5383
DEVELOPMENT AND APPLICATION OF TEST PROCEDURES FOR
SPECIFIC ORGANIC TOXIC SUBSTANCES IN WASTEWATERS
DRAFT FINAL REPORT
RECEIVED
FEB29 1980
LIBRARY, REGION V
AGENCY
by
John W. Rhoades
Richard E. Thomas
Donald E. Johnson
CATEGORY 1: Phthalate Esters
EPA Contract 68-03-2606
SwRI Project 01-5068-101
Prepared for
Mr. James E. Longbottom, Project Officer
Environmental Monitoring & Support Laboratory
Environmental Protection Agency
Cincinnati, Ohio 45268
January 1980
SOUTHWEST RESEARCH
INSTITUTE
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It
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring
and Support Laboratory, U.S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
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CONTENTS
Abstract iv
Figures vi
Tables : vii
1. Introduction 1
2. Conclusions 3
3. Recommendations 5
4. Phase 1 7
Objectives 7
Program Direction 7
Experimental 8
Literature Search 8
Gas Chromatography 9
Extraction Study 13
Preservation Study 25
5. Phase II 37
Objectives 37
Program Direction 37
Liquid-Solid Column Chromatography 38
Wastewater Application 42
Accuracy and Precision 52
Approach 52
Results 53
Summary 56
References 57
Appendices
A. Key References from Literature Search 58
B. Statistical Analyses Phase I Extraction Study 59
C. ANOVA Tables Phase I Extraction Study 66
D. Chromatograms of Wastewater Fractions - Phase II 73
E. Chromatograms of Wastewaters 1 through 5 Single
Fraction Collection - Phase II 76
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FIGURES
Number Page
1. Standard for DMP, DEP and DBP 10
2. Standard for BBP, DEHP and OOP 11
3. Unspiked water extract (100% DCM) 17
vi
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TABLES
Number Page
1. Retention Times 12
2. Sensitivity Values 12
3. Quantities of Spike for Extraction Study 14
4. Extraction Study Results - 100% DCM % Recovery. ... 19
5. Extraction Study Results - 15% DCM in Hexane
% Recovery 19
6. Summary of Extraction Study Analyses 26
7. Results of Preservation Study - DMP, DEP, DMP . . . . 29
8. Summary of Results of Preservation Study 35
9. Recovery {%) from Florisil PR Column 41
10. Recovery (%) from Alumina(N) Column 41
11. Analysis of Wastewater 1 for Phthalates 45
12. Analysis of Wastewater 2 for Phthalates 45
13. Analysis of Wastewater 3 for Phthalates 46
14. Analysis of Wastewater 4 for Phthalates 46
15. Analysis of Wastewater 5 for Phthalates 47
16. Analysis of Wastewater 1 Dosed with Phthalates. ... 48
17. Analysis of Wastewater 3 Dosed with Phthalates. ... 49
18. Analysis of Wastewater 4 Dosed with Phthalates. ... 50
19. Analysis of Wastewater 5 Dosed with Phthalates. ... 51
vii
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SECTION 1
INTRODUCTION
This project was part of an overall program of the Environmental
Protection Agency to establish test procedures for the analysis of
114 toxic substances in wastewaters. These toxic compounds have been
separated into 12 categories as follows:
Category 1: Phthalate Esters
Category 2: Haloethers
Category 3: Chlorinated Hydrocarbons
Category 4: Nitrobenzenes and Isophorone
Category 5: Nitrosamines
Category 6: Dioxin
Category 7: Benzidines
Category 8: Phenols
Category 9: Polynuclear Aromatics
Category 10: Pesticides and PCB's
Category 11: Purgeables
Category 12: Acrolein, Acrylonitrile, Dichlorodifluoromethane
This is the report on Category 1: Phthalate Esters.
The specific compounds studied in this effort were:
1. Dimethyl Phthalate (DMP) 4. Benzyl Butyl Phthalate (BBP)
2. Diethyl Phthalate (DEP) 5. Diethylhexyl Phthalate (DEHP)
3. Dibutyl Phthalate (DBP) - 6. Dioctyl Phthalate (OOP)
It was desirable that common sample treatment for the various
categories be employed, where possible, to minimize cost of analysis
of unrelated compounds in any given water sample. The efforts reported
under the following performance headings were designed to provide
information relative to this common purpose.
The study was conducted in two phases. In Phase I, work was
conducted with clean water and was intended to provide information
1
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which would give direction to Phase II work, conducted on actual wastewaters,
and serve as a comparison base for the information developed.
The ultimate objective was to develop a method, having a maximum
of applicability, which could be published in the Federal Register
and carried out in most analytical laboratories. Other objectives
were to develop accuracy and precision information for the various
steps in the method, to develop procedure variations as required by
a particular wastewater, and to maximize the use of common sample
treatment steps for all of the categories of organic toxic substances
listed above.
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SECTION 2
CONCLUSIONS
The six phthalate esters can be gas chromatographed very well.
Sensitivity quantities (producing a response 10X noise level) for
the electron capture detector range from 20 to 130 picograms. Sensitivity
quantities for the flame ionization detector are roughly 200-700 times
greater than for the electron capture detector.
Extraction of the phthalate esters from clean water at low parts
per billion (yg/L) concentrations when the pH was 2, 7, and 10 was
generally 90 percent or better when using 15 percent DCM in hexane
or 100 percent DCM as the extracting solvent.
At the concentrations mentioned above, some of the conditions
of the preservation study produced notable effects on some of the
phthalate esters. DMP and BBP extraction recoveries were reduced
to 30 percent and 40 percent, respectively, after storage for 7 days
in the dark at pH 10 and 24°C. Storage at 4°C under acid conditions
proved satisfactory.
Recoveries from Florisil and alumina cleanup columns were excellent
if the adsorbent was slightly deactivated by the addition of 3 percent
water.
Five wastewaters were analyzed and none was found to be completely
free of phthalate esters. The number of phthalate esters and amounts
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did vary considerably with the different wastewaters.
The recoveries of the phthalate esters dosed into the wastewaters
was, in general, good. Recoveries of selected phthalate esters was
somewhat reduced with some wastewaters and marked reduction in recoveries
from some wastewaters was noted after storage of the dosed wastewaters
for 7 days at 4°C.
The principal problems encountered with the method are contamination
of glassware and nonlinearity of response of the DMP and DEP with
the electron capture detector.
The method is basically acceptable for use on wastewaters having
a low level of interferences. It does not work well when background
levels are medium to high.
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SECTION 3
RECOMMENDATIONS
The gas chromatographic column used in this effort (1.5 percent
SP-2250 + 1.95 percent SP-2401 on 100/120 mesh Supelcoport), although
satisfactory for separation of all six phthalate esters, is only one
of many GC columns that could be used. The six phthalate esters are
separated primarily on the basis of boiling point and many other column
packings would be satisfactory.
The electron capture detector, being much more sensitive to the
phthalate esters than the flame ionization detector, is recommended
for most wastewater applications. The electron capture detector would
be less desirable with wastewaters contaminated with chlorinated compounds.
Either 15 percent DCM in hexane or pure DCM is satisfactory for
extraction of phthalate esters from wastewaters in the pH range of
2-10. If wastewater is to be stored prior to analysis for phthalate
esters, the water should be adjusted to acidic conditions and the
samples should be stored in a cold (4"C) room. A preliminary evaluation
of the wastewater would still be necessary to ensure that there was
no degradation under these conditions.
Florisil and alumina used in column chromatography must be deactivated
by addition of 3 percent water to insure recovery of all phthalate
esters.
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Great care should be exercised in preparing the reagents and
glassware used in the analysis. Phthalate esters, due to their occurrence
in many manufactured products, are a common source of sample contamination
in the laboratory, and frequent reagent blanks should be run to establish
levels of contamination.
Only chemists experienced with the method and with trace organic
analysis in general should conduct analysis for phthalate esters.
The method is recommended primarily for wastewaters with only
low levels of contamination. The collection of two fractions, instead
of one, from column chromatography cleanup may be helpful in some
cases and is recommended when higher levels of early eluting interferences
are suspected.
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SECTION 4
PHASE I
OBJECTIVES
The objectives of Phase I were limited to:
1. Review of scientific literature for key references relative to
determination of the phthalate esters in water and other matrices.
2. Determination of the best gas chromatographic columns and conditions
for detection of phthalate esters when using the detectors of
interest.
3. Determination of extraction efficiency for phthalate esters from
clean water samples.
PROGRAM DIRECTION
Before experimental work began, representatives of SwRI met in
Cincinnati with the project officer and others of concern in the EPA
Cincinnati laboratories to discuss and decide on certain specific
aspects of the activities in Phase I. In addition to the decisions
reached on GC columns, extraction solvents, and other experimental
details which will be given in subsequent sections of this report,
guidelines were developed as follows:
* Unusual, exotic, or overly expensive items were not desirable.
Simplicity was to be preferred wherever the option existed.
For example, mass spectrometry and WCOT or SCOT columns
were ruled out.
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* Use of chemical treatment to produce a derivative which
cannot be unequivocally related to the parent substance
was not acceptable.
* Quantisation by electronic integration of peak area or by
peak height measurement was acceptable.
EXPERIMENTAL
Literature Search
A comprehensive literature search was made using a terminal linked
to Lockheed Information Systems' DIALOG*. Chemical Abstract Condensates
(CAC) were searched for the years 1972 through the present. Also,
NTIS (1964 to present), Enviroline (1971 to present), and Pollution
Abstracts (1970 to present) were searched. In some cases, both on-
line and off-line prints were obtained, the former in order to rapidly
obtain a small enriched set of references to work with and the latter
to provide comprehensive coverage by retrieval of a larger set of
references.
The literature search as conducted yielded over 150 references.
Examination of titles and in many cases abstracts drastically reduced
the number to those listed in Appendix A as being of interest to this
program. The papers of G. S. Giam and his coworkers at Texas A&M
are of particular interest. The paper by Giam et al. entitled "Sensitive
Method for Determination of Phthalate Ester Plasticizer in Open-Ocean
Biota Samples" (Anal. Chem., Vol. 47, No. 13, November 1975) was probably
the most important product of the literature search. The authors
report several possible laboratory sources of phthalate esters which
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can/could result 1n contamination of samples. They also report the
necessity of deactivation of Florisil to prevent loss of DEHP when
column chromatography employing Florisil is used in a sample cleanup
procedure.
Gas Chromatography
All six (6) phthalates have been chromatographed on two columns.
The primary column (Column 1) is 1.8-m x 4-mm ID glass tubing packed
with 1.5-percent SP-2250 plus 1.95-percent SP-2401 on 100/120 mesh
Supelcoport. The secondary column (Column 2) has 3-percent OV-1 as
its liquid phase but is otherwise the same as Column 1.
Initial investigations with Column 1 indicated that all six phthalates
could be resolved at a column temperature of 200°C. However, the
retention time for OOP was excessive (34 minutes) and the early eluting
phthalates would be difficult to quantitate due to the proximity of
the solvent peak. This would be even more critical on extracts of
wastewater where the early eluters would be more likely to co-elute
with interferences. Therefore, the six phthalates were divided into
two groups of three for chromatography. The "low" temperature (160°C)
group includes DMP, DEP, DBP, while the "high" temperature (225°C)
group includes BBP, DEHP, and DOP. Table 1 gives the retention times
of the six phthalate esters on Column 1. Example chromatograms of
these compounds are shown as Figures 1 and 2.
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. 1 .
1 ; •
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;-4+
^— 7-1
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4
Figure 1. Standard for DMP, DEP and DBP,
10
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•— i lH. r . io-"?i—• - .•
0 2-4 6 8 10 12
BBP - 0.6 ng
OEHP - 6.0 ng
OOP - 9,8 ng
O Colunn 1 - 225'C - 16X - EC
Figure 2. Standard for BBP, DEHP and OOP
11
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TABLE 1. RETENTION TIMES
(Column 1; 60 mL/min)
Phtha
DMP
DEP
OBP
BBP
DEHP
OOP
late Ester
> 160°C
>• 225°C
Minutes
1.9
2.9
12.6
4.1
5.1
9.0
Investigations with Column 2 gave comparable results, leaving
little basis for recommending either column over the other. Both
columns perform satisfactorily, although somewhat greater sensitivities
were obtained with Column 1 (see below).
At the beginning of this work, the electron capture detector
was considered to be the primary detector to be used in the analysis
of phthalates, and the flame ionization detector was designated the
alternate detector. Experimentation confirmed that the electron capture
detector is preferred over the flame ionization detector on the bases
of the greater sensitivity and selectivity. Table 2 gives comparative
sensitivity values for the two detectors.
TABLE 2. SENSITIVITY VALUES
(Amount in nanograms required to produce response 1QX noise level)
Detector
Phthalate Ester
DMP
DEP
DBP
BBP
DEHP
OOP
Electron
Column 1
0.11
0.13
0.02
0.02
0.04
0.11
Capture
Column 2
1.3
1.9
0.3
0.5
0.7
0.9
Flame Ionization
Column 1
18.8
31.3
13.9
15.3
20.3
31.3
12
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The gas chromatographs used in this work were a Hewlett-Packard
5710A with a Ni-63 electron capture detector and a Hewlett-Packard
5710A with a flame ionization detector. Operating conditions of these
instruments were:
ECD FID
Oven temperature variable variable
Detector temperature 300°C 2508C
Carrier gas 5% methane in argon helium
Carrier gas flow rate 60 mL/min 60 mL/min
Flame gases — H2 + air
Extraction Study
The extraction study was initiated to determine the recoveries
of the six phthalates of interest from clean water at pH 2, 7, and
10 using 15 percent dichloromethane (DCM) in hexane and 100 percent
DCM as the extracting solvents.
The water used in the extraction study is a naturally buffered
well water obtained from the SwRI supply line prior to chlorination.
Samples of this water were collected and transported to the laboratory
in empty solvent bottles which had previously contained Burdick and
Jackson solvents. The pH of the untreated water is close to 8. Adjustment
to the required pH's was accomplished by adding strong acid or base
as follows:
pH 2 - 2.5 ml 18N H2S04
pH 7 - 0.1 ml 18N H2S04
pH 10-0.2 ml ION NaOH
The water was found to be very low in electron capture sensitive materials
as determined through comparison of an extract of the water with a
glassware blank. Chromatograms in these instances were very similar,
strongly indicating that the water could not be the source of any
13
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large ir .erferences.
The water was dosed with acetone solutions of the six phthalate
esters, each solution containing 100 times the sensitivity amount
per micro!iter, as determined earlier for each phthalate. By adding
100 vL of the appropriate acetone solution to a liter of water, dosed
water samples for extraction were prepared.
Poor reproducibility, contamination problems and loss of sensitivity
after replacement of a defective EC detector prompted an increase
in dosing levels after two attempts failed to get usable data from
the extraction study. Only the data from the third effort are reported,
as shown in Table 3.
TABLE 3. QUANTITIES OF SPIKE FOR EXTRACTION STUDY
Phthalate
DMP
DEP
DBP
BBP
DEHP
OOP
Dosing Solution
ug/mL
100
100
100
6
60
97.5
Cone, in l Liter
ng/mL
10
10
10
0.6
6.0
9.8
The one-liter samples of water were dosed while in one-liter
Erlenmeyer flasks, then poured into two-liter separatory funnels for
extraction by either 15 percent DCM in hexane or 100 percent DCM in
accordance with the procedure found in the Federal Register, Volume
38, No. 125, Friday, June 29, 1973, page 17320. Three dosed samples
were extracted at pH 2 and 10 with each solvent. At pH 7, four dosed
samples were extracted with each solvent. Thus, 20 samples were extracted
for each group. The final volume of the extract concentrate was always
14
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10 ml, and 10 yL of the concentrate was always Injected into the GC.
At the time' dosing of the samples occurred, 100 uL of the dosing solution
* was added to 10 ml of hexane which then served as the standard against
which all recoveries were measured.
The Federal Register procedure calls for the separated extracts
• to be drained through three to four inches of anhydrous sodium sulfate
into a Kuderna-Danish flask. Frequent plugging of the tube containing
the sodium sulfate was encountered. Usually unplugging could be achieved
• by applying a slight pressure to the top of the tube by means of a
2-ounce rubber bulb. It was discovered that the column of sodium
sulfate could be decreased in height to 3 to 4 centimeters without
* any obvious effect on the analytical results and with a more rapid
delivery of extract into the Kuderna-Danish flask. In the course
of unplugging the sodium sulfate tube, a small amount of water was
at
sometimes forced into the Kuderna-Danish flask. This situation produces
no harmful effects unless the quantity is so great as to cause an
intolerable volume error in determining the final 10-mL volume or
unless the water phase should be accidentally injected into the GC.
When the extracting solvent is 100 percent DCM, it is necessary
to remove essentially all of the DCM prior to analysis. This was
•
done by taking the extract to a volume of 10 to 15 ml, adding 75 to
100 ml of hexane, and then reconcentrating to the final volume. The
DCM extracts usually produced a wider "solvent" peak than those produced
by 15 percent DCM in hexane. This peak broadening was not reduced
when the amount of hexane added before reconcentration was increased
15
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in amounts up to 200 ml. The peak broadening incurred is not a serious
problem, however.
Occasionally, an emulsion resulted during the extraction process.
This occurred usually with the 15 percent OCM in hexane solvent and
at pH 2 and 7. After draining off most of the water phase, the emulsion
could be broken almost always by shaking or swirling the separatory
funnel. When emu!sification occurred, the solvent phase was usually
not as well separated from the water phase as when emu!sification
did not occur, resulting in a higher rate of plugging of the sodium
sulfate tube.
The most serious problem encountered was the inability to obtain
chromatograms of blanks for water, glassware and sodium sulfate which
were devoid of interfering peaks.
The most consistent interfering peaks were observed at the same
retention times as BBP and DEHP. Other interfering peaks with retention
times matching the other four phthalates were present at a lesser
frequency. Figure 3 is a chromatogram (at 225°C) of an unspiked water
extract using DCM. This chromatogram illustrates the magnitude of
the contamination problem.
The interfering peak from Figure 3 was concentrated and analyzed
by GC/MS to determine the nature of the contaminant. This interference
was identified as an adipate, possibly the ethylhexyl.
Elimination of contamination was never completely accomplished
and periodically caused serious problems with the phthalate determinations,
Whether glassware was cleaned and heated to 275°C overnight before
16
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I
-M-
-C Column 1 - 225'C - 8X
zn SBP q: OEHP (?n
EC
OOP
0 2 4 6 8 10 12
Figure 3. Unspiked water extract (100% DCM)
17
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use or was heated for several hours at 450°C before use seemed to
have little effect. Leaving the glassware in a chromic acid solution
(40 mL saturated I^C^Oy - H2S04 solution in 1 liter of water) for
12 to 18 hours and rinsing several times with deionized water, acetone
and petroleum ether prior to use seemed to give the best results.
Early in the study, the sodium sulfate was heated overnight at 450-
500°C before use. Some improvement resulted when this practice was
supplanted by Soxhlet extraction of the sodium sulfate with DCM for
two days.
The sodium sulfate, the holding tube and the glass wool plug
were all rinsed with DCM and hexane immediately before use.
After increasing the phthalate dosing levels, the problem with
contamination became manageable, and the data acquired during the
third extraction effort allowed reliable conclusions to be made with
respect to the extraction efficiencies of the solvents tested.
Percent recovery data were obtained by both computer integrated
peak areas and by peak height measurement. Only data based on peak
height measurement are reported here.
The data acquired in the extraction study are presented in Tables
4 and 5. The data have not been corrected for blank extractions.
Data Analysis—
The results of the extraction study were submitted to statistical
analysis to determine which of the factors affected the recovery efficiency
of the method. The statistical model for these data was a two-way
analysis of variance (ANOVA) defined by:
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TABLE 4. EXTRACTION STUDY RESULTS - 100% DCM % RECOVERY
pH
2
7
10
Extract
1
2
3
Mean
1
2 .
3
4
Mean
1
2
3
Mean
DMP
96
125
67
96
135
143
144
115
134
108
119
113
113
OEP
89
98
71
86
103
106
103
99
103
100
115
111
109
DBP
63
81
40
61
97
95
95
94
95
96
100
88
95
BBP
88
88
90
89
113
110
109
98
109
91
91
90
91
DEHP
102
92 •
101
98
104
99
98
98
100
100
99
112
104
OOP
94
90
96
93
99
97
96
94
97
90
95
94
93
TABLE 5. EXTRACTION STUDY RESULTS - 15% DCM IN HEXANE % RECOVERY
oH Extract
2 1
2
3
Mean
7 1
2
3
4
Mean
10 1
2
3
Mean
OMP
107
105
100
104
116
111
109
104
110
110
110
113
111
DEP
104
101
99
101
101
101
103
101
102
104
103
104
104
OBP
100
87
87
91
104
_
104
97
102
95
93
95
94
BBP
93
92
84
90
_
90
85
94
90
98
94
98
97
OEHP
99
96
127
107
»
101
104
98
101
105
102
112
110
OOP
95
95
91
94
91
92
91
93
92
97
96
99
97
- data not available - contamination (?)
19
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Yijk • u * Ti + pj + Tpij + eijk
where Yjik - observed recovery for solvent system i and pH j on
J replicate k, 1-1,2, j=l,2,3, k=l,2,3 or 1.....4
u - overall mean
TJ - effect due to solvent system i
PJ - effect due to pH j
Tpjj - interactive effect between solvent i and pH j
and Ejjjj - random error associated with Y^
Both solvent and pH are fixed effects, and all F-ratios were calculated
using the mean square for error as the denominator. The 5-percent
significance level was used on all tests.
The interaction between solvent and pH, if significant, implies
that the combination of a particular pH and a particular solvent performed
better (worse) than could be predicted by using the results from the
two factors taken separately. For example, if a difference of 7 percent
is observed between solvents and 6 percent between pH's, then using
the best combination would result in a predicted difference of 13
percent, the sum of the two differences. If the observed difference
were to be 20 percent, then the interaction term in the ANOVA would
be significant. This merely implies that the best estimate of the
expected recovery should be obtained from the results on that particular
combination, not from the pH or solvent means themselves. Interaction
is used in terms of a data analytical rather than physical approach.
A factor or interaction is considered to be significant if an
F-ratio calculated as part of the ANOVA procedure exceeds the corresponding
tabled value at the 5-percent significance level. This implies that
20
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there was more variability among the levels of a factor, e.g., among
the three pH's, than there was among replicate analyses under identical
conditions. In some cases, however, the precision of the individual
analyses allows differences to be termed significant that are too
small to be of any practical importance. This should be considered
in evaluating the results contained in this section.
Separate ANOVA's were conducted for each of the compounds in
the group, and the results were then synthesized to give the recommended
extraction conditions. There are several values in the data set which
appear unusual, either high or low, but none of these were eliminated
or replaced prior to conducting the ANOVA. The rationale behind this
was that there was insufficient information concerning the method
to warrant the use of an outlier test, which may eliminate values
likely to occur in actual use of the procedure. It was noted where
a particular significant result occurred due to one or more of these
values and a qualifying statement was added, where appropriate.
For those factors determined to be significant, a means separation
procedure was used, Fisher's LSD statistic, to determine where the
differences lay. The LSD is calculated according to the formula:
LSD.05 -
where LSD Qg - test statistic at 5-percent significance level
•t.05 (v) - student's value with v degrees of freedom at
the 5-percent level
n^, r\2 - number of observations used to obtain the mean values
and MSE - mean square for error from the ANOVA table.
21
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F tctor means are presented in the statistical summary tables in Appendix
B, along with the LSD values. The results obtained for each of the
phthalate esters and the overall conclusions are summarized below.
DMP—The dimethyl phthalate results are summarized in Table B.I
of Appendix B. The only significant term in the ANOVA was pH, with
pH 2 giving 100 percent recovery with statistically equivalent results
at pH 10, 112.1 percent recovery. These results are considered somewhat
spurious, however, in light of complicating factors. There was considerable
variability in the data, with an overall standard deviation of 13.1
percent recovery, due largely to the 100 percent DCM data. The DCM-
pH 2 results ranged from 67 to 125 percent recovery, and the average"
result of 96 percent recovery seems fortuitous. For the 15 percent
DCM in hexane data, the replicate values and the pH-to-pH values were
fairly consistent, with no real tendency observable. In view of the
data, it is felt that little weight should be given to this result.
Neither the solvent system nor the interaction term was significant,
and no preference for a solvent or solvent-pH combination was indicated
for these data. The conclusion then for the DMP is that no clear
tendency for better recovery was observed for any of the factors in
the experiment.
PEP—The statistical analyses for diethyl phthalate are summarized
in Table B.2 of Appendix B. For these data, both the pH and the pH-
solvent interaction terms were significant. The pH means indicate
that pH's 7 and 10 were equivalent with sample means of 102.1 and
106.2 percent recovery, respectively, and the pH 2 results were significantly
22
-------
lower, with an average of 93.7 percent recovery. The pH 2 data were
affected by one low value with 100 percent DCM but would have indicated
that a different true mean could be expectd even if this value had
been excluded. Similarly, the solvent-pH interaction term indicates
that the low average value for 100 percent DCM-pH 2 was the principal
difference among the means, but this would remain true if the average
of two results, excluding the one low one, was used. The interaction
term revealed that the low pH 2 results are a function of the low
100 percent DCM-pH 2 data and not a true pH effect. The indication
is then that if 100 percent DCM is used as the extraction solvent,
the wastewater should first be adjusted to a neutral-to-basic condition.
If 15 percent DCM in hexane is used, no pH adjustment would be necessary.
DBP—The summary of the statistical analyses on the dibutyl phthalate
data is given in Table B.3 of Appendix B. Both solvent and pH terms
were significant, as well as the solvent-pH interaction. Higher recoveries
were obtained using the 15 percent DCM-hexane system with 95.8 percent
recovery compared to 84.9 percent for the 100 percent DCM. The mean
recoveries for the varying pH's indicate equivalent recoveries at
pH's 7 and 10, 98.0 and 94.5 percent, respectively, with lower overall
recovery at pH 2 of 76.3 percent.
The interaction means indicate, however, that in both of these
cases low recovery with 100 percent DCM-pH 2 was the reason for these
differences. For the remaining solvent-pH combinations, the average
recovery ranged from 91.3 to 101.7 percent, all acceptable results.
As in the previous data, the conclusion is that if 100 percent DCM
23
-------
is used, an adjustment to a neutral-to-basic condition would be required,
but no adjustment of pH need be made if 15 percent DCM in hexane is
used as the extraction solvent.
BBP--The summary of the ANOVA on the benzyl-butyl phthalate data
is shown in Table B.4 of Appendix B. The significant terms were the
solvent, the pH, and the solvent-pH interaction. The preferred solvent
was 100 percent DCM with a recovery of 97.3 percent compared to 93.7
percent for 15 percent DCM in hexane. Better results were obtained
at pH 7 than at pH 2 or 10, 100.6 percent recovery versus 89.2 and
93.7, respectively. The separation of the interaction means, however,
revealed that these differences were influenced by a high average
result for 100 percent DCM-pH 7 and are not truly pH and solvent effects.
All of the solvent-pH combinations were equivalent with the exception
of the 100 percent DCM-pH 7, which had an average recovery of 108.8
percent. The remaining combinations showed recoveries of 88.7 to
96.7 percent and were all acceptable recoveries. The conclusion for
BBP then is that either solvent may be used, but if 100 percent DCM
is used, the pH of the wastewater should be adjusted to a nonneutral
condition.
DEHP—The summary of the statistical analyses on the diethylhexyl
phthalate data is contained in Table B.5 of Appendix B. For these
data there were no significant terms in the ANOVA, and the conclusion
is that neither solvent nor pH affected the recovery of this compound.
The overall mean recovery was 103.1 percent.
D|OP_—The statistical analyses on the dioctyl phthalate data are
summarized in Table B.6 of Appendix B. These data gave one significant
24
-------
term 1n the ANOVA, the solvent-pH interaction term. The best results
were obtained with 100 percent DCM-pH 7 and 15 percent DCM-hexane-
pH 10 with 96.5 and 97.3 percent recovery, respectively. The other
four combinations were equivalent to each other ranging from 91.8
to 93.3 percent recovery so all combinations produced good recoveries.
The preference for those two sets of conditions is slight and may
not be of practical significance.
Summary ~
The results of the analyses on the six compounds are summarized
in Table 6. From this it can be seen that no clear tendency can be
detected for one solvent system to produce superior recoveries or
for the recoveries to be influenced by the pH of the water. The principal
differences occurred when 100 percent DCM was used and a particular
pH gave unacceptable results, especially in the cases of DEP and DBP.
When 15 percent DCM in hexane was used as the extraction solvent,
no differences were detected among the pH's. Another factor in the
evaluation is that the 15 pecent DCM-hexane solvent system produced
more consistent results with fewer of the apparent contaminations
and none of the low recoveries. The conclusion then is that 15 percent
DCM-hexane should be used for extracting the wastewater and no adjustment
of the pH need be made. The mean recoveries obtained with 15 percent
DCM-hexane at the three pH's are shown at the bottom of the summary
table to indicate the recovery obtained.
Preservation Study
The preservation study was conducted to determine the effects
of a 7-day storage period at various conditions on the recovery of
25
-------
TABLE 6. SUMMARY OF EXTRACTION STUDY ANALYSES
Compound
OMP
OEP
OBP
BBP
OEHP
OOP
Solvent
-.*
—
15% DCM-hexane
1002 OCM
~
_w
FACTOR
PH
U*
7,10
7,10
7
—
• <•
-
Solvent x pH
not 1002 DCM-pH 2
not 1002 DCM-pH 2
not 1002 DCM-pH 7
...
1002 DCM-oH 7
Means (152 OCM in Hexane)
COMPOUND
152 DCM-hexane-pH 10
EH
2
7
10
DMP
104.0
110.0
111.0
DEP
101.3
101.5
103.7
DBP
91.3
101.7
94.3
BBP
89.7
89.7
96.7
DEHP
107.31
101.3
109.?!
OOP
93.7
91.8
97.3
* Line indicates no significant difference.
t U indicates data do not lend themselves to ready interpretation.
! Influenced by single high value.
26
-------
the six phthalate esters of interest from dosed water samples. Each
sample consisted of one quart of water dosed with six phthalates,
as in the extraction study (see Table 3). Two replicates for each
of twelve conditions of pH, temperature, and residual chlorine were
prepared for each group as shown in the following model:
pH 2
pH 7
pH 10
4"
0 ppm Cl
2
2
2
C
2 ppm Cl
2
2
2
0 ppm Cl
2
2
2
rc
2 ppm Cl
2
2
2
The 2 ppm residual chlorine level was obtained, where required, by
adding 160 microliters of Mallinckrodt sodium hydrochlorite analytical
reagent (5 percent minimum available Cl). This quantity of hypochlorite
solution was determined by preparing a standard chlorine solution
which was diluted to five different concentrations that were used
to prepare an absorbance versus concentration curve. Storage containers
were one-quart, flint-glass, round, narrow-mouth bottles with aluminum
foil-lined caps. A sample was prepared by filling the bottle about
two-thirds full with pH-adjusted water, adding 160 microliters of
sodium hypochlorite (when required), swirling vigorously, adding the
100 microliters of dosing solution into the vortex, and adding the
remainder of the pH-adjusted water while that already in the bottle
was still swirling. The bottle was capped immediately and stored
in a closed cardboard box at 4°C or 24°C. Care was taken not to slosh
the bottle contents onto the aluminum lining of the cap after closure.
After storage, samples were extracted and analyzed as in the
extraction study.
27
-------
The third extraction study had not been completed when the preservation
study was started. This made it necessary to select an extraction
solvent and extraction pH without the benefit of prior data. The
solvent selected was 15 percent DCM-hexane, Considerations in selecting
this solvent were its proven extraction efficiency with other priority
pollutants (pesticides and RGB's), its relative general use, and the
ideal situation of conforming as many priority pollutant methodologies
to a standard procedure as possible. The preservation study samples
were extracted at their given pH without any prior adjustment.
Data obtained in the preservation study was given in Table 7.
The data presented are uncorrected for values obtained for blanks
at each of the 12 test conditions because of the variation of these
values.
Data Analysis—
The results of the preservation study were examined by submitting
the percent recoveries to an analysis of variance (ANOVA) procedure
to determine which of the factors affected the recovery upon storage.
The ANOVA for these data is based on a 3x2x2 factorial experiment
with two replicates of each combination. The model is presented by:
Y-i-iH ~ ^ "*" P-i "*" "^-i "*" Y|» "*" PT-i-i + PY-Jlx "*" "nf-iU + PTY-HI/ "*" £» -5H
IJI\I I J IV IJ IK JN IJK IJKI
where
Yijkl " °Dserved recovery for pH i, temperature j, chlorine
level k and repetition 1
y - overall mean
P1 - effect of pH i, 1=1,2,3
28
-------
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pTij» p^fk» Tyjk " tw°-fac
-------
for pH, temperature, and/or chlorine taken Individually. The conclusion
is that the means of the combinations should be used to provide the
estimates of percent recovery, and no physical interpretation is implied.
The precision of the analyses allowed, in some cases, relatively small
differences to be termed significant, while there may be no practical
value to the difference between average recoveries in these cases.
DMP—The results for dimethyl phthalate showed that all main
effects and interactions had a significant effect on the recoveries
obtained. The most prominent effects, as determined by the magnitude
of the mean square for each factor, were pH, temperature, and the
pH-temperature interaction. The LSD for the pH factor showed that
all three sample means could be considered to be estimating different
true (population) means. At pH 7, the average recovery was 100.0
percent, while 108.0 percent was obtained for pH 2 and 59.1 percent
for pH 10. The best conditions for this phthalate then was storage
at neutral conditions with basic conditions clearly unacceptable.
The better temperature for storage was 4°C with an average recovery
of 95.8 percent versus 82.2 percent at room temperature.
The pH-temperature combination means gave more information.
At pH's 2 and 7, averages above 100 percent were obtained at room
temperature storage (112.5, 106), while the pH 2-4°C mean was 103.5
and the pH 7-4°C mean was 94.0. The mean recovery for pH 10-4°C was
90.0 percent, an acceptable result, but the recovery for pH 10-24°C
was only 28.2 percent. The average recovery shown above for pH 10
thus was greatly affected by the results from room temperature storage.
31
-------
Clearly then the data Indicate that cold storage is recommended
for preserving samples for DMP, and some preference is noted for neutral
conditions. None of the pH's is unacceptable under storage at 4°C,
but the results at pH 2 were closest to 100 percent, although slightly
above. There was no effect on the results at pH 2 or 7 resulting
from the presence or absence of residual chlorine in the water.
PEP—There were three significant terms in the ANOVA of the diethyl
phthalate data, pH, temperature, and the pH-temperature interaction.
The pH results indicated that better recovery was obtained at pH's
of 2 and 7 (98.5 and 99.1 percent recovery, respectively) than at
pH 10 (90.6 percent recovery). Storage at 4°C produced a significantly
higher average result, but the difference was slight, with 97.9 percent
observed for 4°C and 94.2 percent recovery for 24°C.
The pH-temperature interaction means revealed a similar pattern
to the DMP results. The principal difference among the means was
the low average recovery (82.0 percent) for the samples stored at
pH 10 and at room temperature. Neither the chlorine term nor any
of the interactions involving chlorine were significant, and the conclusion
is that residual chlorine in the water will not affect the results
obtained for DEP upon storage of the samples.
DBF—The dibutyl phthalate results were analyzed according to
the ANOVA model above and gave two significant terms, pH and the pH-
temperature interaction. The best results were obtained under storage
at pH 7 with 90.8 percent recovery on average. The mean for pH 2
(86.5 percent recovery) was indistinguishable from the pH 7 mean using
32
-------
the LSD statistic, but storage at pH 10 produced a significantly lower
result (83.4 percent recovery). As In the previous data, however,
the Interaction term revealed that the principal reason for this difference
was the low result for pH 10-24°C. No effect was noted due to the
presence or absence of residual chlorine in the water, and this does
not appear to be a concern in analyzing for this compound.
BBP—The recoveries for benzyl-butyl phthalate gave three significant
terms in the ANOVA, pH, temperature, and the pH-temperature interaction.
The three pH's produced distinct means for this compound, with storage
at pH 2 giving the best results, 93.8 percent recovery, followed by
pH 7 and pH 10 (72.8 and 60.3 percent recovery, respectively). Higher
results were obtained on average when storage was at 4°C as opposed
to room temperature, with mean recoveries of 84.3 percent and 67.0
percent, respectively. From the pH-temperature interaction, however,
no significant difference was observed between the 4°C and 24°C results
at pH 2 (96.0 and 91.8 percent recovery, respectively). As a result,
for this compound an adjustment to pH 2 prior to storage would remove
the necessity for cold storage. As in the previous analyses, the
significance of the pH-temperature interaction is largely due to the
loss of BBP at pH 10-24°C, and no chlorine effect, either alone or
in combination, was observed in the results.
DEHP—The diethylhexyl phthalate data gave one significant factor
in the ANOVA, the pH of the water. The best results were obtained
when the samples were stored at pH 2, with an average of 100.8 percent
recovery. The average for pH 7, 91.8 percent recovery, was indistinguishable
33
-------
from the pH 2 mean using the LSD statistic, but only marginally.
No other factors or Interactions were significant, and the results
were not affected by either the presence or absence of chlorine.
POP-—The dioctyl phthalate results gave one significant main
effect, pH, and three significant interaction terms, pH-temperature,
pH-chlorine, and temperature-chlorine. The pH results were consistent
with the other compounds, showing best recovery at pH 2 (95.2 percent)
with pH's 7 and 10 lower and equivalent to each other (86.2 and 84.2
percent, respectively). The pH-temperature interaction term revealed
that equivalent results were obtained at pH 2 for either 4° or 24°C,
and at pH 7-4°C. The other combinations were equivalent to each other
but lower.
The two interaction terms involving chlorine provided no information
that would indicate a problem for these analyses. For the pH 2 data,
the same average result (95.2 percent recovery) was obtained for both
0 and 2 ppm residual chlorine in the water, which was the highest
average obtained. For the temperature-chlorine interaction term,
the data indicated that storage at 24°C-0 ppm chlorine gave a lower
average result than the other combinations. However, previous results
indicate a preference for cold storage so the presence or absence
of chlorine, assuming storage at 4°C, is not a problem for this compound.
Summary—
The results of the analyses on the six compounds in this category
are summarized in Table 8. On the basis of the trends shown, the
recommended conditions for storage would specify an adjustment of
34
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35
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the water to acidic conditions, and storage at a temperature of 4°C.
Under these conditions, no Interference can be expected from residual
chlorine up to the 2 ppm level.
Adjustment of wastewater samples to pH 2 may not be practical
under field conditions and may be avoided with minimal effect on the
determination of phthalate esters, provided the samples are stored
at 4°C.
36
-------
SECTION 5
PHASE II
OBJECTIVES
The objectives of Phase II were as follows:
1. Investigation of two liquid-solid chromatographic cleanup procedures,
including the determination of the separation patterns and recoveries
for all of the substances of interest.
2. Application of the method to five wastewater samples from industrial
discharges. This aspect of the work included modifications of
the method as required to analyze the wastewaters. These modifications
would be based on experience gained earlier in the program and
on new developments reported in scientific literature.
3. Generation of accuracy and precision data for all substances
of interest by dosing them into the five wastewaters. Determinations
would be made immediately after dosing and after one week of
storage at the best conditions observed in Phase I.
PROGRAM DIRECTION
Representatives of SwRI met with the project officer in Cincinnati
before Phase II work commenced. Discussion with respect to Phase
II was primarily on the cleanup columns and procedures to be used.
Florisil and alumina were selected as the adsorptive solids. Fractionation
of compounds was limited to that which can be obtained on the above-
37
-------
named solids. Fractionation on secondary columns such as silica gel
or charcoal was considered to be beyond the scope of this program.
DCM was chosen as the extracting solvent to be used with the
wastewater samples.
LIQUID-SOLID COLUMN CHROMATOGRAPHY
The first column chromatography method to be investigated for
cleanup of phthalate esters was based on the work reported by C. S.
Giam et al.^ It was found by Giam et al. that incomplete recoveries
of phthalate esters were obtained from Florisil unless the Florisil
was deactivated with 3 percent water. Two procedures were developed:
one employing 35 grams of Florisil for samples with a high lipid content
and the second procedure using only 10 grams of Florisil for samples
with a low lipid content.
Initial studies using the 10 grams of deactivated Florisil (3
percent fr^O) and the reported solvent system indicated that modification
of the procedure was required. The dimethyl and diethyl phthalates
are more polar than the other phthalates used by Giam and were not
eluted from the column when following the reported procedure.
/
The modified procedure, used in this study, is as follows:
Prepare Florisil (PR) by heating to 400°C overnight. Then add 3 percent
HpO (w/w) and mix thoroughly. Let stand for at least 2 hours. Use
10-mm ID column chromatography tube with glass frit and Teflon stopcock
(Kontes Catalog K420540-0213).
1. Add 10 g of Florisil (3 percent ^0) to the 1-cm ID chromatography
tube and tap to settle.
2. Add =1 cm $0 to top of column.
38
-------
3. Add hexane to tube and pre-elute 40 ml. Discard.
4. Drain until solvent level is even with top of ^SC^.
5. Add sample in =2 ml hexane to top of column.
6. Drain sample onto column (to top of Na2S04).
7. Wash sides of tube with =2-mL hexane and drain into top of ^SO^j.
8. Add hexane to top of column and elute 40 ml of hexane. Discard
this fraction.
9. Drain hexane to top of Na2S04 and add -2 ml 20 percent ether/hexane.
10. Drain new solvent to top of Na2S04. Add more 20 percent ether/hexane.
11. Place a 500-mL Kuderna-Danish evaporator below the column.
12. Collect 100 ml of the 20 percent ether/hexane eluate in the K-D
evaporator.
13. Evaporate the above eluate to <10 ml on a steam bath.
14. Transfer to a solvent-rinsed 4-dram vial with Teflon cap liner.
15. Adjust final volume of concentrate to 10 ml. Evaporate under
nitrogen if necessary.
The second column procedure developed for this program, using alumina
as the adsorbent, is as follows:
Prepare Alumina (Woelm N) by heating to 400°C overnight. Then add
3 percent H20 (w/w) and mix thoroughly. Let stand overnight in sealed
jar. Use 10-mm ID column chromatography tube with glass frit and
Teflon stopcock (Kontes Catalog K420540-0213).
1. Add 10 g of Alumina (3 percent H20) to the 1-cm ID chromatography
tube and tap lightly to settle.
2. Add -1 cm Na2S04 to top of column.
3. Add hexane to tube and pre-elute 40 ml. Discard.
39
-------
4. Drain until solvent level is even with top of ^SO^
5. Add sample in -2 ml hexane to top of column.
6. Drain sample onto column (to top of ^SO^).
7. Wash sides of tube with = 2 ml hexane and drain into top of ^SO^.
8. Add hexane to top of column and elute 35 ml of hexane. Discard
this fraction.
9. Place a 500 ml Kuderna-Danish evaporator below the column.
10. Drain hexane to top of ^$04 and add =2-ml 20 percent ether/hexane.
11. Drain new solvent to top of ^SO^. Add more 20 percent ether/hexane.
12. Collect 140 ml of the 20 percent ether/hexane eluate in the K-D
evaporator.
13. Evaporate the above eluate to -10 ml on a steam bath.
14. Transfer to a solvent-rinsed 4-dram vial with Teflon cap liner.
Rinse twice with hexane and add rinses to vial.
15. Adjust final volume of concentrate to 10 ml. Evaporate under
nitrogen if necessary.
A gel permeation column using Sephadex was considered for use
in this work, but when a computer search of the literature failed
to give any references relative to the use of this substance other
than for removal of lipids, no further use was contemplated. Lipids
are not a problem contaminant with most nondomestic wastewaters.
The cleanup procedures using Florisil and alumina were evaluated
as to the recoveries that could be obtained when doses of the six
phthalates were applied to columns of these adsorbents. Table 9 shows
the recoveries obtained from five replicate samples when using the
Florisil cleanup column. Table 10 shows the recoveries obtained from
40
-------
TABLE 9. RECOVERY* (.%) FROM FLORISIL PR COLUMN
Sampl e
1
2
3
4
5
Avg
DMP
114
115
114
115
117
115
DEP
114
115
114
117
118
116
DBP
99
97
97
98
99
98
BBP
93
92
92
93
94
93
DEHP
98
96
97
96
97
97
DOP
97
94
96
94
95
95
TABLE 10.
RECOVERY*
(*)
FROM ALUMINA(N)
COLUMN
Sample
1
2
3
4
5
Avg
DMP
114
112
114
115
115
114
DFP
108
107
109
111
111
109
DBP
97
97
98
99
97
98
BBP
100
102
99
99
98
100
DEHP
87
85
97
99
84
91
DOP
95
95
98
99
93
96
* Amount added to column in 2 mL hexane.
DMP-15yg, DEP-15yg, DBP-8yg, BBP-lyg, DEHP-8yg, DOP-20yg
41
-------
five replicate s mples using the Alumina N cleanup column. The recoveries
for all six test phthalates from both materials appear to be very
good, averaging 90 percent or better. The high recoveries for DMP
and DEP were unexplained since blank tests showed only small (2-3
percent) interferences or contamination for these two compounds.
A lack of a linear detector response could have been responsible.
WASTEWATER APPLICATION
With the assistance and approval of the project officer, five
wastewaters were procured and analyzed. All samples were put in clean,
one-gallon bottles and shipped unrefrigerated via air to Southwest
Research Institute laboratories. Upon receipt at SwRI, all the wastewater
from a particular source (except Wastewater 2) were pooled, adjusted
to pH 5-7 if necessary, returned to the bottles in which they were
shipped, and stored at 4°C (in the dark) until used. Due to the high
acid content of Wastewater 2 (approximately 48 g/L NaOH required to
neutralize), it was stored at 4°C as received.
In order to develop method improvements and to provide base data
for dosing and recovery experiments and for the accuracy and precision
evaluations to follow, each wastewater was analyzed in triplicate
for each substance of interest in this program.
One liter of wastewaters was extracted three times using 60-mL
DCM for each extraction. The combined extract was dried with ^$04
and placed in a Kuderna-Danish evaporator. The DCM extract was concentrated
to 5-10 ml, 90 ml of hexane was added, and the extract was concentrated
to slightly less than 10 ml in the K-D. The sample was then transferred
42
-------
to small vial and concentrated to 2 ml. This 2-ml concentrated extract
was then subjected to the Florisil cleanup procedure as previously
described. One procedural change, used only with Wastewaters 3, 4,
and 5, was attempted and with some success. In the Florisil cleanup
procedure, instead of collecting a single fraction of 100 ml of eluting
solvent containing all the phthalate esters of interest, a two-fraction
collection was made. Fraction 1 (first 60 ml) contained nearly all
of the OOP and DEHP, most of the DBP and BBP and, in some instances,
much of the early GC-eluting material. Fraction 2 (next 40 ml) contained
all of the DMP, most of the DEP, some DEP and BBP and, in some instances,
reduced amounts of early GC-eluting material(s) which otherwise interfere
with detection of the DMP and DEP. This method of collecting two
fractions instead of one may be of some help in the analysis for DMP
and DEP. For the analysis of the other phthalate esters, it is best
to combine fractions (or collect only the single fraction) before
GC analysis and thus avoid the complications of partial separation
of some of the phthalate esters.
Wastewaters 1 and 2 were analyzed by collection and analysis
of only one fraction. Wastewaters 3,4, and 5 were examined by collection
of the two fractions. Chromatograms of Fraction 2, analyzed for DMP
and DEP only and of combined Fractions 1 and 2, analyzed for the remaining
phthalate esters, are shown 1n Figures D.I and D.2 in Appendix D for
dosed Wastewater 3.
Chromatograms of a standard mixture and Wastewater 1 through
5 (single fraction collection) are shown in Appendix E.
43
-------
The gas chromatography for the wastewater analysis was performed
using Column 1. Samples were analyzed by holding the column at 180°C
for 16 minutes for determination of DMP, DEP, and DBP. The temperature
was then raised rapidly to 220°C to decrease elution time of the BBP,
DEHP, and OOP. All analytical data were obtained using peak height
measurements. All extracts were diluted so compounds of interest
would not go off scale at 8X, thus assuring linearity of the EC detector
for DBP, BBP, DEHP, and DOP. No linear range for DMP and DEP was
found with the EC detector, and analytical data for these two compounds
were obtained from calibration curves.
Tables 11 through 15 show the results of the analysis of raw
Wastewaters 1 through 5.
Tables 16 through 19 show the results of the analysis of Wastewaters
1, 3, 4, and 5 dosed with the six phthalate esters under study.
The data presented in the tables referred to below were used
to calculate a "Method Recovery", i.e., the percent recovered of the
amount added at zero time, and a "Preservation Recovery", i.e., the
percent remaining after seven days of the total amount in the dosed
wastewater at zero time. This was performed as follows:
let k = background (amount found in the undosed wastewater)
let y = quantity added to wastewater
let XQ = quantity found in dosed wastewater at zero time for
replicates 1, 2, or 3.
let XQ av_ = average quantity found in dosed wastewater at zero
time
44
-------
TABLE 11. ANALYSIS OF WASTEWATER 1 FOR PHTHALATES, ug/L
Replicate DMP DEP DBP
1 I* I 157
2 I I 151
3 I I 157
"k" ug/L
Average I I 155
BBP DEHP OOP
8.0 11.6 0
12.8 13.9 0
6.2 13.9 0
9.0 13.1 0
*I - Interfering Peak(s)
TABLE 12. ANALYSIS OF WASTEWATER
2 FOR PHTHALATES, ug/L
Replicate DMP DEP DBP
1 I* 1430 I
2 I 1500 I
3 I 1580 I
"k" ug/L
Average I 1500 I
BBP DEHP OOP
232 I 3260
304 I 3820
304 I 4020
280 I 3700
*I - Interfering Peak(s)
45
-------
TABLE 13. ANALYSIS OF WASTEWATER 3 FOR PHTHALATES, ug/L
Replicate
1
2
3
11 k" ug/L
Average
DMP DEP DBP
0 0 2.4
0 0 1.8
0 '0 2.9
0 0 2.3
BBP
0.8
0.6
1.0
0.8
DEHP
6.6
6.5
6.6
6.6
DOP
0
0
0
0
TABLE 14.
ANALYSIS OF WASTEWATER 4
FOR
PHTHALATES, u<
3/L
Replicate
1
2
3
"k" ug/L
Average
DMP DEP DBP
000
000
000
000
BBP
0.7
0.4
0.9
0.7
DEHP
1.0
1.0
1.0 •
1.0
DOP
0
0
0
0
46
-------
TABLE 15. ANALYSIS OF WASTEWATER 5 FOR PHTHALATES, ug/L
Replicate DMP DEP DBP BBP DEHP OOP
1
2
3
"k" wg/L
Average
0
0
0
0
5.8
5.8
3.8
5.1
1.9
3.7
3.7
3.1
3.2
2.5
2.1
2.6
255
269
285
270
0
0
0
0
47
-------
TABLE 16. ANALYSIS OF WASTEWATER 1 DOSED WITH PHTHALATES
Replicate
Zero time
1
Method recovery.^
2
Method recovery,%
3
Method recovery,%
XQ Avg.
k
Method recovery,% Avg.
7 days at 4°C
1
Preservation recovery,%
2
Preservation recovery,%
3
Preservation recovery,%
Preservation Recovery,
% Average I*
Dose, ug/L
0 0 1500
MP DEP DBP*
* I
1380
82
1360
80
1360
80
1370
* 155 •
81
1390
101
1370
100
1610
118
100
BBP*
86
77
86
77
86
77
86
9.0
77
85
99
80
93
77
90
150
DEHP*
122
69
116
69
117
73
118
13.1
70
111
94
109
92
116
98
150
OOP*
119
79
116
77
116
77
117
0
78
112
96
121
103
116
99
I*
106
94
95
99
* Huge interferences made spiking unfeasible at ug/L levels.
t Extract diluted 500x to see DBP in linear range of EC detector.
4= Extract diluted 50x to see BBP, DEHP, & DOP in linear range of EC detector.
48
-------
TABLE 17. ANALYSIS OF WASTEWATER 3 DOSED WITH PHTHALATES
Replicate
45
DMP*
45
DEP*
Dose,
24
DBPt
uq/L
3
BBPt
24
DEHPt
60
DOPt
Zero Time
1 45 45 25.6 3.8 24.1 46.4
Method Recovery,% 100 100 97 100 73 77
2 45 45 23.2 3.6 20.2 34.4
Method Recovery,% 100 100 87 93 57 57
3 45 45 23.8 4.4 22.2 37.6
Method Recovery,% 100 100 90 120 65 63
Xn Avg. 45 45 24.2 4.0 22.2 39.5
ku 00 2.3 0.8 6.6 0
Method Recovery,%Avg. 100 100 91 124 65 66
7 Days at 4°C
1 36.0 39.3 18.7 3.1 23.4 36.5
Preservation Recovery,£ 80 87 77 79 105 92
2 29.7 38.1 16.4 3.4 24.5 34.4
Preservation Recovery,% 66 85 68 85 110 87
3 32.1 39.3 14.5 3.0 24.6 32.2
Preservation Recovery,% 71 87 60 76 111 82
Preservation Recovery
% Avg. 72 86 68 80 109 87
*DMP & DEP determined from Fraction 2 of Florisil eluate diluted 15x to
come within EC detector calibration range.
fl)BP, BBP, DEHP, & OOP determined from total Florisil eluate (Fracs. 1&2
recombined) diluted 3x to come within linear range of EC detector.
49
-------
TABLE 18. ANALYSIS OF WASTEWATER 4 DOSED WITH PHTHALATES
Dose, pg/L
Replicate
Zero Time
1
Method Recovery, %
2
Method Recovery ,%
3
Method Recovery, %
X0 Avg.
k
Method Recovery, % Avg.
7 Days at 4°C
1
Preservation Recovery, %
2
Preservation Recovery, %
3
Preservation Recovery, %
15
DMP*
12.9
86
13.2
88
13.3
89
13.1
0
88
13.7
105
14.3
109
14.1
108
15
PEP
12.2
81
12.3
82
12.6
84
12.4
0
82
12.9
104
13.2
106
13.1
106
20
DBP
17.6
.88
18.2
91
18.9
95
18.2
0
91
18.2
100
18.6
102
18.1
99
5
BBP
4.2
70
4.6
78
4.7
80
4.5
0.7
76
4.3
96
4.3
96
4.3
96
40
DEHP
19.4
47
19.4
47
23.0
56
20.6
1.0
50
27.9
134
29.5
143
29.3
142
40
POP
18.3
46
19.4
49
21.7
54
19.8
0
50
26.5
134
28.7
145
27.6
139
Preservation Recovery
% Avg. 107 106 100 96 140 139
* All components determined from total extract diluted 1:5 to come in
linear or calibration range of EC detector.
50
-------
TABLE 19. ANALYSIS OF WASTEWATER 5 DOSED WITH PHTHALATES
Dose, yg/L
Replicate
Zero Time
1
Method Recovery, %
2
Method Recovery, %
3
Method Recovery, %
X0 Avg.
u
k
Method Recovery t% Avg.
7 Days at 4°C
1
Preservation Recovery, %
2
Preservation Recovery, %
3
Preservation Recovery, %
Preservation Recovery
% Avg.
50
DMP*
50
100
49
98
47
94
49
0
97
50
102
49
100
48
98
100
50
DEP*
52
94
51
92
50
90
51
5.1
92
49
96
49
96
48
94
95
30
DBF*
18
49
15
40
13
33
15.3
3.1
41
9
59
9
59
10
65
61
30
BBP*
23
68
22
65
19
55
21.3
2.6
63
13
61
15
70
14
65
65
1000
DEHP*
980
71
980
71
980
71
980
270
71
740
76
770
79
780
80
78
150
POP*
140
93
140
93
140
93
140
0
93
110
79
110
79
110
79
79
DMP & DEP determined from Fraction 2 of Florisil eluate diluted 50x
to come within EC detector calibration range.
DBP, BBP, DEHP, & OOP determined from total Florisil eluate
(Fracs. 1&2 recombined) diluted 25x to come within linear range of
EC detector.
51
-------
let Xy avg * average quantity found in dosed wastewater after
seven days for replicates 1, 2, and 3.
Then
"Method Recovery" = (xQ-k)100/y % (calculated for each replicate)
and
"Preservation Recovery" = (x7)(100)/x0 avg % (calculated for
each replicate)
No dosed recovery or preservation data were obtained on Wastewater
2 due to obvious deterioration of the sample.
ACCURACY AND PRECISION
Approach
The accuracy and precision assessment for the method was of a
limited nature due to the number and types of analytical results obtained,
According to the design of the program three replicates of each of
five wastewaters were to be dosed for the compounds of interest and
analyzed, both at a zero time and after seven days storage under the
appropriate conditions. This provided 15 determinations of recovery
of the dose for statistical analyses. However, the five wastewaters
could not be considered equivalent, either in their nature or in the
behavior of the method when applied, so combining the results to form
an overall evaluation of the method was inappropriate. Instead, the
results of each wastewater were treated individually, and no general
statement about the method was attempted.
The accuracy of a method refers to its ability to obtain on average
the true value, within the limits of the variability inherent in the
" 52
-------
method. For this study, the accuracy standard used was the percent
recovery of the amount of the particular phthalate ester dosed Into
the wastewater, with 100 percent representing an accurate method.
Precision relates to the variability among measurements arising from
various sources and is considered separately from the accuracy evaluation.
It 1s possible for a method to be precise, meaning having little variability
among measurements, without being considered accurate. For this study,
the precision measurement used was the range or the difference between
the highest and lowest recoveries. While the range is sensitive to
single extreme values, it was deemed more appropriate for these data
than other measures, given the number of observations available.
The recoveries obtained after seven days storage were examined
separately to evaluate the effects of storage on the observed levels
of these compounds. These recoveries are based on the total amount
present at time zero rather than the dosing level, since it can be
assumed that any degradation would affect both the initial amount
and the dosed amount in equal proportions.
Results
Wastewater 1—
Neither the DMP nor the DEP was dosed into Wastewater 1, either
at time zero or after storage due to the interferences. Consistent
results were obtained for the other four compounds studied, however,
with recoveries from 70 percent for DEHP to 81 percent for DBP. In
each of these cases, the precision of the analyses was good, with
ranges of 2, 0, 4, and 2 percent recovery for DBP, BBP, DEHP, and
DOP, respectively.
53
-------
The preservation recoveries were generally good for the four
higher-boiling phthalates, but more variable than the zero-day analyses.
The recoveries were from a low of 94 percent to a high of 106, with
ranges of 6, 7, 9, and 18. The 106 average for DBP was influenced
by a single value of 118, and the indication is that recovery for
these compounds was not affected by the storage conditions in this
wastewater.
Wastewater 3—
The analyses on this wastewater produced results which ranged
from low to very good, depending upon the particular compound. Results
for both DMP and DEP were very good, with triplicate analyses indicating
100 percent recovery of the spike. The recovery of DBP was 91 percent,
on average, but with a range of 10 percent recovery. The results
for the remaining phthalates were not good in this wastewater, however.
The average recovery of BBP was 104 percent, but the individual recoveries
had a range of 27 percent (93-120). DEHP and OOP were recovered at
65 and 66 percent, respectively, of the dose level with ranges of
16 and 20 percent recovery.
The preservation data were also inconsistent from one compound
to the next. For the six compounds studied, the average recovery
relative to the zero-day analyses went from 68 percent for DBP to
109 percent for DEHP. These analyses were more variable than the
zero-day analyses in the cases of DMP and DBP, equivalent for DEP,
and considerably less variable for the remainder. The ranges of the
triplicate analyses at zero-time were 27, 16, and 20 for DBP, DEHP,
54
-------
and OOP, respectively, at the initial nalyses compared to 9, 6, and
10 for these compounds after seven-days storage.
Wastewater 4—
The analyses on Wastewater 4 for DMP and DEP produced good recovery,
88 and 82 percent, respectively, with ranges of 3 and 2 percent.
Recovery of 91 percent on average was noted for DBP but more variability
with a range of 10 percent recovery. Lower recoveries were obtained
for the remaining three phthalates, with average percent recoveries
of 76, 50, and 50 for BBP, DEHP, and OOP, respectively, with ranges
of 10, 9, and 8.
The preservation data were good for the first four compounds,
going from 96 to 107 percent with ranges from 0 to 4 percent recovery.
For the DEHP and OOP analyses, however, the average recoveries were
140 and 139 percent with ranges of 9 and 11, respectively. These
recoveries are comparable to 70 and 69 percent, respectively, of the
original dosed amount and likely indicate a problem with the initial
analyses.
Wastewater 5—
The results for both DMP and DEP were good in this wastewater,
with average recoveries of 97 and 92 percent, respectively, and ranges
of 6 and 4 percent recovery. The DBP and BBP results were low and
variable, with average recoveries of 41 and 63 and ranges of 16 and
13 percent recovery, respectively. The DEHP and OOP values were consistent
but only 71 percent of the dose was recovered on the DEHP, while 93
percent was recovered on the DOP. For both of these compounds there
was a zero range, with all three analyses showing the same recovery.
55
-------
The preservation recoveries were fairly consistent for all of
the compounds, with ranges of recovery of 0 to 9 percent. However,
the level of recovery could be broken down into three groups: DMP-
DEP, DBP-BBP, DEHP-OOP. The recovery after storage was 95 to 100
for the first pair, 61 to 65 for the second and 78 to 79 for the third.
The 61 percent recovery represents only 25 percent of the initial
dosed amount remaining after seven days and indicates that storage
in this wastewater would not be recommended for these analyses.
Summary
The accuracy and precision evaluations on phthalate esters in
wastewater lead to the following conclusions. There are no assurances
that acceptable data will be obtained when analyzing for phthalate
esters using this method, as in the case of Wastewater 2 where no
usable results were obtainable, and in Wastewater 1, where none were
obtainable for DMP or DEP. The recoveries that can be expected for
the compounds studied ranged from 40 to 100 percent, depending upon
which compound and which wastewater. In general, the precision of
the analyses was acceptable to good, with ranges of less than 10 percent
recovery common.
The overriding conclusion is that the recovery and the ability
to store the water for later analysis are a function of the wastewater.
Storage frequently resulted in significant losses of the study materials
and in less precise determinations and cannot be recommended as a
general rule.
56
-------
REFERENCES
1. Thompson, J. F., Reid, S. F., and Kantor, E. J.y Arch. Environ.
Contam. Toxlcol., 6:143-157, 1977.
2. Standard Methods, 409H, pp. 342-343, Free Available Chlorine
Test, Syringaldazine, and 409A, lodometric Method I.
3. Giam, C. S., Chan, H. S., and Neff, G. S., "Sensitive Method
for Determination of Phthalate Ester Plasticizers in Open-Ocean
Biota Samples," Analytical Chemistry. 47:2225, November 1975.
57
-------
APPENDIX A
KEY REFERENCES FROM LITERATURE SEARCH
C. S, Giam, H. S. Chan, and G. S. Neff, "Sensitive Method for Determination
of Phthalate Ester Plasticizers in Open-Ocean Biota Samples,"
Analytical Chemistry, 47:2225, November 1975.
R. A. Kites and K. Biemann, "Water Pollution. Organic Compounds in
the Charles River, Boston," Science, Pub. 72, Series 178, Issue
4057, pp. 158-160.
R.D.J. Webster and G. Nicklers, "Problems in the Environmental Analysis
of Phthalate Esters," Proc. Anal. Div. Chem. Soc., Pub. 76, Series
13, Issue 11, pp. 333-335.
V. W. Saeger and E. S. Tucker, "Biodegradation of Phthalic Acid Esters
in River Water and Activated Sludge," Applied and Environmental
Microbiology, 31(1), pp. 29-34, January 1976.
C. S. Giam and H. S. Chan, "Control of Blanks in the Analysis of Phthalates
in Air and Ocean Biota Samples," Nat'l. Bur. Stand. (U.S.), Spec.
Pub 442, pp. 701-708, 1976.
58
-------
APPENDIX B
STATISTICAL ANALYSES
PHASE I EXTRACTION STUDY
59
-------
TABLE B-l. STATISTICAL SUMMARY FOR DMP - EXTRACTION STUDY
Anova Table
Source
df
SS
MS
Solvents
pH's
Interaction
Error
Total
Mean
1
2
2
14
19
-112.5
320.00
1679.29
960.29
2391.42
5351.00
320.00
839.65
480.15
170.82
2.87
4.92*
2.81
Cell Means
,
1
116.5
Solvent
2
108.5
pH (LSD- 16-2)
7
1UU.U "
7
122.1
10
112.1
Interaction
(1.2) (1.7) (1.10) (2.2) (2.7) (2.10)
96.0 134.2 113.3 "I5OT lld.O 111.0
Significant at 0.05 level.
60
-------
TABLE B-2. STATISTICAL SUMMARY FOR DEP - EXTRACTION STUDY
Anova Table
Source
Solvents
pH's
Interaction
Error
Total
Mean
df SS MS F
1 33.80 33.80 <1
2 492.16 246.08 6.38*
2 359.49 179.75 4.66*
14 539.75 38.55
19 1425.20
- 100.8
Cell Means
Sol vent
1 2
"99TT 102.1
pH (LSD -4.7)
93.7
Interaction (LSD- 6.7)
(1.2)
86.0
(1.7) ,
102.8
(1.10)
108.7
(2,2)
101.3
(2,7)
101.5
(2,10)
103.7
* Significant at 0.05 level.
61
-------
TABLE B-3. STATISTICAL SUMMARY FOR DBP - EXTRACTION STUDY
Anova Table
Source
Solvents
pH's
Interaction
Error
Total
Mean-
df
1
2
2
13
18
90.0
SS MS
560.49 560.49
1690.11 845.06
860.27 430.14
1072.08 82.47
4182.S5
F
6.80*
10.25*
5.22*
Cell Means
1
Solvent
2
1OT
pH (LSD -10.6)
76.3
7_
98.0
10
94.5
Interaction (LSD- 14.9)
(1.2)
61.3
(1.7)
95.2
(1.10)
94.7
(2.2)
91.3
(2.7)
101.7
(2.10)
94.3
* Significant .at 0.05 level.
62
-------
TABLE B-4. STATISTICAL SUIWARY FOR BBP - EXTRACTION STUDY
Anova Table
Source
Solvents
pH's
Interaction
Error
Total
Mean
df
1
2
2
13
18
- 94.8
SS MS
133.06 133.06
431.28 215.64
546.74 273.37
306.08 23.54
1417.16
F
5.65*
9.16*
11.61
Cell Means
Solvent
pH
Interaction
(1.2)
'88.7""
(1.7)
108.8'
(1.10)
~S077~"
(2.2)
(217)
89.7
(2.10)
1BT7-
* Significant at 0.05 level.
63
-------
TABLE B-5. STATISTICAL SUMMARY FOR DEHP - EXTRACTION STUDY
Anova Table
Source
Total
df
SS
Solvents
pH's
Interaction
Error
1
2
2
. r^ .
143.29
132.19
34.89
fl 25.42
143.29
66.10
17.45
78.88
1.82
<1
<1
18
1335.79
Mean - 103.1
Means
_1
100.5
Solvent
_J
106.0
2
102.8
PH
7
100.3
10
106.7
Interaction
(1.2)
98.3
fl.7)
99.8
(1.101
103.7
(2.2)
107.3
(2.7)
101.3
(2.10)
109.7
* Significant at 0.05 level.
64
-------
TABLE B-6. STATISTICAL SUMMARY FOR DDP - EXTRACTION STUDY
Anova Table
Source
df
SS
MS
Solvents
pH's
Interaction
Error
Total
Mean -94.2
1
2
2
14^
19
1.25
8.54
72.21
63.75
145.75
1.25 <1
4.27 <1
36.11 7.94*
4.55
Cell Means
1
94.5
Solvent
2
94.0
pH
93.5
7
94.1
10
95.2
(1.2)
93.3
(1.7)
96.5
Interaction (LSD a 3.5)
(1.10?
93.0
(2.2)
93.7
(2.7)
91.3
(2.10)
97.3
* Significant at 0.05 level.
65
-------
APPENDIX C
ANOVA TABLES
PHASE I PRESERVATION STUDY
66
-------
TABLE C-T. STATISTICAL SUMMARY FOR DMP - PRESERVATION STUDY
AKOVA Table
Source
PH
Temperature
Chlorine
pH x T
pH x C
Tx C
pH x- T x C
Residual
TOTAL
Mean - 89.0
(2.4)
103.5
df SS MS F
2 10",996.08 5,498.04 2,972*
1 1,107.04 1,107.04 598*
1 22.04 22.04 11.91*
2 6,969.08 3,484.54 1,884*
2 88.08 44.04 23.82*
1 22.04 22.04 11.91*
2 104.08 52.04 28*
10 18.50 1.85
21 19,326.94
Cell Means
pH (LSEH.4)
_L. JL 10
108.0 100.0 59.1
Temperature, °C
_4_ 24
95.3 82.2
pH-Temperature
(2.24) (7.4) (7.24) (10.4) (10.24)
112.5 94.0 106.0 90.0 28.2
* Significant at 0.05 level.
67
-------
TABLE C-2. STATISTICAL SUWARY .FOR DEP - PRESERVATION STUDY
ANOYA Table
Source
PH
Temperature
Chlorine
pH x T
pH x C
Tx C
pH X T x C
Residual
TOTAL
Mean « 96.1
df
2
1
1
2
2
1
2
10..
21
SS
359.08
80.67
4.17
576.08
9.08
10.67
23,08
40.00
1,107.83
MS
179.54
80.67
4.17
288.04
4.54
10.67
14.04
4.00
F
45*
20.17*
1.04
72.01*
1.14
.2.67
3.51
Cell Means
pH (LSD»2.1)
-1— -2— J2_
98.5 99.1 90.6
Temperature. "C
J_ 24
97.9 94.2
pH-Temperature (LSD-3.0)
(2.4)
95.8
(2.24)
101.2
(7.4)
98.3
(7.24)
99.5
(10.4)
99.2
(10.24
82.0
* Significant at 0.05 level.
68
-------
TABLE C-3. STATISTICAL SUMMARY FOR DBP - PRESERVATION STUDY
ANOVA Table
PH
Temperature
Chlorine
pH x T
pH x C
T x C
pH X T x C
Residual
TOTAL
Mean - 86.9
2
86.5
df
2
1
1
2
2
1
2
10
21
SS
219.25
35.04
45.38
907.58
91.75
57.04
30.08
108.50
1,566.62
Cell Means
oH (LSO-4.6)
7
90.3
MS
109.62
35.04
45.38
453.79
45.88
57.04
15.04
18.05
•
10
83.4
F
6.07*
1.94
2.51
25.14*
2.54
3.16-
<1
pH-Temperature (LSD-6.3)
(2.4) (2.24)
84.0 89.0
iL£
87.0
L (7.24)
94.5
(10.4)
• 93.3
(10.24)
73.5
* Significant at 0.05 level.
69
-------
TABLE C-4. STATISTICAL SUMMARY FOR BBP - PRESERVATION STUDY
ANOVA Table
Source
PH
Temperature
Chlorine
pH x T
pH x C
Tx C
pH x T x C
Residual
TOTAL
Mean * 75.6
2
93.8
!L*L (2.24)
96.0 91.8
df
2
1
1
2
2
1
2
.11
22
4
84.
ILS
74.8
SS
4,616.08
1,802.67.
0.67
2,133.58
23.58
6.00
49.75
433.00
9,065.33
Cell Means
pH (LSO-6.9)
7
72.8
Temperature, °C
24
3 67.0
pH-Temperature
1 (7.24)
71.0
MS F
2,308.04 58*
1 ,802.67 45*
0.67 <1
1,066.79 27*
11.79 <1
6.00 <1
24.88 <1
39.68
10
60.3
(10.4) (10.24)
82.3 38.3
* Significant at 0.05 level.
70
-------
TABLE C-5, STATISTICAL SUMMARY FOR.DEHP - PRESERVATION STUDY
ANOVA Table
Source
PH
Temperature
Chlorine
pHxT
pH x C
Tx C
pH x T x C
Residual
TOTAL
Mean * 93.0
df
2
1
1
2
2
1
2
11
22
SS
709.08
287.04
108.38
29.08
57.25
2.04
188.53
681.50
2,062.90
MS
354.54
287.04
108.38
14.54
28.62
2.04
94.29
61.95
F
5.72
4.63
1.75
-------
TABLE C-6. STATISTICAL SUMMARY FOR OOP - PRESERVATION STUDY
ANOVA Table
Source
PH
Temperature
Chlorine
pH x T
pH x C
Tx C
pH X T x C
Residual
TOTAL
Mean • 88.5
df
2
1
1
2
2
1
2
11
22
2
95.2
SS
549.33
88.17
60.17
214.33
212.33
228.17
129.33
254.00
1,735.83
Cell Means
OH (LSD=5.3)
7
86.2
MS
274.67
88.17
60.17
107.17
106.17
228.17
64.67
23.09
10
84.2
F
11.90*
3.82
2.61
4.64*
4.60*
9.88*
2.80
pH-Temperature (LSD*7.5)
(2.4) (2.24)
97.8 92.8
(7.4)
91.5
1LSSI
81.0
(10.4)
82.2
(10.24)
86.2
oH-Chlorine (LSD-7.5)
(2.0) (2.2)
95.2 95.2
(7.0)
80.5
JL2L
92.0
Temperature-Chlorine (LSO=»6
(4.0)
92.0
(4.2)
89.0
III
82
(10.0)
85.2
•1)
.0)
.0
(10.2)
83.2
(24.2)
91.3
Significant at 0.05 level.
72
-------
APPENDIX D
CHROMATOGRAMS OF WASTEWATER FRACTIONS
PHASE II
73
-------
PHTHALATE ESTERS
WASTEWATER »3-OOSED
FRACTION 2
Figure D.I. Chromatogram Wastewater 3 - Fraction 2
74
-------
PHTHAUATE ESTERS
WASTEUATER 13-DOSED
FRACTIONS 1 & 2 COMBINED
Figure D.2. Chromatogram Wastewater 3 - Fractions 1 & 2 Combined
75
-------
APPENDIX E
CHROMATOGRAMS OF WASTEWATERS 1 THROUGH 5
SINGLE FRACTION COLLECTION - PHASE II
76
-------
PHTHAUTE ESTERS
STANDARD
8X Col.
Figure E.I. Standard
77
-------
PHTHALATE ESTERS
UASTEUATER II
Figure E.2. Wastewater 1
78
-------
PHTHALATE ESTERS +
UASTEWATER 12 T
FINAL EXTRACT VOL. 2000 mL ]
5 UL ax col. i i
— DEHP f' i I ~l i
Figure E-3. Wastewater 2
79
-------
PHTHALATE ESTERS
WASTEWATER #3 _
FINAL EXTRACTION VOL. 10W.>-H
5uL 8X Col. 1 !
Figure E.4. Wastewater 3
80
-------
PHTHALATE ESTERS
WASTEWATER 14
FINAL EXTRACT VOL. 20 ml
Figure E.5. Wastewater 4
81
-------
-------
PHTHALATE ESTERS
WASTEWATER 15
FINAL EXTRACT VOL. 500;mL
Figure E.6. Wastewater 5
82
------- |