.85
0.87
Extraction method: Omni-Mix high speed blender, two 5-min extractions with
200 mL of solvent per 100 grams of soil (B 5 X 2). Data taken from Ref. 36.
bStandard deviation: aldrin ±0.03; dieldrin ±0.02 (eight determinations).
Soil 1 (sandy loam), air-dried.
recoveries of DDE, DDT, ODD, lindane, and PCB from five different clay-type
sediments and from five silt materials (Table A-13).
2.2.2.4 Moi sture
Chiba and Morley (36) tried to determine the role of sodium sulfate in
conjunction with the moisture content of soil. Table A-14 shows the signi-
ficance of moisture content when use is made of the 50-percent acetone:n-hexane
system with both blending and tumbling methods. The difference in extraction
efficiency between air-dried soil and soil with a moisture content of 25
percent was significant when sodium sulfate was not added. When sodium sulfate
132
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TABLE A-13. EXTRACTION OF CONTAMINATED BOTTOM MATERIAL3
Extraction
1 + 2 + 3
4b
Sample
Clay 1
Silt 1
Clay 2
Silt 2
Clay 3
Silt 3
Clay 4
Silt 4
Clay 5
Silt 5
Average
Compound
DDE
ODD
DDE
DDE
ODD
DDT
DDE
ODD
DDT
DDE
ODD
DDT
Li ndane
DDE
ODD .
DDT
Li ndane
DDE
DDD
DDT
DDE
DDD
DDT
DDE
DDD
DDT
PCB
PCB
(ng)
88.0
132
60.9
85.4
127
79.5
153
266
454
319
542
1,050
724
4,530
11,100
16,500
1,030
7,680
18,500
24,400
802
990
1,400
601
1,050
1,070
3,530
5,090
(percent)
99.3
100.0
100.0
98.4
100.0
99.1
98.2
97.6
98.6
97.1
98.3
98.5
90.9
98.4
98.4
97.2
83.8
98.7
99.0
98.2
99.5
98.3
98.4
99.0
98.5
98.5
99.9
100.0
97.9
(ng)
0.00
0.00
0.00
0.57
0.00
0.00
1.75
4.16
1.91
3.58
3.83
5.21
22.1
18.8
57.4
133
54.9
22.3
56.8
123
1.87
5.97
9.03
2.37
6.15
9.23
1.59
0.16
(percent)
0.0
0.0
0.0
0.7
0.0
0.0
1.1
1.5
0.4
1.1
0.7
0.5
2.8
0.4
0.5
0.8
4.5
0.3
0.3
0.5
0.2
0.6
0.6
0.4
~"0.6
0.9
0.0
0.0
0.7
(ng)
0.64
0.00
0.00
0.78
0.00
0.76
1.13
2.34
4.31
6.05
5.35
10.5
50.3
55.3
128
347
145
82.7
136
333
2.35
11.5
13.1
3.50
9.64
7.61
0.89
1.50
(percent)
0.7
0.0
0.0
0.9
0.0
0.9
0.7
0.9
0.9
1.8
1.0
1.0
6.3
1.2
1.1
2.1
11.8
1.1
0.7
1.3
0.3
1.1
0.9
0.6
0.9
0.7
0.0
0.0
1.4
aData taken from Ref. 42; the extraction
^Extract from fourth extraction analyzed
cFifth extraction performed after 18-hour
solvent was acetone/hexane.
separately.
exposure to acetone.
133
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TABLE A-14. INFLUENCE OF MOISTURE CONTENT
Extraction
number
13
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Moisture
content 50 g of
(percent) Na2$04
3.1*
3.1 +
3.1
3.1 +
25b
25 +
50
50 +
50
50 +
75
75 +
100
100 +
1.8*
1.8 +
1.8
1.8 +
16. 7C
16.7 +
16.7
16.7 +
Method
Soil 1
85x2
B 5 x 2
Tumbler
Tumbler
B 5 x 2
B 5 x 2
B 5 x 2
B 5 x 2
Tumbler
Tumbler
B 5 x 2
85x2
85x2
B 5 x 2
Soil 2
B 5 x 2
B 5 x 2
Tumbler
Tumbler
85x2
B 5 x 2
Tumbler
Tumbler
Co-
extractants
(mg/g)
0.085
0.260
0.044
0.066
0.130
0.120
0.190
0.120
0.120
0.100
0.140
0.180
0.120
0.140
0.088
0.110
0.072
0.110
0.310
0.910
1.830
1.050d
Al dri n
(ppm)
0.51
0.41
0.42
0.50
0.95
0.40
0.82
0.81
0.84
0.72
0.95
1.00
0.85
0.95
2.35
1.20
1.25
1.55
2.42
2.59
2.59
2.59
Dieldrin
(ppm)
0.47
0.41
0.37
0.39
1.08
0.45
1.00
0.95
1.08
0.71
1.04
1.08
1.08
0.96
0.90
0.40
0.45
0.50
1.24
1.18
1.30
1.18
aAir-dried.
bWater added up to 25, 50, 75, and 100 percent of water-holding
capacity.
GNatural field moisture content.
^Extract showed orange color, probably from rubber gasket material on the
Mason jar. Soil type: sandy loam. Extraction solvent: 50 percent
acetone:n-hexane. Data taken from Ref. 36.
134
-------
was added, the most significant difference was found between 25 and 50 percent
moisture content. The authors also concluded that there may be some risk
involved in the use of sodium sulfate unless the moisture content is measured
precisely. When the moisture content is less than 25 percent, the use of
sodium sulfate may reduce the extraction efficiency.
Williams (37) investigated the effect of water content on Soxhlet
extraction with two silt loam soils. The results were consistent for samples
with a moisture content between 5 percent and 40 percent and were different
from the results obtained with the dry soils (Table A-15).
Saha (41) studied the effect of the addition of 20 percent water to
air-dried soil before extraction and reported considerably increased
extraction efficiencies for methanol, hexane:acetone, hexane:2-propanol, and
benzene:methanol. The amount of water added to the soil before extraction
with hexane:acetone (1:1) resulted in increased recoveries; however, addition
of 40 to 50 percent water reduced the recovery of chlordane residues, and
100 percent water gave poor recoveries (Table A-16).
2.3 EXTRACT CLEANUP
Several types of cleanup techniques are available for the removal of
coextractants from a sample matrix. They are listed below:
Liquid-liquid partitioning
Gel permeation chromatography
Sulfur removal
Liquid-solid chromatography (alumina, Florisil, silica gel)
High-pressure liquid chromatography (HPLC).
Each of these techniques will be discussed in the following sections.
2.3.1 Liquid-Liquid Partitioning
This technique is widely used for removing fats, waxes, and polar
materials. The sample extract is, for example, partitioned with a solvent of
different polarity which is immiscible with the extraction solvent. Thus, if
the extract is in hexane, petroleum ether, or benzene, it will be partitioned
with a polar solvent such as acetonitrile. The waxes and fatty materials will
remain in the nonpolar phase while the pesticides will partition into the
acetonitrile. The polar phase containing the pesticides is diluted with
water, and a sodium sulfate or sodium chloride solution is added to "salt out"
the pesticides from the polar phase back into another nonpolar solvent.
Recovery data for solvent partition of pesticides between one part of hexane
and three parts of polar solvent are given in Table A-17.
Solvent systems such as DMF:hexane (62), DMSO:petroleum ether (63),
and acetone:petroleum ether have been recommended for partitioning to separate
organochlorine pesticides from fat. Although DMF and DMSO were superior in
terms of recovery, and DMSO was the best in removing fat, these two solvent
systems have not been widely used because of emulsion problems (1).
135
-------
TABLE A-15. EFFECT OF WATER ON THE RECOVERY IN PPM OF ORGANOCHLORINE
PESTICIDES FROM TWO SAMPLES OF SILT LOAM SOIL BY
SOXHLET EXTRACTION*
Water added (percent)
Pesticide
10
20
30
40
Soil 1
Dieldrin
4,4'-DDE
2,4'-ODT
4,4'-DDT
0.15
0.054
0.023
0.37
0.18
0.058
0.032
0.41
0.19
0.062
0.032
0.41
0.20
0.068
0.027
0.40
0.20
0.068
0.024
0.40
0.20
0.064
0.021
0.40
Soil 2
Heptachlor
Heptachlor
expoxide
4,4'-DDE
4,4'-DDT
4,4'-DDT
0.007
0.18
0.036
0.031
0.25
0.008
0.24
0.042
0.043
0.34
0.008
0.25
0.044
0.047
0.35
0.008
0.26
0.042
0.047
0.35
0.009
0.24
0.041
0.047
0.36
0.009
0.24
0.042
0.045
0.36
aData taken from Ref. 37.
TABLE A-16. EFFECT OF THE AMOUNT OF WATER ADDED TO AN AIR-
DRIED CLAY LOAM SOIL ON THE RECOVERY OF CHLORDANE
RESIDUES WITH 1:1 HEXANE:ACETONE (MECHANICAL
SHAKER METHOD)a
Recovery Cppm)
Water added
to soil
(percent)
cis-
Chlordane
trans-
Chlordane
Others
Total
0.0
10.0
20.0
30.0
40.0
50.0
100.0
aData taken from Ref. 41.
0.58
0.78
0.88
0.88
0.84
0.75
0.36
0.68
0.92
0.93
0.93
0.90
0.88
0.41
38
08
12
10
98
1.88
0.89
64
78
93
91
72
51
1.66
136
-------
TABLE A-17. SOLVENT PARTITION OF PESTICIDES AND BUTTER OIL BETWEEN
1 PART HEXANE AND 3 PARTS POLAR SOLVENT3
Pesticide
Methanol
Acetonitrile
DMSO
DMF
Al dri n
Butter oil
DDE
DDT
Dieldrin
Endrin
Heptachlor
Heptachlor epoxide
Lindane
73
17
69
77
75
84
83
90
89
50
7.1
56
68
74
87
54
88
96
49
0.3
60
96
85
95
80
97
98
74
11
93
97
93
96
92
97
98
aData taken from Ref. 61.
2.3.2 Gel Permeation Chromatography
In gel permeation Chromatography, the separation mechanism is based on
differences in molecular size. Large molecules are excluded from the pores
of the gel and elute first while smaller molecules which can diffuse into the
pores are eluted last.
Gel permeation Chromatography with Bio-Beads SX-2, SX-3 combined with
cyclohexane, and cyclohexane-methylene chloride as eluants were reported for
the cleanup of fish lipids (64). Other mobile phases included toluene:ethyl
acetate (1:3 v/v) and ethyl acetate.
Bio-Beads SX-3 and toluene:ethyl acetate have been used successfully to
remove lipid interferences from fish extracts for subsequent determination of
Kepone (27).
The elution volumes of organochlorine pesticides and PCBs obtained from
Bio-Beads SX-3 when two different solvent mixtures are used are given in
Table A-18.
2.3.3 Sulfur Removal
Sulfur and organosulfur compounds, if present, will give large peaks in
the solvent area which often mask the region from the solvent peak to the
aldrin peak.
Several methods are available for the removal of sulfur. Shaking with
0.1 to 0.2 mL of metallic mercury (65) is a simple technique, but quite often
it does not remove all the sulfur. Although Goerlitz and Law (65) claimed
that aldrin, dieldrin, heptachlor, lindane, and 4,4'-DDT were not affected by
137
-------
TABLE A-18. ELUTION VOLUMES OF ORGANOCHLORINE PESTICIDES
BY GEL PERMEATION CHROMATOGRAPHYa
Elution volume (mL) Elution volume (mL)
with methylene chloride: with toluene:ethyl
Organochlorine pesticides cyclohexane (15:85) acetate (1:3)
Aldrin
alpha-BHC
alpha-Chlordane
gamma-Chlordane
4,4'-DDD
4,4'-DDE
2,4'-DDT
4,4'-DDT
Dieldrin
Endrin
Heptachlor
Heptachlor epoxide
Hexachlorobenzene (HCB)
Lindane
Methoxychlor
Mi rex
Toxaphene
110->150
150->190
120->170
120->170
160->210
110->160
110->150
120->170
140->180
130->170
110->150
120->170
120->160
160->210
140->200
90->140
120->240
110->130
90->130
100->120
100->120
90->120
110->160
110->130
110->130
110->130
110->130
100->130
110->130
120->150
100->130
100->120
110->130
100->140
PCBs and PBBs
PCB-1016 120->240 NT
PCB-1242 120->240 110->140
PCB-1254 120->240 100->150
PCB-1260 120->240 110->140
Kepone NT _. 100->190
NT — Not tested.
aData taken from Ref. 66.
the addition of Hg to the hexane solutions of these pesticides, Thompson (27)
reported that recovery of heptachlor was only 39.8 percent when Hg was used
for the removal of sulfur (Table A-19).
Activated copper (copper dust treated for removal of surface oxides with
2N HC1) was also used (67). Heptachlor was reported to be degraded by copper
activated with 6 N nitric acid (27); Chau et al. (1) found no degradation for
17 organochlorine pesticides dissolved in isooctane when contacted with
HC1-activated copper for 7 days.
138
-------
TABLE A-19. EFFECT OF EXPOSURE OF ORGANOCHLORINE PESTICIDES
TO VARIOUS SULFUR REMOVAL REAGENTS
Percent recovery
Pesticide
Hg<
Cu*
H2S0
KOH11
TBAC
Aldrin
PCB-1254
alpha-BHC
beta-BHC
gamma-BHC
4,4'-DDD
4,4'-DOE
4,4'-DDMVf
4,4'-DDT
2,4' -DDT
Dieldrin
Endrin
Heptachlor
Heptachlor
95.5
97.1
81. 2d
.
(lindane) 75.7
—
92.1
79. 8d
—
79.1
90.8
39.8
epoxide 69.1
======================
93.3
104.3
98. ^
—
94.8
—
102.9
—
85. id
—
94.9
89.3
5.4
96.6
============
92
—
94
94
92
100
104
100
100
98
0
—
94
83
87
—
59
70
55
95
100
91
95
95
0
—
90
79
104
—
0
6
11
0
4376
190e
0
0
103
—
104
100
73
—
—
—
79
94
95
94
—
—
—
—
97
aRef. 27, level not specified, recovery based on mean of duplicate tests.
Copper dust activated with 6N HN03.
bRef. 43, level 0.02 to 0.1 ppb.
cRef. 45, level 50 ppb, TBA is tetrabutyl ammonium sulfite.
dlsomer not specified in original literature.
eHigh recovery is due to the formation of pesticide by the
decomposition of other.
M.4-DDMV is l-chloro-2,2-bis(4-chlorophenylJethane.
Ahnoff and Josefsson (68) showed that sulfur was-easily removed by
shaking with Raney Nickel or KOH at 120°C for 20 min. This treatment did not
affect the PCBs; however, DDT and ODD were converted to DDE and DDMV
[l-chloro-2,2-bis(4-chlorophenyl)ethane], respectively, and most BHCs were
lost.
An efficient, rapid, nondestructive procedure to remove sulfur according
to the reaction:
2-
(TBA*)2S03
2TBA+
2-
where TBA is the tetrabutyl ammonium ion was reported by Jensen et al. (44).
Results of recovery experiments from spiked sediments are reported in Table A-20.
139
-------
TABLE A-20.
RESULTS OF RECOVERY EXPERIMENTS FROM SPIKED
SEDIMENTS USING TETRABUTYL AMMONIUM SULFITEa
Substance
True
value
n (ppb)
Mean
(percent)
Standard
deviation,
percent of
mean value
4,4'-DDE 7
4,4'-DDD 7
4,4'-DDT 7
2,3',4',5-Tetrachlorobiphenylb 7
2,2',3',4,5,6-Hexachlorobiphenylb 6
2,2',4,4',5,5'-Hexachlorobiphenylb 7
2,3,3',4,4'-Pentachlorobiphenyl& 6
2,2',3,4,4',5'-Hexachlorobiphenylb 6
Hexachlorobenzene 7
Lindane (gamma-BHC) 7
Aldrin 7
Aldrin, prior to evaporization of
extracting solvents 4
Heptachlor epoxide 7
2,4,6-Trichlorophenol 4
2,3,4,6-Tetrachlorophenol 4
Pentachlorophenol 4
50
50
50
50
2.8
50
2.8
2.8
50
50
50
50
50
20
20
50
95
94
94
97
100
98
95
101
94
79
73
95
97
75
82
101
7.2
6.4
5.7
5.5
4.0
5.4
4.0
9.1
3.4
4.4
5.4
1.9
1.2
2.8
1.8
1.9
n - number of replicates.
aData taken from Ref. 44.
b - representative PCB component.
2.3.4 Liquid-Solid Chromatography
The following discussion will emphasize the application of Florisil,
alumina, and silica gel to the cleanup and fractionation of organochlorine
pesticides and PCBs in extracts.
2.3.4.1 Florisil
Florisil is a synthetic magnesium silicate manufactured by the Floridin
Company from magnesium sulfate and sodium silicate. Following precipitation
from solution, it is filtered, dried, and calcinated at 650°C. Examples of
cleanup methods for organochlorine pesticides and PCBs using Florisil are
listed in Table A-21.
Mills (69) introduced the use of Florisil in the cleanup of nonfatty and
fatty food samples for organochlorine pesticides by using a 20-g Florisil
column and by eluting the pesticides with 200 mL of 6 percent ether in
140
-------
TABLE A-21. COLUMN CHROMATOGRAPHIC CLEANUP OF ORGANOCHLORINE PESTICIDES AND
PCBs USING FLORISIL
Column size
(mm)
„
400 x 20
400 x 20
10 m ID
600 x 40
400 x 20
300 x 22
400 x 20
400 x 20
500 x 15
300 x 25
140 x 5
300 x 22
400 x 20
300 x 25
Adsorbent
amounts
1.6 g
20 g
100 mm
10 g
100 g
20 g
20 g
125 nm
75 to
100 mm
30 g
28 g
30 mm
20 g
100 mm
150 mm
Deactlvatlon
Nil
Nil
Nil
2 percent HgO
5 percent HjO
Nil
Nil
5 percent H£0
0.7 percent
H20
Nil
2 percent ^0
Nil
Nil
i
i
Nil
2 percent HgO
Sample
Sediment
extract
Food
Food
Milk
Butterfat
Standards
Butterfat
Soybean oil
Butterfat
Standards
Food
Fish
Water
Food
Fish
Food
Solvent
2 : 4 : 94 methanol : benzene : hexane
1:2:4:93 methanol :acetonitrile:
benzene: hexane
6:94 ether :PE
6:94 ether:PE; 15:85 ether :PE
Hexane
15 to 20 percent methylene chlorine
In PE
6:94 ether :PE; 15:85 ether :PE
30 percent water in acetonltrlle
15:85 methylene ch1oride:PE
6:94 ether:PE; 15:85 ether :PE
PE 5:1 benzene :PE; chloroform
acetone
Hexane; 30:70 methylene chloride:
hexane
6:94 ether:hexane; 15:85 ether:
hexane
20:80 methylene chlor1de:hexane;
50:0.35:49.65 methylene chloride:
acetonitrile:hexane;
50:1.5:48.5 methylene chloride:
acetonltrile.-hexane
10:90 ether :PE
Hexane; 5, 10, 15, 30 percent
methylene chloride in hexane;
5, 10, 30 percent ether In hexane
Pesticide
Kepone
Hultl-OCS
21 DCS
PCBs
Hultl-OCS
Hultl-OCS
Hultl-OCS
DDE. ODD.
DDT, endrln.
dleldrln
Multl-OCS
Multl-OCS
Hultl-OCS
Hultl-OCS
Hultl-OCS
Hultl-OCS
Hultl-OCS
Recovery
(percent)
89 to 109
40 to 100
—
Varies with
congener
100
85 to 100
>80
—
91 to 100
..
94 to 100
96 to 100
>90 except
endrln 60
to 90,
heptachlor
80
96 to 100
_-
Ref.
25
69
77
73
78
74
79
80
81
82
72
83
84
85
86
(continued)
aThe values given represent the amounts in grams or the heights of the adsorbed beds in the columns, as specified.
bAdjust weight of Florisil used according to the lauric acid adsorption value and ascertain that heptachlor epoxlde,
dleldrln, and endrln are being eluted by the appropriate eluant.
cAgN03-coated Florisil was used to separate phthalate esters from organochlorlne pesticides.
OCS - organochlorlne pesticides.
PE - petroleum ether.
-------
TABLE A-21. (concluded)
Column size
(mm)
300 x 25
Adsorbent
amount3
150 mm
Deactlvatlon
10 percent
H20
Sampl e
Food
Solvent
Ether;
Hexane;
Pesticide
Multl-OCS
Recovery
(percent)
~
Ref.
86
300 x 10
20 mm ID
15 mm ID
5 g
100 mm
b
3 9
20 g
Nil
N11 Mater
Not specified Food
Nile
Nil
5, 10, 15, 30 percent
methylene chloride 1n hexane;
5, 10, 30 percent ether 1n hexane
Standards 6:94 ether:PE; 15:85 ether:PE Multl-OCS 94 to 100 87
50:50 ether:PE
Hexane PCBs — 68
20 percent methylene chloride in Multi-OCS — 88
hexane (I);
50 percent methylene chloride;
0.35 percent acetonltrlle;
49.6 percent hexane (II);
50 percent methylene chloride;
1.5 percent acetonitrile;
48.5 percent hexane (III)
Food 3 percent benzene In hexane Multl-OCS 82 to 103 89
Sediment 35:65 ether:hexane; Kepone 77 to 115 47
5:10:85 benzene:methano1:hexane
aThe values given represent toe amounts in grams or the heights of the adsorbed beds In the columns, as specified.
bAdjust weight of Florisil used according to the 1 auric acid adsorption value and ascertain that heptachlor epoxide,
dieldrin, and endrin are being eluted by the appropriate eluant.
cAgN03~coated Florisil was used to separate phthalate esters from organochlorine pesticides.
OCS - organochlorine pesticides.
PE - petroleum ether.
-------
petroleum ether. Later, Johnson (70) extended the cleanup procedure to
dieldrin and endrin by eluting the Fieri si 1 with 15 percent ether in
petroleum ether.
Burke and Mills (71) reported that endosulfan was recovered from the
Florisil column by elution with 30 percent ether in petroleum ether.
To separate PCBs from pesticides, Reynolds and Copper (72) extended the
Florisil procedure by introducing a hexane or a petroleum ether fraction to
the 6 percent and 15 percent ether in petroleum ether.
Mills et al. (84) recommended an elution system which replaced the
ether/petroleum ether mixture for multiple pesticide analysis. The
three eluting mixtures are identified in Table A-22.
Difficulties in obtaining reproducible recoveries and the overlapping of
pesticides between fractions have been reported (74, 75, and 76). This was
attributed to the variation of Florisil among batches because of varying
calcination temperatures and times.
TABLE A-22. MILLS 1972 MODIFIED PROCEDURE FOR ELUTION OF
ORGANOCHLORINE PESTICIDES FROM FLORISILa
Fraction I Fraction II Fraction III
20 percent methylene 50 percent methylene 50 percent methylene
chloride in hexane chloride/0.35 percent chloride/1.5 percent
(200 mL) acetonitrile/49.65 percent acetonitrile/
hexane (200 mL) 48.5 percent hexane
Aldrin Dieldrin
alpha-BHC alpha-Endosulfan
beta-BHC beta-Endosulfan
gamma-BHC Endrin
Chlordane Heptachlor epoxide
2,4'-DDE Methoxychlor
4,4'-DDE
2,4'-DDT
Heptachlor
Hexachlorobenzene
Mi rex
Perthane
PCBs
Toxaphene
Recoveries >90 percent, except for endrin (60 to 90 percent) and
heptachlor (80 percent); data taken from Ref. 84.
143
-------
Burke and Malone (74) studied the calcination conditions used in the
manufacture of Fieri si 1 and reported that l,250°F/3-hour calcination is the
desirable condition for activation. They have found that as the calcination
temperature is increased from 1,000 to 1,400°F, both pesticides and lipid
materials are removed more readily from the column. Similar effects have
been observed for increased calcination time (see Table A-23).
2.3.4.2 Silica Gel
Silica gel has been recommended for the separation of PCBs and
pentachloronaphthalenes from organochlorine pesticides (90 and 91). Armour
and Burke (90) reported separation of PCBs and organochlorine pesticides
when a mixture of silica gel deactivated with 3 percent water and Celite 545
was used. Only aldrin could not be separated from the PCBs, but the other
organochlorine pesticides, including lindane, heptachlor, heptachlor epoxide,
dieldrin, endrin, DDT, DDE, and ODD, were recovered quantitatively with
acetonitrile:hexane:methylene chloride (see Table A-24). With more highly
deactivated silica gel, an overlap of organochlorine pesticides and PCBs was
observed (Figure A-l).
Similar results were reported by Leoni (91) with silica gel deactivated
with 5 percent water (Table A-25). The adsorbent in this case was silica gel
grade 950, 60 to 200 mesh, Davidson code 950-08-08-226, Grace Davidson Chemical,
Baltimore, Maryland. The silica gel was activated for 2 hours at 130°C and was
then partially deactivated with distilled water (5 percent by weight). The
compounds listed in Table A-25 were eluted from the silica gel column with
hexane (20 ml), 60 percent benzene in hexane (8 ml), benzene (8 mL), and 50
percent ethyl acetate in benzene (14 ml).
Biddleman et al. (92) investigated silica gel deactivated with 3.3
percent water for the separation of PCBs, cis- and trans-chlordane, DDE, and
DDT from toxaphene. The chlorinated hydrocarbons were eluted from the silica
gel column in three fractions. Fraction I (50 ml petroleum ether) contained
the PCBs, DDE, and 10 to 30 percent 2,4'-DDT. Fraction II (80 ml petroleum
ether) contained chlordanes, 4,4'-DDT, and 70 to 90 percent 2,4'-DDT. Dieldrin
and 4,4'-ODD were eluted in a third (15 ml dichloromethane) fraction. The
percentages of toxaphene eluted in the three fractions-"Were 10, 30, and 60
percent, respectively. Nonseparation of aldrin from PCBs is not inconvenient
unless significant amounts of PCBs with a low degree of chlorination are
present, since aldrin has a retention time that is different from those of PCBs
with a higher chlorine content.
Acurex has slightly modified this fractionation technique to make it
applicable to all of the organochlorine pesticides listed in Method 8080,
including Kepone. The results are given in Table A-26. Other applications
in which silica gel was used for the cleanup of organochlorine pesticides and
PCB extracts are given in Table A-27.
144
-------
TABLE A-23. PESTICIDE RECOVERIES AND FAT ELUTION FROM
FLORISIL OF DIFFERENT CALCINATIONS
01
Calcination
lindanea
Hepta-
chlor3
Aldrln3
Heptachlor
epoxlde 4,4'-DDTa Dieldrln
Endrln
Fat Residue
(mg)
6 6 6 6 15 6 6 15 6 15 6 15
Temp. Time percent percent percent percent percent percent percent percent percent percent percent percent
(°F) (hr) eluate eluate eluate eluate eluate eluate eluate eluate eluate eluate eluate eluate
1,000
1,100
1,200
1,200
1,200
1,200
1,250
1,250
1,250
1,250
1,300
1,300
1,300
1,300
1,350
1,350
1,350
1,350
1,400
2
2
2
4
8
16
2
4
8
16
2
4
8
16
2
4
8
16
2
99
99
101
97
97
101
98
97
101
100
99
99
101
101
97
102
99
101
98
37
18
11
43
99
102
94
41
92
96
38
81
99
100
95
92
102
101
95
96
95
98
96
101
102
100
97
101
102
96
100
102
103
97
102
'.93
103
99
0
0
0
95
99
103
100
16
101
98
. 95
100
99
100
97
100
99
100
100
90
91
92
2
0
0
0
86
0
0
2
0
0
0
0
0
0
0
0
94
89
97
100
108
98
106
103
106
117
100
95
97
102
100
102
100
99
98
0
0
0
0
23
0
0
0
0
0
0
0
0
40
60
0
100
99
30
0
0
0
95
67
99
101
92
101
97
81
96
100
42
25
101
0
0
55
0
0
0
0
64
8
0
0
0
0
0
2
0
79
90
12
100
99
75
0
0
0
98
21
88
102
98
102
100
79
98
100
7
3
78
0
0
13
0
0
0
0
35
43
1
0
1
0
0
31
0
127
25
136
289
266
86
15
48
102
213
275
239
224
215
272
244
224
267
213
203
291
181
65
80
199
aLindane, heptachlor, aldrln, and 4,4'-DDT were not detected In any of the
15-percent eluates. Data taken from Ref. 74.
-------
TABLE A-24. ELUTION PATTERNS OF ORGANOCHLORINE PESTICIDES AND PCBs USING
ARMOUR/BURKE'S PROCEDURE WITH SILICA GEL/CELITE 545
Fraction II
Column Fraction I 200 mL acetonitrile:hexane:
packing 250 mL petroleum ether methylene chloride (1:19:80)
Silica gel PCB-1254 (97.5 percent) Lindane (96 percent)
(3 percent H20)
plus Celite 545 PCB-1260 (97.5 percent) Heptachlor (93 percent)
Aldrin (101 percent) Heptachlor epoxide
(100 percent)
Dieldrin (104 percent)
Endrin (94 percent)
4,4'-DDE (95 percent)
2,4'-DDT (98 percent)
4,4'-DDT (98 percent)
4,4'-DDD (98 percent)
Note: Celite was mixed with the silicic acid to achieve a faster elution
rate from the column. Silicic acid -- Mallinckrodt, 100 mesh powder,
specially prepared for chromatographic analysis. Activate for
7 hours at 130°C; deactivate with 3 percent water; equilibrate for
15 hours. Celite 545 ~ acid-washed (Johns-Manville).
2.3.4.3 A1umi na
Alumina was used in the cleanup of extracts of organochlorine pesticides
from animal fats and tissues (62), fish (97, 102), food (103, 104), vegetables
(105), water (83, 106), and sediment (42). Table A-28 identifies several of
the applications reported in the literature in which alumina was used for
cleanup of the organochlorine pesticides and PCBs. Besides being an excellent
adsorbent for fat, alumina has been found by Goerlitz and Law (42) to be more
efficient than silica gel and Florisil in the removal of naturally occurring
organic acids, pigments, and other interferences.
A multiple organochlorine pesticides analysis using alumina deactivated
with 5 percent water for cleanup of fats was reported by de Faubert Mauder et
al. (62). Lindane, dieldrin, DDT, DDE, and heptachlor epoxide were eluted from
the column with hexane. Boyle et al. (102) used alumina deactivated with
1.0 to 1.5 percent water and fractionated 13 organochlorine pesticides by
elution with 10 percent ether in hexane and collection of 50 mL-eluate
fractions. Recoveries of various organochlorine pesticides ranged between 91
146
-------
ml petroleum ether eluant
50 100
150
200 250
300
350
Polar
eluant
T
i I i T
Nonreproducible elution
s_
-------
TABLE A-25. ELUTION PATTERNS OF ORGANOCHLORINE PESTICIDES
AND PCBs USING LEONI'S SILICA GEL PROCEDURE
(DEACTIVATED WITH 5 PERCENT WATER)a
Hexane
Compound (20 mL)
60 percent
benzene in
hexane
(8 mL)
Benzene
(8 mL)
delta-BHC
Heptachlor +
Aldrin +
Heptachlor epoxide
Endosulfan I
Dieldrin
Endosulfan II
Endrin aldehyde (no data)
DDT +
alpha-BHC
beta-BHC
gamma-BHC
DDE +
Endrin
DDD (no data)
Endosulfan sulfate (no data)
PCBs +
Chlordane +
Toxaphene (no data)
Kepone (no data)
Methoxychlor
aData taken from Ref. 91.
and 100 percent except for lindane which was only 40 percent. The low
recovery for lindane was attributed to the alkalinity of the alumina since on
acidic and neutral alumina all recoveries were quantitative.
Telling (104) reported significant degradation of beta-BHC,
alpha-endosulfan, and DDT on aluminas of activity higher than 2.5 on the
Brockman scale. Partial separation of PCBs and organochlorine pesticides was
reported on alumina of 2.5 activity (104).
Millar et al. (4) used the alumina procedure developed by
Telling (104) to clean up wastewater extracts containing organochlorine
pesticides and PCBs, and compared the results with those obtained using
Florisil chromatography. The authors concluded that recoveries of spikes
from both columns were excellent, and there was little reason to choose one
148
-------
TABLE A-26. ELUTION PATTERNS OF ORGANOCHLORINE PESTICIDES AND
PCBs USING BIDDLEMAN'S ET AL. SILICA GEL
PROCEDURE (MODIFIED BY ACUREX)
Compound
Fraction
I
(50 mL
hexane)
Fraction
II
(80 mL
hexane)
Fraction
III
(15 mL
methyl ene
chloride)
Fraction
IV
(50 mL
ethyl
acetate)
gamma-BHC
Heptachlor
Aldrin
Heptachlor epoxide
Endosulfan I
Dieldrin
Endosulfan II
Endrin aldehyde
4,4'-DDT
alpha-BHC
beta-BHC
delta-BHC
4,4'-DDE
Endrin
4,4'-DDD
Endosulfan sulfate
PCBs
Chlordane
Toxaphene
Methoxychlor
Kepone
+
+
Note:
With silicic acid (20 g) deactivated with 3 percent water/Celite (5 g),
aldrin elutes in Fraction I (250 mL petroleum ether); heptachlor,
DDE, DDT elute in Fraction II [200 mL acetonitrile:hexane:methylene
chloride (1:19:80)] (90).
cleanup procedure over the other. However, they did observe that pesticides
were much more frequently found in two fractions when an alurflina column was
used, and chlordane and toxaphene were often spread over three fractions.
2.3.5 High-Pressure Liquid Chromatography
The use of HPLC in the cleanup of environmental samples prior to
analysis has significant potential primarily because of the possibility of
149
-------
TABLE A-27. COLUMN CHROMATOGRAPHIC CLEANUP OF ORGANOCHLORINE PESTICIDES AND
PCBs USING SILICA GEL
Column size
(mm)
5 to 22 mm
ID
450 x
400 x
140 x
230 x
i— •
tn
° 6 mm
450 x
300 x
200 x
300 x
300 x
6
22
5
4
ID
7
4.2
10.
10
10
Adsorbent
amount3
1 to 20 g
2 9
20 g silica
gel + 5 g
acid-washed
Celite
30 mm
1 9
2 9
1 9
3 9
80 mm
5 g
Percent
deactlvatlon
(water)
5wL water per
ml hexane
5
3
5
0.5 percent
ether In
benzene
1
3
5
Nil
3
"As Is*
Sample
Soil, plant
tissue, water
Fat, tissue
F1sh
Water
Standards
Water
Standards
Water
Fish
Sediment
Standards
Solvent
Hexane, mixtures of
hexane and benzene,
benzene
Hexane
10:90 ether :hexane
PE
1:19:80 acetonltrlle:
hexane .-methyl ene
chloride
Hexane
15:85 benzene:hexane
50:50 Benzene :hexane
20:80 ether: hexane
Hexane
0.5 to 99.5 ether:
benzene
10:90 benzenerhexane
60:40 benzene :hexane
Hexane
10:90 ether:hexane
Hexane
60:40 benzene: hexane
Benzene
Pentane, benzene
Hexane, benzene
PE
1:19:80 acetonitrile:
hexane rmethylene
chloride
Pesticide
17 individual
DCS
Multi-OCS
PCBs and DDT
analogs
Multi-OCS
PCBs and DDT
analogs
Multi
PCBs and OCS
PCBs and OCS
PCBs and DDT
analogs
PCBs and OCS
PCBs and DDT
analogs
Recovery
(percent)
98 to 100
100
OCS = 80 to 107
93 to 100
—
—
Average 92
95 to 100
OCS = 72 to 118
OCS = 75 to 99
OCS = 80 to 95
Ref.
93 to 95
96
90
83
97
98
99
91
100
42
101
aThe values given represent the amounts in grams or the heights of the adsorbent beds in the columns, as specified.
OCS - organochlorine pesticides.
PE - petroleum ether.
-------
TABLE A-28. COLUMN CHROMATOGRAPHIC CLEANUP OF ORGANOCHLORINE PESTICIDES
AND PCBs USING ALUMINA
Column size
(ram)
__
20 rrni ID
200 x 6
300 x 10
450 x 6
140 x 5
75 x 5
300 x 10
300 x 20
450 x 6
Adsorbent
amount &
10 g
150 ran
2 9
89
2 9
30 mm
1.7 g
80 mm
22 g
3 9
Percent
deactlvation
(water)
5
1 to 1.5
15
15
5
5
1 percent
rnethanol in
benzene
9
10
4
Sample
Animal fats,
tissue
Fish
Food
Vegetable
Fat, tissue
corn oil
Water
Fish,
plankton
Sediment
Food
Water, biota
Solvent
Hexane
10:90 ether :hexane
PE
Hexane
2:98 acetone :hexane
Hexane
Hexane
50:50 benzene :hexane
Hexane
Hexane
Hexane
Hexane
Pesticide
Hulti-OCS
Multi-OCS
Multi-OCS
Multi-OCS
Multi-OCS
Multi-OCS
OCS, PCBs
OCS.PCBs
OCS and PCBs
Multi-OCS
Recovery
(percent)
73 to 94
91 to 100
40 to 90
>90 except
endosulfan 75
97 to 109
94 to 100
—
75 to 99
70 to 124
100
Ref.
62
102
103
105
96
83
97
42
104
106
aThe values given represent'the amounts in grams or the heights of the adsorbent beds in the columns, as
specified.
OCS — organochlorine pesticides.
PE — petroleum ether.
-------
automation. Furthermore, in the case of water samples, the extraction and
cleanup could be accomplished on the same setup. For example, a water sample
can be first pumped through a reversed-phase Microbondapak-Ci8 column where
the various organics including the organochlorine pesticides and PCBs are
absorbed. The pesticides and PCBs are then eluted from the column by
acetonitrile or another organic solvents, and appropriate fractions are
collected for subsequent analysis (107).
Another application involving HPLC was reported by Larose (108). His
system consisted of a liquid chromatograph (LC) equipped with a pellicular
Corasil II column and a refractive index detector; petroleum ether was used
as the mobile phase. Results reported by Larose indicated that fats,
phospholipids, and oil were completely removed, and the recovery of lindane
was better than 90 percent.
2.3.6 Selection of the Cleanup Technique
The choice of which adsorbent to use depends upon many factors, but the
type of matrix and the interfering coextractants present seem to be the most
important ones. Alumina has been found by Law and Goerlitz (83) to be more
efficient than silica gel and Florisil in the removal of fats, naturally
occurring organic acids, and pigments. Silica gel, on the other hand, was
found to remove pigments that could not be removed with either alumina or
Florisil.
When dealing with multicomponent analyses and especially in those cases
where the GC analysis does not allow complete separation of all the
organochlorine pesticides and PCBs, a combination of cleanup techniques may
be required.
Goerlitz and Law (42) used Woelm neutral alumina, activity I (deactivated
with 9 percent water), and silica gel deactivated with 3 percent water to
separate PCBs and polychlorinated naphthalenes (PCNs) which interfere with the
determination of the organochlorine pesticides (Figure A-2).
More polar compounds, such as phthalate esters, were separated from
organochlorine pesticides and PCBs using alumina chromatography (109) as
shown in Figure A-3.
In an attempt to improve the separation of organochlorine pesticides from
PCBs, Berg et al. (110) used a combination of Florisil chromatography and
activated charcoal chromatography (Figure A-4). The first fraction from the
Florisil column was concentrated and applied to the charcoal column.
Heptachlor and aldrin were eluted from charcoal with cyclohexane while the PCBs
were recovered in the benzene fraction.
2.4 GC ANALYSIS
This review will address only the GC analysis with the electron capture
detector (ECD) since this is the most widely used analytical technique in
pesticide and PCB analysis. Basically, these compounds are separated by the
chromatographic column, either packed or capillary, at elevated temperatures,
152
-------
Hexane extract (0.5 nL)
Alumina
0-20 ml fraction A-l
(hexane)
Aldrin
Chlordane
ODD
DDE
DDT
Heptachlor
Lindane
PCBs
Polychlorinated naphthalenes
Toxaphene
l_
Mercury
Silica gel
0-25 ml fraction S-l
(hexane)
Aldrin
PCBs
Polychlorinated naphthalenes
I
20-35 ml fraction A-2
(hexane)
Dieldrin
Endrin
Heptachlor epoxide
25-45 ml fraction S-2
(benzene)
Chlordane
ODD
DDE
DDT
Heptachlor
Lindane
Toxaphene
Figure A-2. Fractionation scheme employing Woelm neutral alumina,
activity I, and silica gel deactivated with 3 percent
water for the separation of organochlorine pesticides
and PCBs. Data taken from Ref. 42.
153
-------
Hexane extract from sample
Evaporate to 0.5 ml
Alumina column
Eluted with 40 ml hexane.
followed by 20 ml benzene9
0-20 mL (fraction A-l)
Treat with Hg to remove S;
evaporate to 0.5 mL
20-35 ml (fraction A-2)
Dieldrln
Endrln
Heptachlor epoxlde
35-55 nL (fraction A-3)
Silica gel column
i
Silica gel column
Elute with 30 ml hexane,
followed by 20 ml benzene9
0-25 ml (fraction S-l)
Aldrin
PCB's
PCN's
HI rex
25-45 ml (fraction S-2)
Chlordane
ODD
DOE
ODT
Heptachlor
Lindane
Toxaphene
0-20 ml (fraction AS-1)
Discard
aApproximately 5 ml of eluting liquid is retained by column
20 ml eluent (A-3).
followed by 20 ml of
10 percent acetone
in benzene9
20-35 ml (fraction AS-2)
Fhthalate esters
Dimethyl
Diethyl
Oiallyl
Oi-n-butyl
Di-n-hexyl
Butyl benzyl
Di-2-ethylhexyl
Diphenyl
Di-n-octyl
Figure A-3. Fractionation of organochlorine pesticides, PCBs, PCNs and
phthalate esters using neutral alumina, activity I, and
silica gel deactivated with 0.3 percent water (109).
-------
Sample extract
Florisil* column
in
CO
to
9
in
Petroleum ether fraction
Containing heptachlor,
alpha-BHC, 4,4'-DDE,
aldrin, mi rex, photomirex,
PCBs
Mini-charcoal/foam column
Cyclohexane Benzene
fraction fraction
Heptachlor,
aldrin
PCBs,
alpha-
BHC,
4, 4 '-DDE
6 percent diethyl ether in
petroleum ether fraction
4,4'-DDD, 4,4'-DDT, 2,4'-DDT,
lindane, cis-and
trans-chlordanes,
4,4'-methoxychlor, heptachlor
epoxide
15 percent diethyl ether in
petroleum ether fraction
Endrin, alpha-endosulfan,
dieldrin
.^.50 percent'diethyl ether in
petroleum ether fraction or
100 percent CHC13
beta-endosulfan
Figure A-4. Florisil/charcoal column
pesticides and PCBs.
fractional on of organochlorine
155
-------
and the compounds are detected with an ECD. Other detectors, such as the
microcoulometric or the modified Hall electrolytic conductivity detector,
have also been used (1). Examples of organochlorine pesticide analysis by
HPLC have been reviewed by Chau (1).
2.4.1 Gas Chromatographic Columns
Several factors should be considered when selecting columns for the GC
determination of organochlorine pesticides and PCBs.
° The operating parameters should yield maximum resolution and detector
response at minimal retention time.
° The stationary phase should be stable and not bleed during the GC
analysis.
° The columns should stand up well under sustained periods of
injection loading.
Columns of single and mixed stationary phases, both nonpolar and polar
packed and capillary columns have been used in the analysis of organochlorine
pesticides. Some of the more common ones are listed below. Table A-29 gives
the relative retention times for several organochlorine pesticides.
1.5 percent OV-17 + 1.95 percent QF-1 (or OV-210)'On Chromosorb W,
HP, 100 to 120 mesh, 200°C and 60 mL/min carrier flowrate. By using
this column, 4,4'-DDT is eluted in 20 min or less. This is an
efficient column which completely resolves all common organochlorine
pesticides except the partially resolved pairs beta-BHC and
heptachlor, alpha-chlordane and alpha-endosulfan, 4,4'-DDO and
beta-endosulfan, and 4,4'-DDE and dieldrin (Figure A-5).
4 percent SE-30 + 6 percent OV-210 (or QF-1) on Chromosorb W, HP,
80 to 100 mesh, 200°C, 70 mL/min flowrate. This column gives no
separation of lindane and beta-BHC but does give good separation
of 2,4'-DDT and 4,4'-DDD (Figure A-6).
4 percent OV-101 + 6 percent OV-210 on Chromosorb W, HP, 80 to
100 mesh, 2008C, 80 mL/min flowrate. This column has retention
characteristics very similar to the above 4-percent SE-30 +
6-percent OV-210 (or QF-1) column and provideVgood separation of
many organochlorine pesticides. Also, on-column decomposition of
endrin is much reduced with this column when compared, for example,
with the 4-percent DC-11 + 6-percent QF-1 column. Therefore, endrin
and photoendrin can be resolved without excessive column
preconditioning, and the detection limit for endrin is lowered.
This column also gives sharp peaks and, thus, improved sensitivity
for polar compounds such as 1-hydroxychlordene (decomposition product
of heptachlor in water) and l-hydroxy-3-chlorochlordene.
5 percent OV-210 (or QF-1) on Chromosorb W, HP, 100 to 120 mesh,
180°C, 60 mL/min flowrate. This packing gives complete separation
of all common BHC isomers and fair separation among 4,4'-ODD,
4,4'-DDT, and heptachlor epoxide (Figure A-7).
156
-------
TABLE A-29.
RELATIVE RETENTION TIMES OF SOME COMMON
ORGANOCHLORINE PESTICIDES
Pesticide
Column la Column & Column 3C Column 4d Column 5e Column 6f Column 79 Column 8h Column
in
alpha-BHC
gamma -BHC
beta-BHC
Heptachlor
Aldrln
Heptachlor epoxlde
gamma -Chlordane
alpha-Chlordane
alpha-Endosulfan
4,4'-ODE
Dleldrln
Endrln
2, 4 '-DOT
4, 4 '-ODD
beta -Endosul fan
4, 4' -DDT
Methoxychlor
a1.8 m x 4 mm ID,
b1.8 m x 4 mm ID,
C1.8 m x 4 mm ID,
dl,8 m x 4 mm ID,
e2.0 m x 4 mm ID,
fl,8 m x 4 mm ID,
91.8 m x 4 mm ID,
hl,8 m x 4 mm ID,
.Ref. 27.
'1.8 m x 4 mm ID,
Ref. 27.
1.
4
4
3
3
3
10
1.
5
0.53
0.68
0.78
0.80
1.00
1.55
1.69
1.85
1.94
2.23
2.38
2.91
3.16
3.47
3.55
4.17
8.20
0.51
0.61
0.62
0.83
1.00
1.38
1.48
1.60
1.70
1.71
2.00
2.27
2.20
2.35
2.52
2.83
3.96
5 percent OV-17 + 1
percent
percent
percent
percent
percent
percent
0.48
0.59
0.60
0.84
1.00
1.45
1.51
1.66
l.RO
1.85
2.15
2.45
2.40
2.64
2.75
3.20
4.58
0.44
0.51
0.48
0.81
1.00
1.22
1.39
1.53
1.51
1.75
1.75
2.03
2.32
2.21
2.03
2.86
4.21
.95 percent OV-210,
SE-30 + 6 percent OV-210,
OV-101 1+ 6
OV-101,'200
OV-17, 198°
OV-225, 200
DC -200 on
6 percent OV-17 + 6
percent
DC-200 + 7.
percent OV-210
°C; data taken
C; data taken
°C; data taken
Chromosorb W,
200°C
, 200°
from
0.45
0.61
0.74
0.79
1.00
1.52
1.79
1.99
1.98
2.53
2.46
3.10
3.77
4.06
3.87
4.95
10.38
200°C; data
; data taken
0.
1.
—
0.
1.
2.
2.
2.
2.
3.
3.
3.
4.
7.
7.
7.
14.
94
18
90
00
14
66
66
47
00
34
97
24
06
09
07
81
0.40
0.48
0.43
0.80
i.no
1.24
—
—
1.56
1.88
1.84
2.06
2.54
2.39
2.11
3.18
4.93
0.54
0.70
0.84
0.83
1.00
1.69
—
—
2.14
2.32
2.65
3.22
3.23
3.80
4.00
4.46
8.5
0.46
0.58
0.59
0.81
1.00
1.47
__
—
1.88
1.95
2.24
2.5fl
2.58
2.82
2.93
3.37
5.30
taken from Ref. 1.
from Ref.
C; data taken from Ref
Ref. 1.
1.
. 1.
from Ref. 1.
from
HP, 80
.4 percent OV-210 on
5 percent OF-1
Ref. 1.
to 100 mesh
Chromosorb
on Chromosorb W,
, 190°C; data taken
W, HP, 80
HP, 80 to
from Ref.
to 100 mesh, 190°C;
100 mesh,
27.
data taken from
190°C; data taken
from
-------
5
Figure A-5. 1.5 percent OV-17 + 1.95 percent OF-1.
Figure A-6. 4 percent SE-30 + 6 percent OV-210.
Figure A-7. 5 percent OV-210.
158
-------
3 percent DEGS on Gas Chrom P, 80 to 100 mesh, 195°C, 70 mL/min
flowrate. This gives excellent separation of 8HC isomers as well as
other commonly found organochlorine pesticides. The unusual elution
pattern (beta-BHC after 2,4'-ODT, 4,4'-DDT before 4,4'-DDD) makes it
useful for the confirmation of peaks (Figure A-8).
Other packing materials (10 percent DC-200; 1.6 percent OV-,17 +
6.4 percent OV-210; 5 percent DC-200 + 7.5 percent QF-1) have been used for
the organochlorine pesticides (Figures A-9 through A-ll).
Resolution efficiencies for organochlorine pesticides and their related
compounds with wall-coated open tubular columns (WCOT) and with support-coated
open tubular (SCOT) glass capillary columns were compared with packed columns
(111). The resolution index which is defined as the ratio of peak height to
half-width of peak of standard injected increased in the order: packed « WCOT
< SCOT.
A GC/EC chromatogram of the organochlorine pesticides and PCBs obtained
at Acurex on the fused-silica capillary column DB-5 (J&W Scientific, Rancho
Cordova, CA) is shown in Figure A-12. Although not all the organochlorine
pesticides were resolved from the mixture of PCB-1016 and -1260 on the fused-
silica capillary column, the excellent resolution of the capillary column
compared to the packed column (Figure A-13) resulted in the separation of over
50 components. Except for 4,4'-DDE and dieldrin, all compounds listed in Table
A-30 have been resolved on the DB-5 fused-silica capillary column. Complete
separation of 4,4'-DDE and dieldrin was achieved by Acurex on another fused-
silica capillary column coated with SPB-608, and by Cooke and Ober (112) on an
OV-17 + QF-1 capillary column.
2.4.2 Problems With Chromatography
2.4.2.1 Overloading
Symptoms of injection overloading include the gradual depression of the
response, peak tailing, lowered column efficiency and resolution, and the
on-column decomposition of certain compounds such as 4,4'-DDT. Figure A-14
provides an example of column overload with fatty materials (18 injections of
25 mg fat).
2.4.2.2 On-Column Decomposition of 4,4'-DDT and Endrin
Decomposition of 4,4'-DDT during the GC analysis is quite common and has
been related to some problems in or adjacent to the front end of the column
(113). Data from 19 laboratories performing organochlorine pesticide analyses
are summarized in Table A-31. Severe decomposition of 4,4'-DDT in Laboratory 8
was related to the fact that the injection insert tube was not changed for 3
weeks prior to analysis.
Endrin decomposition has been reported to occur (Table A-32) whenever
analysis was performed on a freshly prepared column (113). Silylation was
reported to help in reducing endrin decomposition (Figure A-15).
159
-------
a;
•o
r-
CM
1
Figure A-8. 3 percent DEGS.
Figure A-9. 10 percent DC-200.
Figure A-10.
1.6 percent OF-17 +
6.4 percent OV-210.
160
Figure 11.
5 percent DC-200 +
7.5 percent OF-1.
-------
in
CM
in
9
u>
TT
Figure A-12. GC/EC chromatogram of composite organochlorine pesticide and
PCB-1016/1260 standard (30 m x 0.25 mm ID DB-5 fused-
silica capillary column; 100 to 200 pg pesticides and
1,000 pg PCBs injected).
-------
ro
Figure A-13.
GC/EC chromatogram of composite organochlorine pesticide and
PCB-1016/1260 standard (1.8 m x 4mm ID packed 1.5 percent
OV-17 + 1.95 percent OV-210; 25 to 50 pg pesticides and
500 pg PCBs injected).
-------
TABLE A-30. SUMMARY OF PESTICIDE RETENTION TIMES (MIN)
ON TWO FUSED-SILICA CAPILLARY COLUMNS3
Compound DB-5b SPB-608C
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
Endosulfan I
4,4'-DDE
Dieldrin
Kepone
Endrin
4,4'-DDD
Endosulfan II
4, 4 '-DDT
Endrin aldehyde
Endosulfan sulfate
Methoxychlor
gamma-Chlordane
alpha-Chlordane
6.52
7.28
7.49
8.19
9.83
11.02
12.43
13.72
14.68
14.68
16.72
15.46
15.80
16.20
17.59
16.51
17.39
19.77
13.26
13.82
9.46
11.33
10.97
12.73
12.46
13.76
15.98
17.40
18.36
18.60
D
19.96
20.53
20.69
21.72
21.90
22.54
24.90
16.70
17.31
D = degraded on-column.
aData obtained at Acurex.
bTemperature program: 160°C (hold 2 min) to 270°C
(hold 1 min) at 5°C/min.
cTemperature program: 160°C (hold 2 min) to 290°C
(hold 1 min) at 5°C/min; nitrogen at 20 psi.
2.5 CONFIRMATION OF COMPOUND IDENTITY
Several techniques are available for the confirmation of compounds
detected with the GC detectors. These include the multi-column confirmation
technique, chemical derivatization followed by reanalysis of the extract,
confirmation by gas chromatography/mass spectrometry (GC/MS), and
fractionation of the extract followed by analysis.
Although the multi-column technique has been, and still is, a widely
practiced procedure, there have been reports which claim that misinterpretation
of pesticide identity occurred when the retention time was the only criterion
163
-------
(a)
(b)
Q
O
t~\
1
_
^^
a
o
—
«±
t\
^-
a>
cvj
(C)
(d)
Figure A-14. Chromatograms illustrating column overloading and subsequent
rejuvenation:
a. Freshly prepared column.
b. After 18 consecutive injections of 25 mg fat.
c. After 18 consecutive injections of fat, a clean glass insert was
installed in the injection port, and the system was requilibrated for
30 min.
d. Complete rejuvenation of the system after overnight conditioning.
164
-------
TABLE A-31. ON-COLUMN DECOMPOSITION OF 4,4-DDTd
en
Lab.
No.
14
17
12
1
4
15
19
5
13
9
3
6
16
10
2
7
18
8
======
Col umn
age
(days)
21
12
49
47
257
144
38
70
186
100
34
70
545
253
71
108
4
141
:========:
OV-17 +
Est. total
No. of
injections
0
0
e
20
800
1,300
146
250
1,500
300
—
125
3,000
320
400
400
45
200
QF-1
Glass
insert
changed
(days)
21
1
1
6
10
1
1
1
1
1
1
2
5
21
5
5
-i-b
23
Total
decomposition
(percent)
None
None
None
None
Neglig.
Neglig.
Neglig.
1.4
2.2
3.1
3.1
3.4
3.7a
8.5a
14.0
14.0
15. Oa
— c
Lab
No.
5
17
15
12
6
14
13
7
16
4
2
10
9
18
11
1
8
3
Col umn
age
(days)
126
13
142
49
70
46
186
73
e
254
71
254
110
4
5
57
141
35
SE-30
Est. total
No. of
injections
500
75
1,300
e
125
150
1,500
60
3,000
800
400
460
150
None
30
20
200
e
+ QF-1
Glass
insert
changed
(days)
1
1
1
1
2
1
1
5
5
10
5
22
1
— b
5
0
23
1
Total
decomposition
(percent)
None
None
None
None
None
Neglig.
Neglig.
Neglig.
3.2a
6.0
6.0
7.0
8.6a
11. Oa
13. Oa
22. Oa
25. Oa
— C
Breakdown to 4,4'-DDD and 4,4'-DDE.
^Using on-column injection.
cCould not assess because of noise in baseline.
dData taken from Ref. 113.
eUnknown.
-------
TABLE A-32. ON-COLUMN DECOMPOSITION OF ENDRINa
OV-17 + QF-1
SE-30 + QF-1
CPl
Lab.
No.
19
12
7
13
15
14
9
16
2
8
4
10
5
3
1
17
11
18
6
Est. total
No. of
injections
146
d
400
1,500
1,300
0
300
3,000
400
200
800
320
250
20
0
30
45
125
Col umn
silylation
No
No
No
Yes
Yes
No
Yes
No
No
d
No
No
Yes
No
No
No
No ',
No
No
Total
decomposition
(percent)
Neglig.
Neglig.
Neglig.
6
6
7.5
7.7
8
9
9
15
15
17
18
51
55
— b
— b
— c
Lab
No.
6
7
15
9
13
10
2
4
5
12
16
14
8
11
1
18
17
3
Est. total
No. of
injections
125
60
1,300
150
1,500
460
400
800
500
d
3,000
150
200
30
20
0
75
d
Col umn
silylation
No
No
Yes
Yes
Yes
No
No
No
No
No
No
No
d
No
Yes
No
No
No
Total
decomposition
(percent)
None
Neglig.
3.6
4.4
6.0
7.0
7.5
10
11
11
11
12
14
15
26
— b
— b
— b
aData taken from Ref. 113.
bDid not allow chromatogram to run for full retention of last breakdown peak.
cBreakdown peaks obscured by noisy baseline.
^Unknown.
-------
Before si'lylation
(a)
§
20 24 28 32 36 40 44
After silylation
(b)
8 12 16 20 24 28 32 36 40
Figure A-15.
Reduction in
silylation.
decomposition of endrin resulting from column
167
-------
used in the Identification. Therefore, two and three columns have been
required to confirm the compounds when GC is used for analysis.
Chemical derivatization techniques have been used intensively for
confirmation in pesticide residue work and will be addressed only briefly
here since it is really beyond the scope of this project.
Confirmation of pesticide identity by GC/MS has been reported; this
technique can only be applied to those samples in which the pesticide
concentration is above approximately 5 ug/L for water and 50 ng/g for soil
samples.
Fractionation of extracts using silica gel, alumina, Florisil, etc., has
been addressed in Section 4.3.
Chemical Derivatization
A summary of the type of reactions used for the confirmation of organo-
chlorine pesticides by chemical derivatization-GC techniques is presented in
Table A-33. Applications of alkali dehydrochlorination for DDT and analogs are
given in Table A-34, and summaries for chemical derivatization procedures for
endrin/dieldrin, aldrin, the chlordane group, BHCs, and endosulfans are given
in Tables A-35 through A-39.
In selecting the most appropriate method(s) for routine work,
consideration should be given to those procedures that allow unambiguous
confirmation of pesticide identity and that circumvent interference(s) in the
quantitative analysis. Furthermore, the method should use routine
instrumentation, should be easy to standardize, and should not include
fractionation (the fractionation of an extract is not desirable since it
increases the number of analyses).
2.6 STABILITY OF PESTICIDE SOLUTIONS
A study intended to identify the magnitude of solvent evaporation under
normal laboratory conditions was reported by Hodgson (148 and 149). The
factors studied include choice of solvent, type of container and closure,
solution volume, and storage temperature. Long-term chemical stability of
dilute standards was assessed under four storage conditions: freezer at
-15°C; refrigerator at 3°C; ambient temperature in the dark, and ambient
temperature on the bench top exposed to fluorescent and natural light. The
conclusions of this study are summarized below.
° The evaporation rates of solvents from 10-mL glass-stopper
volumetric flasks at ambient temperatures showed a direct
relationship with the boiling point temperatures and with the ratios
of vapor pressures to surface tensions. Table A-40 lists the
experimental values for the evaporation rate and the ratios of vapor
pressures to surface tensions. Isooctane and toluene are recommended
as solvents for the organochlorine pesticides.
168
-------
TABLE A-33. TYPE OF REACTIONS USED FOR THE CONFIRMATION OF ORGANOCHLORINE
PESTICIDES BY CHEMICAL DERIVATIZATION — GC TECHNIQUES
Type of reaction
Reagent
Pesticides
Dehydrochlori nati on
to olefins
Reductive dechlorination
Addition
Epoxide rearrangement
Oxidation
Intramolecular Rx
(base-catalyzed)
Intramolecular Rx
(acid-catalyzed)
Cleavage of sulfite
moiety
KOH or NaOH
t-BuOK
CrCl2
Bromination
Chi orination
t-BuOCl/t-BuOH
t-BuOCl/AcOH
Peracids
H2S04
H2S04/Ac20
BCl3/2-Chloro-
ethanol
HCl/ZnCl2
Cr03
KOH/ROH
t-BuOK
H2S04/A1203 (on
SM reaction)
H2S04 in solution
H2S04/A1203 (SM
reaction)
LiAlH4 followed
by silylation or
acetylation
Ac20/H2S04 (in
solution)
Ac20/H2S04/Al203
(SM reaction)
DDT and analogs
DDT, analogs, and
chlordanes
Heptachlor + endrin
Aldri n
Aldrin
Aldrin
Aldrin
Aldrin
heptachlor
heptachlor
chlordene
Endrin + dieldrin
Endrin + dieldrin
Endrin + dieldrin
Endrin + dieldrin
DDE + endrin +
heptachlor
alpha- + beta-endosulfans,
Endrin cyclic ketones
(degradation products
of endrin)
Endosulfans
Endrin
Endosulfans
Endosulfans
Endosulfans
169
-------
TABLE A-34. APPLICATIONS OF ALKALI DEHYDROCHLORI NATION FOR DDT AND ANALOGS
Compounds
Reagents
Reaction conditions
Known
Interference Reference
4,4-'DOT
4,4'-DDT, 2,4'-DDT, 4,4'-DDD,
perthane
4,4'-DDT and ODD, 2,4'-DDT
4, 4 '-ODD and DDT, methoxychlor
4,4'-DDT + ODD, 2,4'-DDT
2 percent NaOH 1n
ethanol (10 mL)
2 percent NaOH in
ethanol (10 mL)
0.5 N alcoholic KOH,
(0.5 ml)
K2C03 solid
2 percent KOH 1n
Reflux, 20 min
Reflux, 20 m1n
Room temperature, 5 min
Flash heater (240°C),
RxGLC
77°C, 15 min
Chlordanes
Chlordanes
Chlordanes
Chlordanes
Chlordanes
114
115
103
116
82
4,4'-and 2,4'-DDT
4,4'-DDT, 2,4'-DDT, 4,4'-DOD,
methoxychlor
4,4'-OOT, 2,4'-OOT, 4,4'-OOD,
methoxychlor, perthane
4,4'-DDT, 2,4'-DDT, 4,4'-DDD,
methoxychlor
4,4'-DDT and ODD, Chlordanes
2,4'- and 4,4'-DOT
2,4'-DDT; 2,4'-ODD perthane,
methoxychlor
4,4'- and 2,4'-DDT, ODD,
perthane, methoxychlor
(and other pesticides)
4,4'- and 2,4'-DDT, DOD
perthane, methoxychlor
DDT, DOD isomers, perthane,
methoxychlor, Chlordanes
95 percent ethanol
5 ml
5 percent NaOH in ? ml
Anhydrous NaOMe
NaOH + KOH on Gas
Chrom Q
2 percent KOH in
95 percent ethanol
t-BuOK/t-BuOH
200 rag/2 mL
NaOH, pH 8
t-BuOK/t-BuOH
200 mg/2 ml
2 percent KOH in
95 percent ethanol
2mL
2 percent KOH in
ethanol (1 mL)
A1203/KOH 200 g/20 g
AloOs/t-BuOK 100 g/
20 g
Reflux, 30 min Chlordanes 53
Shake with 1 mL sample Chlordanes 117
extract in hexane
(1 min or 20 min) at
room temperature
Alkaline pre-column Chlordanes 118
(200°C, ?) RxGLC
80°C, 12 min Chlordanes 119
Reflux (82 to 85°C) — 120,121
30 min
30°C, 10 m1n Chlordanes 122
50°C or reflux 30 min — 123
Reflux at 100°C 15 min Chlordanes 124
Reflux and heat Chlordanes . 125
(15 min) to 0.2 mL
Room temperature or -- 126
75°C, 1 hr and 3 hr
75°C, 1 hr Chlordanes 126
170
-------
TABLE A-35. SUMMARY OF CHEMICAL CONFIRMATION PROCEDURES FOR ENDRIN AND
OIELDRIN
Compounds
Reagents
Reaction conditions
Known
Interference Reference
EndMn, dleldrln
EndMn
Endrln, dfeldrln
Endrln
0.5 ml (1:2, HBr Ac20)
4 mL (15 g Cr03 in
50 mL HOAc)
0.5 mL (0.1 g ZnCl2
2 mL HC1, 25 mL abs.
EtOH)
0.5 mL H2S04
room temperature, 30 m1n
77°C, 12 min
100°C, 10 min
room temperature, 10 to
15 m1n
01e1dr1n 1n
some GC
column
103
82
127
121
Endrln, dleldrln
Endrln (heptachlor)
Endrln
Endrln (heptachlor)
Two endrln photode-
gradation products
Endrln dleldrln
Endrln, dleldrin
(endosulfans,
heptachlor epoxlde)
H2S04/HOAc
Acidic CrCl2 sol. in
acetone
A1203/H2SOA/H20
(100 g/10 mL/5 mL)
A1203/H2S04/H20
(50 g/10 mL/5 mL)
t-BuOK/t-BuOH followed
by silylation or
acetylatlon
BCl3/2-Cl£tOH
HCl/Ac20 (20 mL/10 mL)
100°C, 30 m1n
55 to 60°C, 45 min
room temperature, 1-1/2 hr
95 to 100°C, 1-1/2 hr
65 to 70°C, 1-1/2 hr
90°C, 2 hr
90°C, 10 min (endrln only)
room temperature, 30 min
128
129
130,131
132
133
134
135
TABLE A-36. SUMMARY OF CHEMICAL CONFIRMATION PROCEDURES FOR ALDRIN
Compounds
Reagents
Reaction conditions
Known
Interference Reference
Aldrin C12/CHC13 Room temperature, 5 min — 103
Aldrin, (heptachlor, CrOi/HOAc 77°C, 12 min Oieldrin 82
DDE)
Aldrin Peracetic acid Reflux, 3 hr Dieldrin 136
Aldrin m-chloroperbenzoic add/ 43 to 45°C, 1-1/2 hr Dleldrln 137,138
hexane
Aldrin Monoterephthallc acid/ Room temperature, 20 min Oieldrin 120
hexane
Aldrin, (heptachlor) t-BuOCl/HOAc 100°C, 10 min — 121
Aldrin Chloroacetlc anhydride/ 56 to 60°C, 2 hr Not 139
HBr investigated
171
-------
TABLE A-37.
SUMMARY OF CHEMICAL CONFIRMATION PROCEDURES FOR THE CHLOROANE
GROUP
ro
Compounds
Reagents
Reaction conditions
Known
Interference
Reference
Heptachlor epoxide
Heptachlor, (Aldrln, DDE)
Heptachlor
Heptachlor
Heptachlor, heptachlor
epoxide, c1s- and
trans-chlordane
(DDT + DDD isomers)
Heptachlor (and endrln)
Heptachlor, heptachlor
epoxide, els- and
trans-chlordane
(DDT + DDD Isomers,
perthane, methoxycnlor)
Heptachlor, endosulfans,
endrln
Heptachlor, endrln
Heptachlor, heptachlor
epoxide, endrln,
dieldrin, endosulfans
Heptachlor, heptachlor
epoxide ,
0.5 ml HBr/Ac20
Cr03/HOAc
AgOAc/HOAc
Ag2C03/50 percent aq.
EtOH, then
sllylation or
acetylatfon
t-BuOK/t-BuOH, then
sllylation or
acetylatlon
CrCl2 aq. acidic sd.
Al203/t-BuOK
24
(50 g/10 ml)
A1203/H2S04/H20
(50 g/10 mL/5 mL)
0.5 ml HCl/Ac20
UV In petroleum ether
(traces of MeOH)
120°C, 30 m1n OP room
temperature, 30 mln
77°C, 12 rain
Reflux, 30 m1n
Reflux, 45 m1n
Reflux, 30 mln
55 to 60°C, 45 mln
70 to 75°C, 1 hr or
room temperature
overnight
100°C, 1-1/2 hr
95 to 100°C, 1-1/2 hr
Room temperature,
30 mln
30 mln
2- and 3-chloro-
chlordene, Nonachlor
Chlordene
2- and 3-chloro-
chlordene
Heptachlor + octachlor
epoxide derivatives
on some column; UV
derivative of
heptachlor epoxide
and parent epoxide
separable on OV-101
column
103
82
140
140
120,121
129
126
141
132
Not fully Investigated 135
142
-------
TABLE A-38. CHEMICAL CONFIRMATION OF BHC ISOMERS
Compounds
BHC isomers
(all)
BHC Isomers
(alpha, beta, gamma)
Reagents
2 percent KOH 1n
ethanol
0.1 g NaOMe in
MeOH (2 ml)
Reaction conditions
100CC, 15 m1n,
GC column: 200°C
50°C, 15 min,
GC column: 200 °C
Known
Interference
BHC Isomers mutually
interfere
BHC Isomers mutually
interfere
Reference
124,125
143
TABLE A-39. SUMMARY OF CHEMICAL CONFIRMATION PROCEDURES FOR ENDOSULFANS
Compounds
alpha- and
beta-endosulfan
alpha- and
beta-endosulfan,
(heptachlor)
alpha- and
beta-endosulfan
(heptachlor epoxide,
endMn, dieldrln)
Reagents
L1A1H4/THF, then
sllylatlon or
acetylation
Ac20/HOAc/trace of
H2S04
2 percent KOH in EtOH
Al203/Ac20/H2S04
(50 g/IO mt/5 ml)
AL203/H2S04
(50 g/10 mL)
AL203/H2S04
(50 g/10 mL)
0.5 mL HCl/Ac20
Reaction
conditions
60°C, 1 hr
100"C, 3/4 hr
Reflux, 12 min
100 ±5"C, 2 hr
95°C, 1 hr
1008C, 1-1/2 hr
Room temperature
30 min
Known
Interference Reference
144
--
144
Not Investigated 145
146
147
141
Not investigated 135
173
-------
TABLE A-40. EVAPORATION RATES FROM 10-mL GLASS-STOPPER
VOLUMETRIC FLASKS AND VAPOR PRESSURE/SURFACE
TENSION RATIOS FOR SOME COMMON ORGANIC
SOLVENTS3
Solvent
Vapor pressure/
surface tension
ratio, torr
(cm/dyne)
Evaporation
rate
(mL/wk ± SD)
Pentane
Ethyl ether
Methyl ene chloride
Hexane
Acetone
Chloroform
Methanol
Ethyl acetate
Acetonitrile
Benzene
Ethanol
Isooctane
Heptane
Isopropanol
Toluene
n-Decane
33.1
32.4
16.0
8.45
8.01
7.32
5.65
3.97
3.79
3.38
2.73
2.68
2.31
2.11
1.02
0.04
— _
0.63 ± 0.17
0.25 ± 0.10
0.15 ± 0.07
0.22 ± 0.01
—
0.076 ± 0.17
—
—
0.096 ± 0.0013
—
0.058 ± 0.022
—
—
0.045 ± 0.0021
—
=======================================
aData taken from Ref. 148.
The wrapping of Teflon tape on the outside of the joint between the
glass stoppers and the neck of the volumetric flask did not reduce
the evaporation rate.
The average evaporation loss for 10-mL glass-stopper volumetric
flasks filled with hexane and stored at ambient temperature, +3°C,
and -15°C were 0.15, 0.06, and 0.02 mL/week, respectively.
The evaporation rates of hexane and isooctane from prescription
bottles were the lowest when compared to volumetric flasks,
multivials, septum bottles, and small vials. Caps with a hard-liner
backing underneath the Teflon liner gave the best seal. Figure A-16
gives the comparative evaporation rates of hexane and isooctane
from different containers at ambient temperature.
Solutions should be stored in a refrigerator or freezer to reduce
solvent losses.
Containers should be sealed tightly to reduce evaporation from
around the cap seal.
174
-------
en
Volumetric flames
Hexane
Iso-octane
Prescription bottles
Hexane
u . . . . , Iso-octane
Multivial
Hexane with ampule glass in
neck
Hexane without ampule glass
in neck
Serum bottle
Hexane without punctured
septum
Hexane with punctured septum
Small vials
Hexane
Iso-octane
(C
IT
u:
9
u:
L, . I .1 j
i nil
H 1
1
1
1
u i i
1 — r* i i i i i i i i i i i i i i i i i
0.01 0.03 0.05 0.07 0.09 0.11 0.13 0.15 0.17 0.
Evaporation rate (mL/wk)
Figure A-16.
Comparative evaporation rates (mL/wk) of hexane and
isooctane from different containers at ami bent temperature.
The range of data for each container is plotted. Veritical
lines represent different container volumes (i.e.,
volumetric flasks and prescription bottles) or different
container/cap configurations (i.e., small vials). Data
taken from Ref. 148.
-------
« Decomposition of less than 10 percent was reported for 24
organochlorine pesticides and PCBs at four different storage
conditions and for a 2-year period (Table A-41). Increase in
compound concentration after a period of 10 months was attributed
to solvent evaporation.
The authors also make the following recommendations for quality control
practices:
° Primary reference standards should be stored in a desiccated
container in the freezer.
° Concentrated stock solutions should be made fresh every year and
should be stored in a freezer.
8 The working solutions should be made fresh every 6 months (by
dilution of stock solutions) and should be stored in a refrigerator
when not in use. They should be replaced sooner if evaporation is
evident or suspected.
176
-------
TABLE A-41. ORGANOCHLORINE-IN-ISOOCTANE SOLUTIONS AND 2-YEAR STABILITY TEST
RESULTS*
Degradation at storage condition0
Compound
Concentration
Soln.b (pg/uL)
Freezer
-15°C
Refrigerator
+3°C
Dark
24°C
Light
24'C
Aldrin B
PCB-1016 E
PCB-1254 F
beta-BHC C
gamma-BHC (lindane) C
Chlordane (tech) G
Chlordeconed K
Chlordene D
DCPA B
4,4'-DDD B
4,4'-DDE A
2,4'-DDT A
4,4'-DDT A
Dieldrin B
Endosulfan I C
Endosulfan II C
Endrin C
HCB C
Heptachlor A
Heptachlor epoxide A
1-Hydroxychlordene D
Methoxychlor F
Mi rex A
trans-Nonachlor D
Oxychlordane D
PCNB A
Toxaphene H
9.9
100
200
16.2
6.0
80.4
19.5
5.3
15,
30,
22.0
36.5
47.8
20.6
26.0
30.0
50.0
5.0
8.9
15.0
15.8
71.4
48.3
14.0
13.7
7.8
452
aData taken from Ref. 149.
^Components of pesticide mixtures.
cStable (+) or unstable (-).
^Solvent 1 percent methanol - benzene.
177
-------
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124. Krause, R. T. Quantitative Dehydrochlorination of Perthane Residues
and Effect of Alcoholic Potassium Hydroxide on Other Pesticides.
J. Assoc. Off. Anal. Chem. 55: 1042 (1972).
125. Young, S. J. V., and Burke, J. A. Micro Scale Alkali Treatment for Use
in Pesticide Residue Confirmation and Sample Cleanup. Bull. Environ.
Cont. Toxicol. 7: 160 (1972).
126. Chau, A., S. Y., and Lanouette, M. Confirmation of Pesticide Residue
Identity. II. Derivative Formation in Solid Matrix for the
Confirmation of DDT, ODD, Methoxychlor, Perthane-,'Cis- and
Transchlordane, Heptachlor and Heptachlor Epoxide Pesticide
Residues by Gas Chromtography. J. Assoc. Off. Anal. Chem. 55: 1058
(1972).
127. Wiencke, W. W., and Burke, J. A. Derivatization of Dieldrin and Endrin
for Confirmation of Residue Identity. J. Assoc. Off. Anal. Chem. 52:
1277 (1969).
128. Chau, A. S. Y. Some Reactions of CrCl2 on Organochlorine Pesticides
and Their Utilization in Chemical Confirmative Tests. Presented to the
Eastern Canada Seminar on Pesticide Residue Analysis. Ottawa, Ontario
(May, 1970).
188
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129. Chau, A. S. Y., and Cochrane, W. P. Chromous Chloride Reduction. VI.
Derivative Formation for the Simultaneous Identification of Heptachlor
and Endrin Pesticide Residues by Gas Chromatography. J. Assoc. Off.
Anal. Chem. 54: 1124 (1971).
130. Chau, A. S. Y. Confirmation of Pesticide Residue Identity. III.
Derivative Formation in Solid Matrix for the Confirmation of Endrin
by Gas Chromatography. Bull. Environ. Cont. Toxicol. 8: 169
(1972).
131. Chau, A. S. Y. Solid Matrix Derivatization Technique. I.
Derivative Formation in Solid Matrix for the Confirmation of Endrin
by Gas Chromatography. In: Proc. 7th Pesticide Residue Analysis
Seminar (Western Canada), Department of Agriculture, Saskatoon
(1972).
132. Chau, A. S. Y. Confirmation of Pesticide Residue Identity. VII. Solid
Matrix Derivation Procedure for the Simultaneous Confirmation of
Endrin and Heptachlor Residues in the Presence of Large Quantities
of Polychlorinated Biphenyls. J. Assoc. Off. Anal. Chem. 57: 585
(1974).
133. Chau, A. S. Y. Confirmation of Pesticide Residue Identity. I.
Derivative Formation for the Confirmation of Photo Products of Endrin,
Hexachloro and Pentachloro-ketone Pesticide Residues by Gas
Chromatography. J. Assoc. Off. Anal. Chem. 55: 519 (1972).
134. Woodham, D. W., Loftis, C. D., and Collier, C. W. Identification of
the Gas Chromatographic Dieldrin and Endrin Peaks by Chemical
Conversion. J. Agric. Food Chem. 20: 163 (1972).
135. Musial, C. J., Peach, M. E., and Stiles, D. A. A Simple Procedure for
the Confirmation of Residues of ct- and B-Endosulfan, Dieldrin, Endrin,
and Heptachlor Epoxide. Bull. Environ. Cont. Toxicol. 16: 98 (1976).
136. Noren, K. Determination of Aldrin Residues in Vegetables by the
Chemical Conversion of Aldrin to Dieldrin. Analyst (London) 93: 39
(1968).
137. Osadchuk, M., and Romach, M. Problems in Interfering Responses in the
Identification and Quantisation of Pesticide Residues. Presented to
the Eastern Canada Seminar on Pesticide Residue Analysis, Guelph,
Ontario, 1968; McLeod, H., Ed. Analytical Methods for Pesticide
Residue in Foods. Food and Drug Directorate, Ottawa, Ontario, Canada.
138. Osadchuk, M., and Wanless, E. B. Identification and Quantitative
Estimation of Aldrin Residues in the Presence of Interfering Materials
in Samples of Plant and Animal Origin. J. Assoc. Off. Anal. Chem. 51:
1264 (1968).
189
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139. Chau, A. S. Y., and Wilkinson, R..0. Unpublished results (1970).
140. Cochrane, W. P., and Chau, A. S. Y. Note on Gas Chromatographic
Identification of Heptachlor Pesticide Residues by Derivative
Formation. J. Assoc. Off. Anal. Chem. 51: 1267 (1968).
141. Chau, A. S. Y., and Terry, K. Confirmation of Pesticide Residue
Identity. VI. Derivative Formation in Solid Matrix for Confirmation of
Heptachlor and Endosulfan Isomers. J. Assoc. Off. Anal. Chem. 57:
395 (1974).
142. Ward, P. M. Confirming Heptachlor and Heptachlor Epoxide in Food
Samples by Gas-Liquid Chromatography of Their Photoderivatives.
J. Assoc. Off. Anal. Chem. 60: 673 (1977).
143. Cochrane, W. P., and Maybury, R. B. Chemical Confirmation of BHC
Isomers: Comparison of Alkaline Reactions in Solution and by Gas
Chromatographic Pre-Column. J. Assoc. Off. Anal. Chem. 56: 1324
(1973).
144. Chau, A. S. Y. Derivative Formation for the Confirmation of
Endosulfan by Gas Chromatography. J. Assoc. Off. Anal. Chem. 52:
1240 (1969).
145. Greve, P. A., and Wit, S. L. Rapid Identification Method for
Endosulfan from GLC Peak Shifts Under the Influence of Alkali.
J. Agric. Food Chem. 19: 372 (1971).
146. Chau, A. S. Y., and Terry, K. Confirmation of Pesticide Residue
Identity, IV. Derivative Formation in Solid Matrix for the Confirmation
of a- and 0-Endosulfan by Gas Chromatography. J. Assoc. Off. Anal.
Chem. 55: 1228 (1972).
147. Chau, A. S. Y. Confirmation of Pesticide Residue Identity. V.
Alternative Procedure for Derivative Formation in Solid Matrix for the
Confirmation of a- and e-Endosulfan by Gas Chromatography. J. Assoc.
Off. Anal. Chem. 55: 1232 (1972).
148. Hodgson, D. W., and Watts, R. R. Accuracy of Pesticide Reference
Standard Solutions. Part I. Factors Affecting Organic Solvent
Evaporation. J. Assoc. Off. Anal. Chem. 65: 89 (1982).
149. Hodgson, D. W., Thompson, J. F., and Watts, R. R. Accuracy of Pesticide
Reference Standard Solutions. Part II. Chemical Stability Under Four
Storage Conditions. J. Assoc. Off. Anal. Chem. 65: 94 (1982).
190
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APPENDIX B
METHOD 8080*
ORGANOCHLORINE PESTICIDES AND PCBs
1. Scope and Application
1.1 This method provides procedures for the determination of certain
organochlorine pesticides and polychlorinated biphenyls (PCBs) in liquid and
solid sample matrices. The following substances can be determined by this
method:
Parameter
alpha-BHC
beta-BHC
gamma-BHC (Lindane)
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-ODD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
Storet No. CAS No.
39337
39338
39340
34259
39410
39330
39420
39350
34361
39320
39380
39390
34356
39310
34366
39018
34351
39300
NA
39400
34671
39488
39492
39496
39500
39504
39508
319-84-6
319-85-7
58-89-9
319-86-8
76-44-8
309-00-2
1024-57-3
57-74-9
959-98-8
72-55-9
60-57-1
72-20-8
33212-65-9
72-54-8
7421-93-4
143-50-0
1031-07-8
50-29-3
72-43-5
8001-35-2
12674-11-2
1104-28-2
11141-16-5
53469-21-9
12672-29-6
11097-69-1
11096-82-5
NA — Storet number not available at the time of protocol preparation.
*Revised May 1986
191
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1.2 This is a gas chromatographic (GO method applicable to the
determination of the substances listed above. When this method is used to
analyze samples for any or all of these substances, compound identification
should be supported by at least one additional qualitative technique. This
method describes analytical conditions for a second gas chromatographic
column that can be used to confirm the measurements made with the primary
column. Retention time information obtained on the two gas chromatographic
columns is given in Table B-l.
1.3 The sample extraction and concentration steps in this method are
those specified in Methods 3510 and 3520 for liquid matrices, and 3540 and
3550 for solid matrices.
1.4 Limitations: When both organochlorine pesticides and PCBs are
present, fractionation of the extract by using silica gel chromatography is
recommended. Toxaphene and chlordane, which are both multicomponent
mixtures, cannot be satisfactorily separated by using silica gel chromatography
and, therefore, may interfere with the quantification of certain organochlorine
pesticides.
1.5 The method detection limit (MDL) for each parameter is listed in
Table B-l. The MDLs for the components of a specific sample may differ from
those listed in Table B-l because the MDLs are dependent upon the nature of
interferences in the sample matrix.
1.6 This method is restricted to use by or under the supervision of
analysts experienced in the use of a gas chromatograph and in the
interpretation of gas chromatograms.
2. Summary of Method
2.1 A measured volume or weight of sample (100 mL to 1 L for liquids,
10 g for solids) is extracted by using the appropriate sample extraction
technique specified in Methods 3510, 3520, 3540, and 3550. Liquid samples
are extracted at neutral pH with methylene chloride by using either a
separatory funnel (Method 3510) or a continuous liquid-liquid extractor
(Method 3520). Solid samples are extracted with hexarre-acetone (1:1) by
using either Soxhlet extraction (Method 3540) or sonication (Method 3550).
After cleanup, the extract is analyzed by gas chromatography with electron
capture detection.
2.2 The method provides a silica gel column fractionation procedure and
an elemental sulfur removal procedure to aid in the elimination of
interferences that may be encountered. A procedure for gel permeation
chromatography cleanup is also provided for samples that contain high
amounts of liquids and waxes.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing hardware
may introduce discrete artifacts which result in elevated baselines causing
192
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TABLE B-l. GAS CHROMATOGRAPHIC RETENTION TIMES AND METHOD DETECTION
LIMITS FOR THE ORGANOCHLORINE PESTICIDES AND PCBs
Retention Time
(min)
Compound
alpha-BHC
beta-BHC
gamma -8HC (Lindane)
delta -BHC
Heptachlor
Aldrin
Heptachlor epoxlde
gamma -Chi ordane
Endosulfan I
4, 4 '-ODE
DleldHn
Endrln
Endosulfan II
4, 4 '-ODD
Endrln aldehyde
Kepone
Endosulfan sulfate
4, 4 '-DOT
4,4'-Methoxychlor
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
DB-5<»
12.29
13.13
13.37
14.14
15.91
17.16
18.60
19.48
19.94
20.83
20.91
21.71
22.05
22.38
22.75
23.01
23.64
23.79
25.94
mr
mr
mr
mr
mr
mr
mr
mr
SPB-6086
9.46
11.33
10.97
12.73
12.46
13.76
15.98
16.70
17.40
18.36
18.60
19.96
20.69
20.53
21.90
0
22.54
21.72
24.90
mr
mr
mr
mr
mr
mr
mr
mr
MOLC
Liquid
(ug/L)
0.035
0.023
0.025
0.024
0.040
0.034
0.032
0.037
0.030
0.058
0.044
0.039
0.040
0.050
0.050
NO
0.035
0.081
0.086
NA
0.54
NA
NA
NA
NA
NA
0.90
Solid
(ug/Kg)
1.9
3.3
2.0
l.l
2.0
2.2
2.1
1.5
2.1
2.5
I
3.6
2.4
4.2
1.6
NO
3.6
3.6
5.7
NA
57
NA
NA
NA
NA
NA
70
Liquid: reagent water; Solid: sandy loam soil.
mr — multiple peak response.
D — degrades on column.
NA — data not available.
NO ~ not detected at 0.1 ug/L 1n liquid matrix
or 10 ug/kg 1n solid matrix.
I — unable to determine due to Interference.
aTemperature program: 100°C (hold 2 min) to 160°C at
15°C/m1n, then at 5°C/m1n to 270°C; helium at 16 ps1.
"Temperature program: 160°C (hold 2 m1n) to 290°C
(hold 1 min) at 5°C/m1n; nitrogen at 20 psi.
CMDL is the method detection limit. MDL was determined
from the analysis of seven replicate aliquots of each
matrix processed through the entire analytical method
(extraction, silica gel cleanup, and GC/EC analysis).
MDL » t/n.i 0,99) x sn where t/n_i n 99) is the
student s t value appropriate for a'99$ confidence
interval and a standard deviation with n-1 degrees of
freedom, and SO is the standard deviation of the seven
replicate measurements.
193
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misinterpretation of gas chromatograms. These materials must therefore be
demonstrated to be free from interferences under the conditions of the
analysis by running method blanks. Specific selection of reagents and
purification of solvents by distillation in all-glass systems may be
required. Pesticide-grade or distilled-in-glass solvents are suitable for
pesticide residue analysis without further purification. Each new batch of
solvents should be checked for possible interferences as follows:
concentrate to 1 mL the amount of solvent equivalent to the total volume to
be used in the analysis. Inject 1 to 2 uL of the concentrate into a gas
chromatograph equipped with an electron capture detector set at the lowest
attenuation. If extraneous peaks are detected that are greater than 10 pg
on-column, the solvent must be purified either by redistilling or by passing
it through a column of highly activated alumina (acidic or basic alumina,
activated at 300° to 400°C) or Florisil.
3.2 Interferences coextracted from the samples will vary considerably
from waste to waste. While general cleanup techniques are provided as part
of this method, unique samples may require additional cleanup approaches to
achieve desired sensitivities.
3.3 Glassware must be scrupulously clean. Clean all glassware as soon
as possible after use by rinsing with the last solvent used, followed by
thorough washing of the glassware in hot, detergent-containing water. Rinse
with tap water, distilled water, acetone, and finally pesticide-quality
hexane. Heavily contaminated glassware may require treatment in a muffle
furnace at 400°C for 15 to 30 min. Some high-boiling materials, such as
PCBs, may not be eliminated by this treatment. Volumetric glassware should
not be heated in a muffle furnace. Glassware should be sealed and stored in
a clean environment immediately after drying or cooling to prevent any
accumulation of dust or other contaminants. Store the glassware by inverting
or by capping with aluminum foil.
A chromic acid wash is not satisfactory for the cleaning of glassware
contaminated with organochlorine pesticides, most of the organochlorine
pesticides are stable when exposed to chromic acid at room temperature for
many days. Some pesticides such as heptachlor and aldrin react with chromic
acid to form heptachlor epoxide and dieldrin.
3.4 Interferences by phthalate esters can pose a major problem in
pesticide analysis. These materials elute in Fractions III and IV of the
silica gel cleanup procedure. Their presence can usually be minimized by
avoiding contact with any plastic materials.
3.5 The presence of elemental sulfur will result in large peaks and
often mask the region from the solvent peak to the aldrin peak in the gas
chromatogram. Several cleanup techniques for removal of sulfur have been
reported (i.e., metallic mercury (2), activated copper (3), activated Raney
Nickel (4), and tetrabutyl-ammonium sulfite (TBA) (5). The TBA procedure
works well for the removal of elemental sulfur; for most organochlorine
pesticides and PCBs, almost quantitative recoveries were obtained (Table B-2).
Recoveries from actual samples may be lower. Since the recovery of endrin
194
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TABLE B-2. RECOVERIES OF THE ORGANOCHLORINE PESTICIDES AND PCBs
USING THE TBA PROCEDURE (STANDARDS ONLY, IN HEXANE)
Compound
Percent Recovery
Without sulfur
addeda
With sulfur
addedb
alpha-BHC
beta-BHC
gamma-BHC (Lindane)
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
gamma-Chlordane
Endosulfan I
4,4'-DDE and Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDT
4,4'-Methoxychlor
Toxaphene
PCB-1016
PCB-1260
110
106
112
100
103
104
107
98
108
91
87
99
103
10
107
94
86
85
93
108
87
103
99
108
86
97
99
101
100
106
103
89
99
106
8
63
93
90
68
83
88
68
aThe values given are the averages of two determinations.
bSingle determination.
aldehyde is drastically reduced, this compound must be determined prior to
sulfur cleanup.
3.6 Waxes and lipids can be removed by gel permeation chromatography
(6). Extracts containing high amounts of lipids are viscous and may even
solidify at room temperature.
4. Safety
4.1 The toxicity and carcinogenicity of most of the compounds to be
determined by this method have not been precisely defined; therefore, each
compound should be treated as a potential health hazard, and exposure of
personnel to these compounds must be reduced to the lowest possible level. The
195
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laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should also be made
available to all personnel involved in chemical analysis.
4.2 Primary standards of pesticides and PCBs should be prepared in a
hood. A NIOSH/MESA-approved toxic gas respirator should be worn when the
analyst handles high concentrations of these toxic compounds.
5. Apparatus and Materials
5.1 Glassware (see also Methods 3510, 3520, 3540 and 3550 specifications)
5.1.1 Drying column: chromatographic column, approximately 150 mm
long x 25 mm ID, with coarse frit filter disc.
5.1.2 Kuderna-Danish (K-D) apparatus
5.1.2.1 Concentrator tube: 10 ml, graduated. Calibration
must be checked at the 1.0- and 10.0-mL level. Ground glass stopper (size
19/22 joint) is used to prevent evaporation of extracts.
5.1.2.2 Evaporative flask: 50 ml. Attach to concentrator
tube with springs.
5.1.2.3 Snyder column: Three-ball macro (Kontes K503000-
0121 or equivalent).
5.1.2.4 Boiling chips: Approximately 10/40 mesh. Heat to
400°C for 30 min or Soxhlet-extract with methylene chloride.
5.2 Water bath: heated, with concentric ring cover, capable of
temperature control (±2°C). The bath should be used in a hood.
5.3 Gel permeation chromatography (GPC) cleanup device
5.3.1 Automated system
5.3.1.1 Gel permeation chromatograph, Analytical Biochemical
Labs, Inc., GPC Autoprep 1002, or equivalent, including:
5.3.1.2 25 mm ID by 600-700 mm glass column packed with 70 g
Bio-Beads SX-3.
5.3.1.3 Syringe, 10 ml with luer lok fitting.
5.3.1.4 Syringe filter holder and filters — stainless steel
and TFE, Gelman 4310, or equivalent.
5.3.2 Manual system assembled from parts (6)
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5.3.2.1 24 mm ID by 600-700 mm heavy-wall glass column
packed with 70g Bio-Beads SX-3.
5.3.2.2 Pump: Altex Scientific, Model No. 1001A,
semi preparative, solvent metering system. Pump capacity: 28 mL/min.
5.3.2.3 Detector: Altex Scientific, Model No. 153, with
254 nm UV source and 8-uL semi preparative flowcell (2-mm pathlengths).
5.3.2.4 Microprocessor/controller: Altex Scientific,
Model No. 420, Microprocessor System Controller, with extended memory.
5.3.2.5 Injector: Altex Scientific, Catalog No. 201-56,
sample injection valve, Tefzel, with 10-:mL sample loop.
5.3.2.6 Recorder: Linear Instruments, Model No. 385,
10-in. recorder.
5.3.2.7 Effluent Switching Valve: Teflon slider valve,
3-way with 0.060 in. ports.
5.3.2.8 Supplemental Pressure Gauge with Connecting Tee:
U.S. Gauge, 0 to 200 psi, stainless steel. Installed as a "downstream"
monitoring device between column and detector. Flowrate was typically
5 mL/min of methylene chloride. Recorder chart speed was 0.50 cm/min.
5.4 Gas chromatograph: An analytical system complete with gas
chromatograph suitable for on-column and split/splitless injection, and all
required accessories including syringes, analytical columns, gases, electron
capture detector, and recorder/integrator or data system.
5.4.1 Column 1: 30 m x 0.25 or 0.32 mm ID fused-silica capillary
column chemically bonded with SE-54 (DB-5 or equivalent).
5.4.2 Column 2: 30 m x 0.25 mm ID fused-silica capillary column
SPB-608 (Supelco, Inc.), 0.25 pm coating thickness.
5.5 Chromatographic column for silic gel: 200 mm x 11 mm ID, glass
column.
6. Reagents
6.1 Reagent water: Reagent water is defined as water in which an
interferent is not observed at the MDL of the parameters of interest.
6.2 Preservatives
6.2.1 Sodium hydroxide (ACS): 10 N in distilled water.
6.2.2 Sulfuric acid (ACS) (1 + 1): mix equal volumes of
concentrated sulfuric acid with distilled water.
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6.3 Acetone, hexane, isooctane, methylene chloride: pesticide quality
or equivalent.
6.4 Ethyl acetate, methanol, 2-propanol: nanograde.
6.5 Sodium sulfate (ACS): granular, anhydrous. Purify by heating at
400°C for 4 hours in a shallow tray.
6.6 Silica gel PR grade (100/200 mesh): before use, activate at least
16 hours at 130° to 140°C. Deactivate with water (3.3 percent by weight).
Equilibrate for 1 hour after addition of the water.
6.7 Tetrabutyl ammonium sulfite reagent: a solution of 3.39 g
(0.01 mol) tetrabutyl ammonium hydrogen sulfate in 100 ml of water is
extracted with three 20-ml portions of hexane (to remove impurities), and
then 25 g anhydrous sodium sulfite (analytical grade) are added.
6.8 Stock standard solutions (1.0 ug/uL): stock standard solutions can
be prepared from pure standard materials or can be purchased as certified
solutions. The NBS Office of Standard Reference Materials developed the
Standard Reference Material 1583, chlorinated pesticides in
2,2,4-trimethylpentane certified for five chlorinated pesticides: gamma-BHC,
delta-BHC, aldrin, 4,4'-DDE, and 4,4'-DDT. An uncertified concentration is
provided for heptachlor epoxide. The concentrations range from approximately
0.8 to 1.9 ug/g; the corresponding concentrations in ug/mL at 23°C are also
provided.
6.8.1 Prepare stock standard solutions by accurately weighing about
0.0100 g of pure compound. Dissolve the compound in isooctane and dilute to
volume in a 10-tnL volumetric flask. If compound purity is 96 percent or
greater, the weight can be used without correction to calculate the
concentration of the stock standard.
6.8.2 Beta-BHC, dieldrin, and Kepone are not adequately soluble in
isooctane; methanol, acetone, or benzene should therefore be used for the
preparation of the stock standard solutions of these compounds.
6.8.3 Transfer the stock standard solutions into sealed screw-cap
bottles or ground-glass-stoppered reagent bottles. Store at 4°C and protect
from light. Stock standard solutions should be checked frequently for signs
of degradation or evaporation especially just prior to preparing calibration
standards.
6.9 Corn Oil Solution: 200 mg/mL in methylene chloride.
7. Sample Collection, Preservation, and Handling
7.1 Grab samples must be collected in appropriately cleaned glass
containers, and the sampling bottles must not be prewashed with the sample
before collection. Composite samples should be collected in refrigerated
198
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glass containers in accordance with the requirements of the program.
Automatic sampling equipment must be free of tygon and other potential
sources of contamination.
7.2 Liquid samples must be iced or refrigerated from the time of
collection until extraction. Chemical preservatives should not be used in
the field unless more than 24 hours will elapse before delivery to the
laboratory. If the samples will not be extracted within 48 hours of
collection, the sample should be adjusted to a pH range of 6.0 to 8.0 with
sodium hydroxide or sulfuric acid.
7.3 Stability of the organochlorine pesticides and PCBs in soil has not
been systematically investigated. Storage of soil samples at room
temperature should be avoided since degradation of some organochlorine
pesticides has been reported to occur (7). Losses of lindane and aldrin,
after incubation of spiked soil samples at 25°C under aerobic conditions,
were noticeable 7 days after the initiation of the experiment, and only 35 to
40 percent of the two pesticides were found at day 30 of the experiment.
Deep-freezing at -10°C or -20°C appears to be the most suitable method for
storage of solid matrices since it has the widest range of application,
causes the least changes in the samples, and makes the addition of
preservatives unnecessary.
7.4 All samples must be extracted within 7 days of collection and must
be completely analyzed within 30 days of extraction.
8. Procedures
8.1 Sample Preparation
8.1.1 Extraction. Extract water samples at a neutral pH with
methylene chloride as a solvent by using a separatory funnel (Method 3510) or
a continuous liquid-liquid extractor (Method 3520). Extract solid samples
with hexane:acetone (1:1) by using either the Soxhlet extraction
(Method 3540) or sonication procedures (Method 3550).
8.1.2 Spiked samples are used to verify the "applicability of the
chosen extraction technique to each new sample type. Each sample must be
spiked with the compounds of interest to determine the percent recovery and
the limit of detection for that sample. Spiking of water samples should be
performed by adding appropriate amounts of pesticide and PCB compounds,
dissolved in methanol, to the water sample immediately prior to extraction.
After addition of the spike, mix the samples manually for 1 to 2 min. Typical
spiking levels for water samples are 1 to 10 ug/L for samples in which pesti-
cides and PCBs were not detected, and 2 to 5 times the background level in
those cases where pesticides and PCBs are present. Spiking of solid samples
should be performed by adding appropriate amounts of pesticides and PCB com-
pounds, which are dissolved in methanol, to the solid samples. The solid
sample should be wet prior to the addition of the spike (at least 20 percent
moisture) and should be mixed thoroughly with a glass rod to homogenize the
material. Allow the spike to equilibriate with the solid for 1 hour at room
temperature prior to extraction. Transfer the whole portion that was spiked
199
-------
with the test compounds to the extraction thimble for Soxhlet extraction
(Method 3540) or proceed with the sonication extraction in the case of Method
3550.
8.2 Cleanup/Fractionation
8.2.1 Cleanup procedures may not be necessary for a relatively
clean sample matrix. If PCBs are known to be present in the sample, then the
silica gel fractionation is recommended. Elemental sulfur, which may appear
in certain sediments and industrial wastes and which interferes with the
electron capture gas chromatography of certain pesticides, can be removed by
the technique described in Section 8.3.
8.2.2 Silica gel column cleanup/fractionation
8.2.2.1 Place a weight of silica gel (normally 20 g)
activated for at least 16 hours at 130°C into a glass jar and deactivate it
with reagent water to bring the moisture content to 3.3 percent. Mix the
contents of the glass jar thoroughly and equilibrate for 6 hours. Store the
deactivated silica gel in a sealed glass jar inside a desiccator. Transfer
a 3-g portion into an 11-mm ID glass column and top it with 2 to 3 cm of
anhydrous sodium sulfate.
8.2.2.2 Add 10 mL hexane to the top of the column to wet and
rinse the sodium sulfate and silica gel. Just prior to exposure of the
sodium sulfate layer to air, stop the hexane eluate flow by closing the
stopcock on the chromatographic column. Discard the eluate.
8.2.2.3 Transfer the sample extract (2 mL) onto the column.
Rinse the extract vial twice with 1 to 2 ml hexane and add each rinse to the
column. Elute the column with 80 ml hexane (Fraction I) at a rate of about
5 mL/min. Remove the collection flask and set it aside for later
concentration. Elute the column with 50 ml hexane (Fraction II) and collect
the eluate. Perform a third elution with 15 mL methylene chloride
(Fraction III) and a fourth one with 50 mL ethyl acetate (Fraction IV). The
elution patterns for the organochlorine pesticides and PCBs are shown in
Table B-3. If it is known that Kepone is not present," then Fractions III and
IV may be combined. This should be done only in those cases where matrix
coextractants do not interfere with the analysis of pesticides present in
Fraction III.
8.2.2.4 Concentrate the fractions as described in
Methods 3510, 3520, 3540, and 3550. Methylene chloride is exchanged for
hexane (Fraction III), and the ethyl acetate in Fraction IV is exchanged for
benzene-methanol (98:2). The exchange step is performed by adding 50 mL of
the solvent/solvent mixture specified above and by concentrating the extract
using a Kuderna-Danish apparatus. When the apparent volume of the liquid
reaches 1 mL, remove the Kuderna-Danish apparatus and allow it to drain and
cool for at least 10 min. Proceed with the nitrogen blowdown technique.
Losses of the more volatile organochlorine pesticides (lindane, heptachlor,
200
-------
TABLE B-3. DISTRIBUTION AND PERCENT RECOVERIES OF THE ORGANOCHLORINE
PESTICIDES AND PCBs IN THE SILICA GEL COLUMN FRACTIONS a,b,c,d,e
ro
O
Fraction I
Fraction II
Fraction III
Fraction IV
Total Recovery
Compound
alpha-BHCf
beta-BHC
gamma -BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor epoxide
Endosulfan I
4,4'-DDE
Oieldrln
Endrin
Endosulfan II
4,4'-DDDf
Endrin aldehyde
Kepone
Endosulfan sulfate
4,4'-DDTf
4,4'-Methoxychlor
PCB-1016
PCB-1260
Technical
chlordane
Toxaphene
Cone. 1
109 (4.1)
97 (5.6)
86 (5.4)
86 (4.0)
91 (4.1)
14 (5.5)
Cone. 2
118
104
94
87
95
22
(8.7)
(1.6)
(2.8)
(6.1)
(5.0)
(5.3)
Cone. 1
86 (13.4)
19 (6.8)
r5 (2.4)
Cone. 2
73 (9.1)
39 (3.6)
17 (1.4)
Cone. 1
82 (1.7)
107 (2.1)
91 (3.6)
92 (3.5)
95 (4.7)
95 (5.1)
96 (6.0)
85 (10.5)
97 (4.4)
102 (4.6)
81 (1.9)
93 (4.9)
15 (18.7)
99 (9.9)
29 (5.0)
73 (9.4)
Cone. 2 Cone. 1
74
98
85
83
88
87
87
71
86
92
76
82
8.7
82
37
84
(8.0)
(12.5)
(10.7)
(10.6)
(10.2)
(10.2)
(10.6)
(12.3)
(10.4)
(10.2)
(9.5) 29 (4.2)
41 (7.9)
(9.2)
(15.0)
(10.7) 9.0 (4.8)
(5.1)
(10.7)
Cone. 2 Cone. 1
82
107
91
92
109
97
95
95
86
96
85
97
102
33 (4.7) 110
73 (12.0) 41
93
101
5.7 (3.0) 108
86
91
62
88
(1.7)
(2.1)
(3.6)
(3.5)
(4.1)
(5.6)
(4.7)
(5.1)
(5.4)
(6.0)
(10.5)
(4.4)
(4.6)
(3.0)
(7.9)
(4.9)
(5.3)
(10.0)
(4.0)
(4.1)
(3.3)
(12.0)
Cone. 2
74 (8.0)
98 (12.5)
85 (10.7)
83 (10.6)
118 (8.7)
104 (1.6)
88 (10.2)
87 (10.2)
94 (2.8)
87 (10.6)
71 (12.3)
86 (10.4)
92 (10.2)
109 (5.8)
73 (12.0)
82 (9.2)
82 (23.7)
88 (5.8)
87 (6.1)
95 (5.0)
98 (1.9)
101 (10.1)
aE1uant composition:
Fraction I - 80 ml hexane.
Fraction II - 50 ml hexane.
Fraction III - 15 mL methylene chloride.
Fraction IV - 50 ml ethyl acetate.
Concentration 1 is 0.5 ug per column for BHCs, heptachlor, aldrin, heptachlor epoxide, endosulfan I; 1.0 ug per column for
dieldrin, Kepone, endosulfan II, 4,4'-DDT, endrin aldehyde, 4,4'-DDD, 4,4'-DOE, endrin, and endosulfan sulfate; 5 ug per column for
4,4'-methoxychlor and technical chlordane; 10 ug per column for toxaphene, PCB-1016,and PCB-1260.
cFor concentration 2 the amounts spiked are 10 times as high as those for concentration 1.
dThe values given represent the average recoveries of three determinations; the numbers in parentheses are the standard deviations;
recovery cutoff point is 5 percent.
eData obtained with standards, as indicated in footnotes b and c, dissolved in 2 mL hexane.
fit has been found that because of batch to batch variation in the silica gel material these compounds cross over in two fractions,
and the amounts recovered in each fraction are difficult to reproduce.
-------
aldrin) have been reported (8) when sample extracts were concentrated to a
volume of 0.5 mL or less by a stream of air or nitrogen; losses increased as
the residual volume approached dryness and were relatively greater when
smaller amounts of pesticides were present.
8.3 Elemental Sulfur Removal — Shake the hexane extract (2 mL) with
2-propanol (1 mL) and the TBA-sulfite reagent (1 mL) for at least 1 min. Add
approximately 100 mg crystalline sodium sulfite. If the sodium sulfite
disappears, more sodium sulfite is added in 100-mg portions until a solid
residue remains after repeated shaking. Water (5 mL) is added, and the test
tube is shaken for another minute; this process is followed by
centrifugation. Finally, the hexane layer is transferred to another vial for
analysis.
8.4 Gel Permeation Chromatography Cleanup
8.4.1 Packing the column: Place 70 g of Bio-Beads SX-3 in a 400-mL
beaker, cover the beads with methylene chloride, and allow them to swell
overnight (before packing the columns). Transfer the swelled beads to the
column and pump methylene chloride through the column from bottom to top at
5.0 mL/min. After approximately 1 hour, adjust the pressure on the column to
7 to 10 psi and pump an additional 4 hours to remove all air from the column.
Adjust the column pressure periodically, as required, to maintain 7 to
10 psi.
8.4.2 Calibration of the column: Load 5 mL of the corn oil solution
into sample loop No. 1, and 5 mL of the pesticide-PCB standard into loop No 2.
Inject the corn oil and collect 10 mL fractions (i.e., change fraction at 2-min
intervals) for 36 min. Inject the pesticide-PCB standard and collect 15-mL
fractions for 60 min. Determine the corn oil elution pattern by evaporation of
each fraction to dryness with a gravimetric determination of the residue.
Analyze the pesticide-PCB fractions by gas Chromatography and plot the concen-
tration of each component in each fraction versus total eluant volume (or time)
from the injection points. Choose a "dump time" which allows ^85 percent
removal of the corn oil and >85 percent recovery of the pesticide-PCB com-
pounds. Choose the "collect time" that will extend to at least 10 min after
the elution of the pesticide-PCB compounds. Wash the column for at least 15
min between samples. Typical parameters selected are ^the dump time, 30 min
(150 mL), the collect time, 36 min (180 mL), and the wash time, 15 min (75 mL).
The column can also be calibrated by the use of a 254 nm UV detector in place
of gravimetric and GC analyses of fractions. Measure the peak areas at various
elution times to determine appropriate fractions.
The SX-3 Bio-Beeds column may be reused for several months even if
discoloration occurs. System calibration usually remains constant over this
period of time if the column flowrate remains constant.
8.4.3 Prefilter or load all extracts via the filter holder to avoid
particulates that might cause flow stoppage. Load one 5.0-mL aliquot of the
extract onto the GPC column. Use sufficient clean solvent after the extract
to transfer the entire aliquot into the loop. Between extracts, purge the
sample loading tubing thoroughly with clean solvent. Process the extracts by
using the dump, collect, and wash parameters determined from the calibration
202
-------
and collect the cleaned extracts in 250-mL amber bottles. Concentrate the
extracts as described in Section 8.2.2.4.
9. Gas Chromatography
9.1 Gas chromatographic conditions. The recommended gas chromatographic
columns and operating conditions for the GC/EC instrument are as follows:
8 Column 1: temperature program from 100°C (hold 2 min) to 160°C
at 15°C/min, then at 5°C/min to 270°C; carrier gas helium at
16 psi; injector temperature 225°C; detector temperature 300"C.
0 Column 2: temperature program from 160°C (hold 2 min) to 290°C
(hold 1 min) at 5°C/min; carrier gas nitrogen at 20 psi;
injector temperature 225°C; detector temperature 300°C.
9.2 Table B-l gives the retention times and the MDLs that can be
achieved by this method for the organochlorine pesticides and PCBs. Examples
of the separations achieved with the DB-5 fused-silica capillary column are
shown in Figures B-l to B-10.
9.3 Calibration
9.3.1 Establish the gas chromatographic operating parameters
equivalent to those indicated in Section 9.1. The gas chromatographic system
can be calibrated by using the external standard technique (Section 9.3.2) or
the internal standard technique (Section 9.3.3).
9.3.2 External standard calibration procedure
9.3.2.1 For each parameter of interest, prepare calibration
standards at a minimum of three concentration levels by adding volumes of one
or more stock standards to a volumetric flask and by diluting to volume with
isooctane. One of the external standards should be at a concentration near
but above the MDL. The other concentrations should correspond to the
expected range of concentrations found in real samples or should define the
working range of the detector.
9.3.2.2 By using injections of 1 to 2 uL of each calibration
standard, tabulate area responses against the amounts injected. The results
can be used to prepare a calibration curve for each parameter. Alternatively,
the ratio of the response to the amount injected, defined as the calibration
factor (CF), can be calculated for each parameter at each standard concentra-
tion. If the relative standard deviation of the calibration factor is less
than 15 percent over the working range, linearity through the origin can be
assumed, and the average calibration factor can be used in place of a
calibration curve.
203
-------
VI
1. alpha-BHC
2. beta-BHC
3. gaima-BHC (Lindane)
4. delta-BHC
5. Heptachlor
6. Aldrln
7. Heptachlor epoxlde
8. gamma-Chlordane
9. Endosulfan I
10. 4.4'-DDE
11. Dleldrln
12. Endrln
13. Endosulfan II
14. 4.4'-ODD
15. Endrln aldehyde
16. Kepone
17. Endosulfan sulfate
18. 4.4'-DOT
19. 4.4'-Methoxychtor
Figure B-l. Gas chromatogram of the organochlorine pesticide standard; column:
30 m x 0.25 mm ID DB-5 fused-silica capillary. Temperature
program: 100°C (hold 2 min) to 160°C at 15°C/min5 then at
5°C/min to 270°C; carrier gas He at 16 psi.
-------
Figure B-2.
Gas chromatogram of the toxaphene standard; column: 30 m x 0.25 mm ID
DB-5 fused-silica capillary. Temperature program: 100°C (hold
2 min) to 160°C at 15°C/min, then at 5°C/min to 270°C; carrier
gas He at 16 psi.
-------
o
•I
•01
- o
IT)
ro a:|
• ul
I
IO
r-
o
•j
10
-o
CD ro
t r~
Figure B-3.
Gas chromatogram of the PCB-1016 standard; column: 30 m x 0.25 mm ID
DB-5 fused-silica capillary. Temperature program: 100°C (hold
2 min) to 1608C at 15°C/min, then at 5°C/min to 270°C; carrier
gas He at 16 psi.
-------
o
I-
\
-o
o>
rn
\ —
E
O ••
ON
in
ON
•O J3
0» CO
o =
0>
tC. j
r-
IMIO— aoo-j- r- ^r^o
-------
•o
V
OJ
tO
CD
r^
in
-o o-
o •
to o
•o
T
0-
to
ON
vL
in •
— T — r-
in r^(M 10 ' ^r CN r- -^
o- — • inoN i^ M o- in OK
oo-to OK in
CO
r-ni
T —
in OK
•oco
mo)
Figure B-5. Gas chromatogram of the PCB-1232 standard; column: 30 m x 0.25 mm ID
DB-5 fused-silica capillary. Temperature program: 100°C (hold
2 min) to 160°C at 15°C/min, then at 5"C/min to 270°C; carrier
gas He at 16 psi.
-------
in
10
in in
ro in
•o —
03 o- —
I
in—
inrxi
o r-
o
O- G
• rooi
o-r-
in— (MM3 M 0- -O -O
.— ruin— *OG»3inarT
-o- inf
CM^. . I. . -
-------
o
\
z
o- r-
r- to
o 10
J
oo
(M
i
i
ID T
Q'* —• *o o*' **&• *t ^oin
i tooin oor-iMn intoni inoo
U-.(MO^> MTO- -00
tori- .....
ry kr ^rTUin
iloi (\ir>ior>j
.01
JvMAjlvA.Jj
dim
in
01
-o
Figure B-7. Gas chromatogram of the PCB-1248 standard; column: 30 m x 0.25 mm ID
DB-5 fused-silica capillary. Temperature program: 100°C (hold
2 min) to 160°C at 15°C/min, then at 5°C/min to 270°C; carrier
gas He at 16 psi.
-------
o
H-
Figure B-8. Gas chromatogram of the PCB-1254 standard; column: 30 m x 0.25 mm ID
DB-5 fused-silica capillary. Temperature program: 100°C (hold
2 min) to 160°C at 15°C/min, then at 5°C/min to 270°C; carrier
gas He at 16 psi.
-------
o
(-
X
E in
r
•3>Q:
• ul
Figure B-9. Gas chromatogram of the PCB-1260 standard; column: 30 m x 0.25 mm ID
DB-5 fused-silica capillary. Temperature program: 100°C (hold
2 min) to 160°C at 15°C/min, then at 5°C/min to 270°C; carrier
gas He at 16 psi.
-------
U'l
CO
r--
OJ
Ein
\ —
£
O ••
O-
<5fL I
•Ull
t
•J-
r-o • -OD
— -TV
loin— —
-o
•
Figure B-10.
Gas chromatogram of the technical chlordane standard; column:
30 m x 0.25 mm ID DB-5 fused-silica capillary. Temperature
program: 100°C (hold 2 min) to 160°C at 15°C/min, then at
5°C/min to 270°C; carrier gas He at 16 psi.
-------
9.3.2.3 The working calibration curve or the calibration
factor must be verified on each working day by the measurement of one or more
calibration standards. If the response for any parameter varies from the
predicted response by more than ±20 percent, the test must be repeated with a
fresh calibration standard. Alternatively, a new calibration curve or
calibration factor may be prepared for that parameter.
9.3.2.4 For multicomponent mixtures (toxaphene and PCBs),
match retention times of peaks in the standards with peaks in the sample.
Quantify 8 to 10 major peaks, add peak areas, and calculate as total response
in the sample versus total response in the standard.
9.3.2.5 Technical chlordane is a mixture of alpha-chlordane
(13 percent), gamma-chlordane (18 percent), heptachlor (8 percent), chlordene
(19 percent), and other compounds from the chlorination of chlordene. The sum
of the concentrations of alpha-chlordane and gamma-chlordane can be used to
estimate the concentration of chlordane.
calpha + cgamma
^technical =
0.31
where ^technical = estimated concentration of technical chlordane
Calpha = measured concentration of alpha-chlordane
Cgamma = measured concentration of gamma-chlordane.
9.3.3 Internal standard calibration procedure. To use this
approach, the analyst must select one or more internal standards similar in
analytical behavior to the compounds of interest. The analyst must further
demonstrate that the measurement of the internal standard is not affected by
method or matrix interferences. Because of these limitations, no internal
standard applicable to all samples can be suggested.
9.3.3.1 Prepare calibration standards, at a minimum of three
concentration levels for each parameter of interest by" adding volumes of one
or more stock standards to a volumetric flask. To each calibration standard,
add a known constant amount of one or more internal standards, and dilute to
volume with isooctane. One of the calibration standards should be at a
concentration near but above the MDL. The other concentrations should
correspond to the expected range of concentrations found in real samples or
should define the working range of the detector.
9.3.3.2 By using injections of 1 to 2 uL of each calibration
standard, tabulate the area responses against the concentration for each
214
-------
compound and internal standard. Calculate response factors (RF) for each
compound as follows:
RF = (AsCis)/(AisCs)
where:
As = Response for the parameter to be measured
AJS = Response for the internal standard
C-js = Concentration of the internal standard in ng/uL
Cs = Concentration of the parameter to be measured in ng/uL.
If the RF value over the working range is constant with less than ±15 percent
relative standard deviation, the RF can be assumed to be invariant, and the
average RF can be used for calculations. Alternatively, the results can be
used to plot a calibration curve of response ratios, As/Ais against RF.
9.3.3.3 The working calibration curve or RF must be verified
on each working day by the measurement of one or more calibration standard.
If the response for any parameter varies from the predicted response by more
than 20 percent, the test must be repeated with a fresh calibration
standard. Alternatively, a new calibration curve must be prepared for that
compound.
9.4 Identify compounds in the sample by comparing the retention times of
the peaks in the sample chromatogram with those of the peaks in standard
chromatograms. The retention time window used to make identifications is
based upon measurements of actual retention time variations over the course
of 10 consecutive injections (Table B-4). Three times the standard deviation
of a retention time window can be used to calculate a suggested window size.
9.5 If the response of a peak exceeds the working range of the system,
dilute the extract and reanalyze.
10. Quality Control
10.1 Before processing any samples, the analyst should demonstrate
through the analysis of a distilled water method blank that all glassware and
reagents are interference-free. Each time a set of samples is extracted or
there is a change in reagents, a method blank should be processed as a
safeguard against laboratory contamination. The blank samples should be
carried through all stages of the sample preparation, cleanup, and analysis
steps.
10.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to validate the precision of
the sampling technique. Laboratory replicates (minimum of two per batch of
20 samples per matrix) should be analyzed to validate the precision of the
analysis. Fortified samples (minimum of one per batch of 20 samples per
215
-------
TABLE B-4. REPRODUCIBILITY OF RETENTION TIMES
OF THE ORGANOCHLORINE PESTICIDES
FOR 10 CONSECUTIVE INJECTIONS
================================================
Retention Time
Compound Reproducibi1i ty
SD (min)a
alpha-BHC 0.010
beta-BHC 0.009
gamma-BHC 0.011
delta-BHC 0.011
Heptachlor 0.008
Aldrin 0.009
Heptachlor epoxide 0.009
gamma-Chlordane 0.012
Endosulfan I 0.010
4,4'-DDE 0.008
Dieldrin 0.008
Endrin 0.007
Endosulfan II 0.006
4,4'-DDD 0.008
Endrin aldehyde 0.007
Kepone 0.011
Endosulfan sulfate 0.008
4,4'-DDT 0.008
4,4'-Methoxychlor 0.007
Toxaphene 0.004-0.006b
PCB-1016 0.042-0.104°
PCB-1260 0.035-0.040b
SD — Standard deviation.
aNumber of determinations is 10 except^
for Kepone which is 8.
bValue determined for 3 major peaks
for each mixture.
matrix) should be analyzed to validate the sensitivity and accuracy of the
analysis. If the fortified waste samples do not indicate sufficient
sensitivity to detect less than or equal to 1 M9/9 of sample, then the
sensitivity of the instrument should be increased or the extract should be
subjected to additional cleanup. The fortified samples should be carried
through all stages of the sample preparation and measurement steps. Where
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as mass spectroscopy should be used.
216
-------
10.3 The analyst must, on an ongoing basis, demonstrate through the
analyses of quality control check standards that the operation of the gas
chromatograph is under control. The frequency of the check standard analysis
should be equivalent to 10 percent of the samples analyzed. The check standard
will give an indication of how good the calibration standards are. If the
recovery of any compound found in the check standard is less than 80 percent,
the laboratory performance is judged to be out of control, and the problem
must be corrected. A new set of calibration standards should be prepared and
analyzed.
10.4 The analyst must, on an ongoing basis, demonstrate through the
analysis of standards that the silica gel fractionation scheme is
reproducible. Batch-to-batch variation in the composition of the silica gel
material and slight changes in the moisture content may cause a change in
the distribution patterns of the organochlorine pesticides. Compounds found
to exhibit such behavior include alpha-BHC, gamma-chlordane, 4,4'-DDT, and
4,4'-DDD.
10.5 Whenever compounds are found in more than one fraction, add up the
concentrations of the various fractions if the final volumes of the fractions
are identical. It is up to the analyst to decide whether the cut-off point
should be 5 or 10 percent of the concentration in the fraction where the
compound is expected to elute.
11. Method Performance
11.1 The MDL is defined as the minimum concentration of the test
compound that can be measured and reported with 99 percent confidence that
the value is above zero. The MDL concentrations listed in Table B-l were
obtained by using reagent water and sandy loam soil. Details on how to
determine the MDL are given in Reference 9. The MDL actually achieved in a
given analysis will vary as it is dependent on instrument sensitivity and
matrix effects.
11.2 This method has been tested in a single laboratory by using clean
hexane and liquid and solid waste extracts which were fortified with the test
compounds at three concentrations. Single-operator precision, overall
precision, and method accuracy were found to be related to the concentration
of the compound and the type of matrix. For exemplification, results of the
single-laboratory method evaluation are given in Tables B-5 and B-6.
217
-------
TABLE B-5. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE
PESTICIDES AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (LIQUID
WASTE NO. 1 EXTRACT)
ro
i—>
CO
Average
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor
epoxide
gamma-Chlordane
Endosulfan
4,4'-DDE
Dieldrin
Endrin
Endosulfan
4, 4 '-ODD
I
II
Fraction I Fraction II
hexane hexane
(80 ml) (50 ml)
57 ± 2.5 (4.4)
90 ± 11 (12)
92 ± 9.2 (10)
85 ± 7.2 (8.5) 10 ± 9.2 (92)
95 ± 16 (17)
33 ± 4.0 (15)
Endrin aldehyde
Kepone
Endosulfan
4,4'-DDT
sulfate
88 ± 18 (21)
4,4'-Methoxychlor
PCB-1016
PCB-1260
118 ± 9.8 (8.3)
100l± 18 (18)
4
SO (RSD)a'b'c
Fraction III
methylene
chloride
(15 ml)
22
90
90
90
89
88
82
65
79
43
i
83
75
4
±
t
t
±
±
±
±
±
Fraction IV
ethyl acetate
(50 ml)
9.2 (42)
3.1 (3.4)
4.0 (4.4)
11 (12)
4.1 (4.6)
3.8
4.3
3.1
7.1
(4.
(5.
(4.
(9.
3)
3)
7)
0)
* 16 (37)
±
4
4.0
4.6
(4.
(6.
113; 223C
8)
1)
Total recovery
79
90
90
90
90
92
89
95
88
95
82
65
79
76
1
113
83
88
75
118
100
4
4
±
±
4
±
±
±
4
4
±
4
j
±
4
±
4
4
10 (13)
3.1 (3.4)
4.0 (4.4)
11 (12)
11 (12)
9.2 (10)
4.1 (4.6)
8.0 (8
3.8 (4
16 (17)
4.3 (5
3.1 (4
7.1 (9
16 (21)
223C
4.0 (4
18 (21)
4.6 (6
9.8 (8
18 (18)
.4)
.3)
.3)
.7)
.0)
.8)
.1)
.3)
1 -- Unable to determine the recovery because of Interference.
aThe values given represent the average percent recoveries from three replicate determinations
standard deviation; the numbers in parentheses are the relative standard deviations.
bThe amounts spiked are 15,000, 30,000, and 150,000 ng per 2 mL extract per column for the
organochlorine pesticides and PCB-1016/PCB-1260, respectively.
cDuplicate determinations.
one
-------
TABLE B-6. ELUTION PATTERNS AND AVERAGE RECOVERIES OF THE ORGANOCHLORINE
PESTICIDES AND PCBs AFTER SILICA GEL CHROMATOGRAPHY (NBS
SEDIMENT EXTRACT)
t—»
IO
Average i-
Compound
alpha-BHC
beta-BHC
gamma-BHC
delta-BHC
Heptachlor
Aldrin
Heptachlor
epoxlde
gamma-Chlordane
Endosulfan
4, 4 '-DDE
Oieldrin
Endrin
Endosulfan
4, 4 '-ODD
I
II
Fraction I
hexane
(80 ml)
70 ± 7.7 (11)
65 i 4.6 (7.1)
71 ± 3.2 (4.5)
76 i 7.1 (9.3)
Endrin aldehyde
Kepone
Endosulfan
4, 4 '-DDT
sulfate
61 ± 7.9 (13)
4,4'-Methoxychlor
PCB-1016
PCB-1260
104 ± 2.5 (2.4)
95i± 7.5 (7.9)
• SD
(RSD)a-b
Fraction III
Fraction II methylene Fraction IV
hexane chloride ethyl acetate
(.50 mL)
55 ± 6.1 (11) 20
94
89
92
91
10 i 2.0 (20)
88
85
87
81
36 ± 2.0 (5.6) 49
71
86
99
(15
±
±
±
±
±
±
±
±
±
±
±
±
±
1
3
4
5
5
5
9
6
4
1
9
5
mL)
.7
.0
.1
.2
.7
.1
.4
.4
.5
.2
.2
.0
17
(8.7)
(3.2)
(4.6)
(5.6)
(6.3)
(5.8)
(11)
(7.3)
(5.5)
(2.4)
(13)
(5.8)
(17)
(50 mL) Total
75
94
89
92
70
65
91
81
88
76
85
87
81
85
71
0 0
86
61
99
104
95
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
recovery
6.0
3.0
4.1
5.2
7.7
4.6
5.7
4.9
5.1
7.1
9.4
6.4
4.5
3.1
9.2
5.0
7.9
(8.0)
(3.2)
(4.6)
(5.6)
(ID
(7.1)
(6.3)
(6.1)
(5.8)
(9.3)
(11)
(7.3)
(5.5)
(3.6)
(13)
(5.8)
(13)
* 17 (17)
±
±
2.5
7.5
(2.4)
(7.9)
aThe values given represent the average percent recoveries from three replicate determinations ± one
standard deviation; the numbers in parentheses are the relative standard deviations.
bThe amounts spiked are 3,000, 6,000, and 30,000 ng per 2 mL extract per column for the organochlorlne
pesticides and PCB-1016/PCB-1260, respectively.
-------
REFERENCES
1. Development and Application of Test Procedures for Specific Organic Toxic
Substances in Wastewaters. Category 10 - Pesticides and PCBs Report for
EPA Contract 68-03-2606.
2. Goerlitz, D. F., and L. M. Law. Note on Removal of Sulfur Interferences
from Sediment Extracts for Pesticide Analysis. Bull. Environ. Contam.
Toxicol. 6: 9 (1971).
3. Blumer, M., Removal of Elemental Sulfur from Hydrocarbon Fractions.
Anal. Chem. 29: 1039 (1957).
4. Ahnoff, M., and B. Josefsson, Cleanup Procedures for PCB Analysis on
River Water Extracts. Bull. Environ. Contam. Toxicol. 13: 159 (1975).
5. Jensen, S., L. Renberg, and L. Reutergardh. Residue Analysis of
Sediment and Sewage Sludge for Organochlorines in the Presence of
Elemental Sulfur. Anal. Chem. 49: 316-318 (1977).
6. Wise, R. H., D. F. Bishop, R. T. Williams, and B. M. Austern. Gel
Permeation Chromatography in the GC/MS Analysis of Organics in Sludges.
U.S. EPA, Municipal Environmental Research Laboratory, Cincinnati,
Ohio 45268.
7. Pionke, H. B., G. Chesters, and D. E. Armstrong. Extraction of
Chlorinated Hydrocarbon Insecticides from Soil. Agron. J. 60: 289
(1968).
8. Burke, J. A., P. A. Mills, and D. C. Bostwick. Experiments with
Evaporation of Solutions of Chlorinated Pesticides. J. Assoc. Off.
Anal. Chem. 49: 999 (1966).
9. Glazer, J. A., et al. Trace Analyses for Wastewaters. Environ. Sci.
and Technol. 15: 1426 (1981).
220
-------
APPENDIX C
METHOD 3510, 3520, 3540, AND 3550 PROTOCOLS
(PROPOSED REVISIONS)
METHOD 3510 — SEPARATORY FUNNEL LIQUID-LIQUID EXTRACTION
1. Scope and Application
1.1 Method 3510 is a liquid-liquid extraction procedure that uses the
standard separatory funnel techniques. The sample and the extraction solvent
must be immiscible to allow recovery of the target compounds. Since this
method is used in conjunction with other SW-846 methods, subsequent cleanup and
detection methods that will be used to analyze the extract are given elsewhere.
2. Summary of Method
A measured volume of sample is adjusted to a specific pH and is
extracted with the appropriate solvent in a separatory funnel. Methylene
chloride should be employed unless the method specifies a different solvent.
Samples are extracted three consecutive times; the extracts are combined
and dried with anhydrous sodium sulfate and are then concentrated in a
Kuderna-Danish apparatus.
3. Interferences
3.1 A method blank should be performed for the compounds of interest
prior to the use of this method. The level of interferences must be below
the method detection limit before the actual samples are extracted.
3.2 If emulsion formation prevents acceptable extract recovery with
the separatory funnel extractions, continuous liquid-liquid extraction
{Method 3520) should be used.
3.3 Procedures for the removal of interfering compounds coextracted
with the target compounds are described in the referring analytical methods.
4. Apparatus
4.1 Separatory funnel: 2-liter, with Teflon stopcock.
4.2 Drying column: chromatographic column approximately 400 mm long by
20 mm ID, with coarse frit. The substitution of a small pad of disposable
Pyrex glass wool for the frit will help prevent cross-contamination.
221
-------
4.3 Concentrator tube: Kuderna-Danish, 10 ml graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked at the volumes
employed in the test. Ground-glass stoppers should be used to prevent
evaporation of extracts.
4.4 Evaporative flask: Kuderna-Danish, 500 ml (Kontes K-5700010500 or
equivalent). Attach to the concentrator tube with springs.
4.5 Snyder column: Kuderna-Danish, three-ball macro (Kontes
K-503000-0121 or equivalent).
4.6 Snyder column: Kuderna-Danish, two-ball micro (Kontes
K-569001-0219 or equivalent).
4.7 Vials: glass, 10 to 15 ml capacity, with Teflon-lined screw caps.
4.8 Pyrex glass wool: prerinse glass wool with appropriate solvents
to ensure its cleanliness.
4 9 Silicone carbide boiling chips: approximately 10/40 mesh. Heat the
chips to 400°C for 30 min or Soxhlet-extract them with methylene chloride.
4.10 Water bath: heated, with concentric ring cover, capable of
temperature control (±2°C). The bath should be used in a hood.
4.11 Nitrogen evaporation device (N-Evap by Organomation Associates,
Inc., South Berlin, MA, or equivalent)
4.12 pH indicator paper covering the pH range from 1 to 14.
5. Reagents
5.1 The specific reagents to be employed in this method may be listed
under the referring analytical method that will be used to analyze the
extract. Check the analytical method for the specific extraction solvent.
If a specific extraction solvent is not listed for the compound(s) of
interest, methylene chloride shall be used.
5.2 The solvent of choice should be appropriate for the method of
measurement to be used, and it should give an analyte-to-solvent partition
coefficient of at least 1 to 1000.
5.3 Sodium sulfate (ACS): granular anhydrous (purified by heating at
400"C for 4 hr in a shallow tray).
5.4 'Sodium hydroxide (ACS): 10 N in distilled water.
5.5 Sulfuric acid (1:1): mix equal volumes of concentrated ^$04 (ACS)
with distilled water.
5.6 Distilled water.
5.7 Methylene chloride: pesticide quality or equivalent.
222
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6. Sample Collection, Preservation, and Handling
6.1 Adhere to those procedures specified in the referring analytical
methods for collection, preservation, and handling.
7. Sample Extraction
7.1 Use a 1-liter graduated cylinder to measure out a 1-liter portion
and place it into a 2-liter separatory funnel. If less than 1 liter of
sample is available, or if high concentrations are anticipated, use a smaller
volume of sample.
7.2 Check the pH of the sample with wide-range pH paper and adjust it
to that indicated in the referring method.
7.3 Add 60 ml of the appropriate extraction solvent to the separatory
funnel and extract the sample by shaking the funnel for 2 min with periodic
venting to release excess pressure.
7.4 Allow the phases to separate for a minimum of 10 min. If the
emulsion interface between layers is more than one-third the size of the
solvent layer, the analyst must employ mechanical techniques to complete the
phase separation. The optimum technique depends upon the sample but may
include stirring, filtration of the emulsion through glass wool, or
centrifugation.
7.5 Collect the extract into a 250-mL Erlenmeyer flask and then repeat
the extraction two more times with fresh portions of solvent.
7.6 Combine the three extracts and discard the extracted waste
properly if no further extractions are to be performed.
7.7 Dry the extract by passing it through a column of anhydrous sodium
sulfate. Collect the dried extract in a Kuderna-Danish evaporative
concentrator equipped with a 10-mL collection ampule.
7.8 Add 1 or 2 clean boiling chips to the flask-arid attach a three-ball
Snyder column. Prewet the Snyder column by adding about 1 mL solvent to the
top. Place the Kuderna-Danish apparatus on a steam or hot water bath so that
the concentrator tube and the entire lower rounded surface of the flask are
bathed in hot water or vapor. Adjust the vertical position of the apparatus
and the water temperature as required to complete the concentration in
15 to 20 min. At the proper rate of distillation, the balls of the column will
actively chatter, but the chambers will not flood. When the apparent volume
of liquid reaches 1 ml, remove the Kuderna-Danish apparatus and allow it to
drain for at least 10 min while cooling.
7.9 Remove the Snyder column and add 50 ml of the solvent specified in
the referring analytical method if a solvent exchange step is required.
Concentrate the extract as before. When the apparent volume of liquid
223
-------
reaches 1 mL, remove the Kuderna-Danish apparatus and allow it to drain and
to cool for at least 10 min.
7.10 Remove the Snyder column and rinse the flask and its lower joint
with 1 to 2 ml of the solvent specified in the referring analytical method
into the concentrator tube.
7.11 Nitrogen blowdown technique
7.11.1 Place the concentrator tube in a warm-water bath (35°C),
and evaporate the solvent to 0.5 ml by using a gentle stream of clean, dry
nitrogen (filtered through a column of activated charcoal.
Caution: The extract must never be allowed to become dry.
7.11.2 Dilute the extract to 1 ml with solvent and proceed with
the cleanup or analysis as described in the referring analytical method.
8. Quality Control
8.1 Comprehensive quality control procedures are specified for each
target compound in the referring analytical method.
8.2 Before processing any samples, the analyst must analyze a reagent
water blank to demonstrate that interferences from glassware are under
control. Each time a set of samples is extracted or reagents are changed, a
reagent water blank must be processed as a safeguard against laboratory
contamination.
8.3 By fortifying distilled water or another liquid similar to the
sample matrix, the analyst should demonstrate that the compound(s) of
interest is being quantitatively recovered before applying this method to
actual samples.
224
-------
METHOD 3520 — CONTINUOUS LIQUID-LIQUID EXTRACTION
1. Scope and Application
1.1 Method 3520 is a liquid-liquid extraction procedure designed to
extract organic compounds from liquid samples in a continuous extraction
apparatus. This method is available as an alternative to Method 3510
which is a separatory funnel extraction procedure. Compared to standard
separatory funnel techniques, Method 3520 has the advantage of minimizing
emulsion formation. The sample and the extraction solvent must be immiscible
to allow recovery of the target compounds. Subsequent cleanup and detection
methods are described in the referring analytical methods.
1.2 Method 3520 is designed for extraction solvents with greater
density than the sample. Continuous extraction devices are available for
extraction solvents that are less dense than the sample. The analyst must
demonstrate the effectiveness of any such automatic extraction device before
employing it in sample extraction.
2. Summary of Method
A measured volume of sample is adjusted to a specific pH and is placed
into a continuous liquid-liquid extractor. The extraction pH and solvent are
those specified in the referring analytical method. Samples are extracted
for 16 hours; the extract is collected, dried with anhydrous sodium sulfate,
and concentrated in a Kuderna-Danish apparatus. In some cases, the sample pH
is adjusted after the first extraction, and continuous extraction is carried
out for another 16 hours to recover an additional class of compounds.
3. Interferences
3.1 A method blank should be performed for the compounds of interest
prior to the use of this method. The level of interferences must be below
the method detection limit before the actual samples are extracted.
3.2 Procedures for the removal of interfering compounds coextracted
with the target compounds are described in the referrfng analytical methods.
4. Apparatus
4.1 A continuous liquid-liquid extractor equipped with Teflon or glass
connecting joints and stopcocks requiring no lubrication (Hershberg-Wolfe
type, Ace Glass Company, Vineland, NJ, P/N 6841-10, equivalent).
4.2 Drying column: chromatographic column approximately 400 mm long
by 20 mm ID, with coarse frit. Substitution of a small pad of disposable
Pyrex glass wool for the frit will help prevent cross-contamination.
4.3 Concentrator tube: Kuderna-Danish, 10 mL graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked at the volumes
225
-------
employed in the test. Ground-glass stoppers should be used to prevent
evaporation of extracts.
4.4 Evaporative flask: Kuderna-Danish, 500 mL (Kontes K-5700010500 or
equivalent). Attach to the concentrator tube with springs.
4.5 Snyder column: Kuderna-Danish, three-ball macro (Kontes
K-503000-0121 or equivalent).
4.6 Snyder column: Kuderna-Danish, two-ball micro {Kontes
K-569001-0219 or equivalent).
4.7 Vials: glass, 10 to 15 ml capacity, with Teflon-lined screw caps.
4.8 Pyrex glass wool: prerinse glass wool with appropriate solvents
to ensure its cleanliness.
4 9 Silicone carbide boiling chips: approximately 10/40 mesh. Heat
them to 400°C for 30 min or Soxhlet-extract them with methylene chloride.
4.10 Water bath: heated, with concentric ring cover, capable of
temperature control (±2°C). The bath should be used in a hood.
4.11 Nitrogen evaporation device (N-Evap by Organomation Associates,
Inc., South Berlin, MA, or equivalent)
4.12 pH indicator paper covering the pH range from 1 to 14.
5. Reagents
5.1 The specific reagents to be employed in this method may be Listed
under the referring analytical method that will be used to analyze the
extract. Check the analytical method for the specific extraction solvent.
If a specific extraction solvent is not listed for the compound(s) of
interest, methylene chloride shall be used.
5.2 The solvent of choice should be appropriate-for the method of
measurement to be used, and it should give an analyte-to-solvent partition
coefficient of at least 1 to 1000.
5.3 Sodium sulfate (ACS): granular anhydrous (purified by heating at
400°C for 4 hours in a shallow tray).
5.4 Sodium hydroxide (ACS): 10 N in distilled water.
5.5 Sulfuric acid (1:1): mix equal volumes of concentrated ^$04 (ACS)
with distilled water.
5.6 Distilled water.
5.7 Methylene chloride: pesticide quality or equivalent.
226
-------
6. Sample Collection. Preservation, and Handling
6.1 Adhere to those procedures specified in the referring analytical
methods for collection, preservation, and handling.
7. Sample Extraction
7.1 Use a 1-liter graduated cylinder to measure out a 1-liter portion
and place it into a continuous extractor. If less than 1 liter of sample is
available, or if high concentrations are anticipated, use smaller volumes of
sample and add organics-free water to bring the sample volume to 1 liter.
7.2 Refer to Figure C-l to aid in understanding the procedures
described in Sections 7.3 through 7.7 for setting up the continuous
extractor. The analyst is reminded that the following steps apply to
sample/solvent systems where the solvent is more dense than the sample.
7.3 Place 150 mL of the extraction solvent in the extractor and 350 ml
of the extracting solvent in the 500-mL distilling flask. Add several
boiling chips to the distilling flask.
7.4 Add the sample to the continuous extractor. More organics-free
water may be needed to ensure proper operation.
7.5 Turn on the cooling water and the heating mantle and extract the
sample for 16 hours.
7.6 Let the system cool and remove the extract contained in the 500-mL
distilling flask.
7.7 If an additional extraction is to be performed, adjust the sample
pH accordingly. Attach a 500-mL distilling flask containing 350 mL of
extraction solvent, add several boiling chips, and proceed from step 7.5.
7.8 Dry the extract by passing it through a column of anhydrous sodium
sulfate. Collect the dried extract in a Kuderna-Danish evaporative
concentrator equipped with a 10-mL collection ampule. ~*
7.9 Add 1 or 2 clean boiling chips to the flask and attach a three-ball
Snyder column. Prewet the Snyder column by adding about 1 mL solvent to the
top. Place the Kuderna-Danish apparatus on a steam or hot-water bath so that
the concentrator tube and the entire lower rounded surface of the flask are
bathed in hot water or vapor. Adjust the vertical position of the apparatus
and the water temperature as required to complete the concentration in 15 to
20 min. At the proper rate of distillation, the balls of the column will
actively chatter, but the chambers will not flood. When the apparent volume
of liquid reaches 1 mL, remove the Kuderna-Danish apparatus and allow it to
drain for at least 10 min while cooling.
7.10 Remove the Snyder column and add 50 mL of the solvent specified in
the analytical method if a solvent exchange step is required. Concentrate
227
-------
HMting M»ntU
Figure C-l. Continuous liquid-liquid extractor.
228
-------
the extract as before. When the apparent volume of liquid reaches 1 ml,
remove the Kuderna-Danish apparatus and allow it to drain and to cool for at
least 10 min.
7.11 Remove the Snyder column and rinse the flask and its lower joint
with 1 to 2 ml of the solvent specified in the referring analytical method
into the concentrator tube.
7.12 Nitrogen blowdown technique
7.12.1 Place the concentrator tube in a warm-water bath (35°C),
and evaporate the solvent to 0.5 mL by using a gentle stream of clean,
dry nitrogen (filtered through a column of activated charcoal).
Caution: The extract must never be allowed to become dry.
7.12.2 Dilute the extract to 1 mL with solvent and proceed with
the cleanup or analysis as described in the referring analytical methods.
8. Quality Control
8.1 Comprehensive quality control procedures are specified for each
target compound in the referring analytical method.
8.2 Before processing any samples, the analyst must analyze a reagent
water blank to demonstrate that interferences from glassware are under
control. Each time a set of samples is extracted or reagents are changed, a
reagent water blank must be processed as a safeguard against laboratory
contamination.
8.3 By fortifying distilled water or another liquid similar to the
sample matrix, the analyst should demonstrate that the compound(s) of
interest is being quantitatively recovered before applying this method to
actual samples.
229
-------
METHOD 3540 — SOXHLET EXTRACTION
1. Scope and Application
Method 3540 is a procedure for extracting organic compounds from solids
such as soils and sludges. The Soxhlet extraction procedure ensures intimate
contact of the sample matrix with the extraction solvent. Since this method
is used in conjunction with other SW-846 methods, subsequent cleanup and
detection methods that will be used to analyze the extract are given
elsewhere.
2. Summary of Method
A known amount of the solid sample is mixed with anhydrous sodium
sulfate, is placed in an extraction thimble or between two plugs of glass
wool, and is extracted with an appropriate solvent in a Soxhlet extractor.
The extract is then dried and concentrated and is either cleaned-up further
or is analyzed directly by use of the referring analytical method.
3. Interferences
3.1 A method blank should be performed for the compounds of interest
prior to the use of this method. The level of interferences must be below
the method detection limit before the actual samples are extracted.
3.2 Procedures for the removal of interfering compounds coextracted
with the target compounds are described in the referring analytical method that
will be used to analyze the extract.
4. Apparatus
4.1 Soxhlet extractor: 40 mm ID, with 500-mL round bottom flask.
4.2 Rheostat-controlled heating mantle.
4.3 Drying column: chromatographic column approximately 400 mm long x
20 mm ID, with coarse frit. Substitution of a small pad of disposable Pyrex
glass wool for the frit will prevent cross-contamination.
4.4 Concentrator tube: Kuderna-Danish, 10 ml graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked at the volumes
employed in the test. Ground-glass stoppers should be used to prevent
evaporation of extracts.
4.5 Evaporative flask: Kuderna-Danish, 500 ml (Kontes K-5700010500 or
equivalent). Attach to the concentrator tube with springs.
4.6 Snyder column: Kuderna-Danish, three-ball macro (Kontes
K-503000-0121 or equivalent).
230
-------
4.7 Snyder column: Kuderna-Danish, two-ball micro (Kontes
K-569001-0219 or equivalent).
4.8 Vials: glass, 10 to 15 ml capacity, with Teflon-lined screw caps.
4.9 Pyrex glass wool: prerinse glass wool with appropriate solvents
to ensure its cleanliness.
4.10 Silicone carbide boiling chips: approximately 10/40 mesh. Heat
them to 400°C for 30 min or Soxhlet-extract them with methylene chloride.
4.11 Water bath: heated, with concentric ring cover, capable of
temperature control (±2°C). The bath should be used in a hood.
4.12 Nitrogen evaporation device (N-Evap by Organomation Associates,
Inc., South Berlin, MA, or equivalent).
4.13 Apparatus for determining percent moisture.
4.13.1 Drying oven
4.13.2 Desiccator.
4.13.3 Porcelain crucibles.
4.14 Vacuum filtration apparatus.
4.14.1 Buchner funnel.
4.14.2 Filter paper, Whatman No. 41, or equivalent.
4.15 Amber bottles, 250 ml.
5. Reagents
5.1 The specific reagents to be employed in this method may be listed
under the referring analytical methods that will be usB'd to analyze the
extract. Check the analytical method for the specific extraction solvent.
5.2 Sodium sulfate (ACS): granular anhydrous (purified by heating at
400"C for 4 hours in a shallow tray).
5.3 Solvent(s): soil samples shall be extracted by using either of the
following solvent systems.
5.3.1 Acetone/hexane, 1:1 v/v, ACS reagent grade only.
5.3.2 Toluene/methanol, 10:1 v/v, ACS reagent grade only.
231
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6. Sample Collection, Preservation, and Handling
6.1 Adhere to those procedures specified in the referring analytical
methods for collection, preservation, and handling.
7. Procedure
7.1 Blend 10 g of the solid sample with an equal weight of anhydrous
sodium sulfate and place the mixture in either a glass or paper extraction
thimble. The extraction thimble must drain freely for the duration of the
extraction period. The use of a glass-wool plug above and below the sample is
also acceptable.
7.2 Place 300 mL of the extraction solvent into a 500-mL round-bottom
flask containing a few boiling chips. Attach the flask to the extractor, and
extract the solids for 16 hours.
7.3 Allow the extract to cool after the extraction is complete. Rinse
the condenser with the extraction solvent and drain the Soxhlet apparatus
into the collecting round-bottom flask. Filter the extract and dry it by
passing it through a 4-in column of sodium sulfate which has been washed
with the extraction solvent. Collect the dried extract in a 500-mL
Kuderna-Danish (K-D) flask fitted with a 10-mL graduated concentrator tube.
Wash the extractor flask and sodium sulfate column with 100 to 125 mL of the
extraction solvent.
7.4 Add 1 or 2 clean boiling chips to the flask, and attach a
three-ball Snyder column. Prewet the Snyder column by adding about 1 mL
solvent to the top. Place the Kuderna-Danish apparatus on a steam- or hot-
water bath so that the concentrator tube and the entire lower rounded surface
of the flask are bathed in hot water or vapor. Adjust the vertical position
of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 min. At the proper rate of distillation, the balls
of the column will actively chatter, but the chambers will not flood. When
the apparent volume of liquid reaches 1 mL, remove the Kuderna-Danish
apparatus and allow it to drain for at least 10 min while cooling.
7.5 Remove the Snyder column and add 50 mL of the solvent specified in
the analytical method if a solvent exchange step is required. Concentrate
the extract as before. When the apparent volume of liquid reaches 1 mL,
remove the Kuderna-Danish apparatus and allow it to drain and to cool for at
least 10 min.
7.6 Remove the Snyder column and rinse the flask and its lower joint
with 1 to 2 mL of the solvent specified in the referring analytical method
into the concentrator tube.
7.7 If GPC cleanup is not required, proceed to Paragraph 7.8.; if GPC
is required, refer to Method 8080 for instructions on how to perform it.
232
-------
7.8 Nitrogen blowdown technique
7.8.1 Place the concentrator tube in a warm-water bath (35°C), and
evaporate the solvent to 0.5 ml by using a gentle stream of clean, dry nitrogen
(filtered through a column of activated charcoal).
Caution: The extract must never be allowed to become dry.
7.8.2 Dilute the extract to 1 mL with solvent and proceed with the
cleanup or analysis as described in the referring analytical method.
7.9 Moisture determination: Immediately after weighing the sample for
extraction, weigh 5 to 10 g of the sample into a tared crucible. Determine
the percent moisture by drying overnight at 105°C. Allow to cool in a
desiccator before weighing. Concentrations of individual analytes will be
reported relative to the dry weight of soil or sediment.
weight of sample - weight of dry sample
1 x 100 = percent moisture
weight of sample
8. Quality Control
8.1 Comprehensive quality control procedures are specified for each
target compound in the referring analytical method.
8.2 Before processing any samples, the analyst must perform a method
blank to demonstrate that interferences from glassware are under control.
Each time a set of samples is extracted or reagents are changed, a method
blank must be performed as a safeguard against laboratory contamination.
8.3 By fortifying clean soil or another solid similar to the sample
matrix, the analyst should demonstrate that the compound(s) of interest
is being quantitatively recovered before applying this method to actual
samples.
233
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METHOD 3550 ~ SONICATION EXTRACTION
1. Scope and Application
1.1 Method 3550 is a procedure for extracting organic compounds from
solids such as soils and sludges. The sonication process ensures intimate
contact of the sample matrix with the extraction solvent. Since this method is
used in conjunction with other SW-846 methods, subsequent cleanup and
detection methods that will be used to analyze the extract are given
elsewhere.
2. Summary of Method
A known amount of sample of the solid waste is ground, is mixed with the
extraction medium, and then is dispersed into the solvent by using
sonication. The extract is dried with anhydrous sodium sulfate and is
concentrated in a Kuderna-Danish apparatus. The resulting solution may be
cleaned-up further or may be analyzed directly by using the appropriate
technique.
3. Interferences
3.1 A method blank should be performed for the compounds of interest
prior to the use of this method. The level of interferences must be below
the method detection limit before the actual samples are extracted.
3.2 Procedures for the removal of interfering compounds coextracted
with the target compounds are described in the referring analytical method that
will be used to analyze the extract.
4. Apparatus and Materials
4.1 Apparatus for grinding: if the sample will not pass through a 1-mm
standard sieve or cannot be extruded through a 1-mm opening, it should be
processed into a homogeneous sample that meets these requirements. Fisher
Mortar Model 155 Grinder, Fisher Scientific Co., Catalogue Number 8-323, or
an equivalent brand and model, is recommended for sample processing. This
grinder should handle most solid samples except gummy, fibrous, or oily
materials.
4.2 Sonication: a horn-type sonicator equipped with a titanium tip
should be used. The following sonicators, or equivalent brands and models,
are recommended: Sonifer/Cell Disrupter, Model W-350, Ultrasonics Inc., or
Sonic Dismembrator, Model 300, Fisher Scientific Co., Catalog
Number 15-338-40.
4.3 Drying column: chromatographic column approximately 400 mm long
by 20 mm ID, with coarse frit. Substitution of a small pad of disposable
Pyrex glass wool for the frit will help prevent cross-contamination.
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4.4 Concentrator tube: Kuderna-Danish, 10 mL graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked at the volumes
employed in the test. Ground-glass stoppers should be used to prevent
evaporation of extracts.
4.5 Evaporative flask: Kuderna-Danish, 500 ml (Kontes K-5700010500 or
equivalent). Attach to the concentrator tube with springs.
4.6 Snyder column: Kuderna-Danish, three-ball macro (Kontes
K-503000-0121 or equivalent).
4.7 Snyder column: Kuderna-Danish, two-ball micro (Kontes
K-569001-0219 or equivalent).
4.8 Vials: glass, 10 to 15 ml capacity, with Teflon-lined screw caps.
4.9 Pyrex glass: prerinse glass wool with appropriate solvents to
ensure its cleanliness.
4.10 Silicone carbide boiling chips: approximately 10/40 mesh: Heat
them to 4008C for 30 min or Soxhlet-extract them with methylene chloride.
4.11 Water bath: heated, with concentric ring cover capable of
temperature control (±2°C). The bath should be used in a hood.
4.12 Nitrogen evaporation device (N-Evap by Organomation Associates,
Inc., South Berlin, MA, or equivalent)
4.13 Apparatus for determining percent moisture.
4.13.1 Drying oven.
4.13.2 Desiccator.
4.13.3 Porcelain crucibles.
4.14 Vacuum filtration apparatus.
4.14.1 Buchner funnel.
4.14.2 Filter paper, Whatman No. 41, or equivalent.
4.15 Amber bottles, 250 ml.
5. Reagents
5.1 The specific reagents to be employed in this method may be listed
under the referring analytical method that will be used to analyze this
extract. Check the analytical method for the specific extraction solvent.
If a specific extraction solvent is not listed for the compound(s) of
interest, methylene chloride shall be used.
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5.2 Sodium sulfate (ACS): granular anhydrous (purified by heating at
400°C for 4 hours in a shallow tray).
5.3 Sodium hydroxide (ACS): 10 N in distilled water.
6. Sample Collection, Preservation, and Handling
6.1 Adhere to those procedures specified in the referring analytical
methods for collection, preservation, and handling.
7. Sample Extraction
7.1 Grind or subdivide the solid sample so that it either passes
through a 1-mm sieve or can be extruded through a 1-mm hole. Introduce
sufficient sample into the grinding apparatus to yield at least 10 g after
grinding.
7.2 Transfer 50 g of the soil/sediment to a 100-mL beaker. Add 50 ml
water and stir for 1 hr. Determine the pH of the sample with a glass
electrode and a pH meter while stirring.
7.3 Weigh 10.0 g of suitably dispersed material into a 75-mL glass
flask, adjust the pH as required, and add 30 ml of an appropriate solvent.
Sonicate with agitation for approximately 3 min. Filter the resulting
suspension by using vacuum filtration, or centrifuge and decant the
extract. Reextract the solid residue with an additional 30-mL portion of
solvent. Repeat the extraction a third time and use fresh solvent each time.
7.4 After the extraction is complete, filter the extract and dry it by
passing it through a 4-in column of sodium sulfate which has been washed
with the extraction solvent. Collect the dried extract in a 500-mL
Kuderna-Danish flask fitted with a 10-mL graduated concentrator tube and a
three-ball Snyder column. Wash the extractor flask and sodium sulfate column
with 100 to 125 ml of the extraction solvent.
7.5 Add 1 or 2 clean boiling chips to the flask, and attach a
three-ball Snyder column. Prewet the Snyder column by-"adding about 1 ml
solvent to the top. Place the Kuderna-Danish apparatus on a steam or hot
water bath so that the concentrator tube and the entire lower rounded surface
of the flask are bathed in hot water or vapor. Adjust the vertical position
of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 min. At the proper rate of distillation, the balls
of the column will actively chatter, but the chambers will not flood. When
the apparent volume of liquid reaches 1 ml, remove the Kuderna-Danish
apparatus and allow it to drain for at least 10 min while cooling.
7.6 Remove the Snyder column and add 50 mL of the solvent specified in
the analytical method if a solvent exchange step is required. Concentrate
the extract as before. When the apparent volume of liquid reaches 1 ml,
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remove the Kuderna-Danish apparatus and allow it to drain and to cool for at
least 10 min.
7.7 Remove the Snyder column, rinse the flask and its lower joint with
1 to 2 ml of the solvent specified in the referring analytical method into
the concentrator tube.
7.8 If GPC cleanup is not required, proceed to paragraph 7.9; if GPC
cleanup is required, refer to Method 8080 for instructions on how to perform
it.
7.9 Nitrogen blowdown technique.
7.9.1 Place the concentrator tube in a warm-water bath (35°C), and
evaporate the solvent to 0.5 ml by using a gentle stream of clean, dry nitrogen
(filtered through a column of activated charcoal.
Caution: The extract must never be allowed to become dry.
7.9.2 Dilute the extract to 1 ml with solvent and proceed with the
cleanup or analysis as described in the referring analytical method.
7.10 Moisture determination: Immediately after weighing the sample for
extraction, weigh 5 to 10 g of the sample into a tared crucible. Determine
the percent moisture by drying overnight at 105°C. Allow to cool in a
desiccator before weighing. Concentrations of individual analytes will be
reported relative to the dry weight of soil or sediment.
weight of sample - weight of dry sample
x 100 = percent moisture
weight of sample
8. Quality Control
8.1 Comprehensive quality control procedures are" specified for each
target compound in the referring analytical method.
8.2 Before processing any samples, the analyst must analyze a solvent
blank to demonstrate that interferences from glassware are under control.
Each time a set of samples is extracted or reagents are changed, a solvent
blank must be processed as a safeguard against laboratory contamination.
8.3 By fortifying clean soil or another solid similar to the sample
matrix, the analyst should demonstrate that the compound(s) of interest
is being quantitatively recovered before applying this method to actual
samples.
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