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EPA-R2-73-277
August 1973
CURRENT PRACTICE IN GC-MS ANALYSIS
OF ORGANICS IN WATER
by
Ronald G. Webb
Arthur W. Garrison
Lawrence H. Keith
John M. McGuire
Southeast Environmental Research Laboratory
College Station Road
Athens, Georgia 30601
Project 16020 GHP
Program Element 1B1027
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.25
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND MONITORING
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
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EPA Review Notice
Mention of commercial products and trade names
is for information only and does not constitute
endorsement.
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ABSTRACT
Experiences during five years of evaluating the appli-
cation of gas chromatography-mass spectrometry (GC-MS)
to wastewater analysis at the Southeast Environmental
Research Laboratory have resulted in the selection of
recommended practices for such applications. Liquid-
liquid extraction with solvents such as methylene
chloride and chloroform removed greater than 50 percent
of compounds found in pulp mill and petrochemical waste
at concentrations of 2 yg/£ to 20 ug/£. The Kuderna-
Danish evaporator was the most effective means of con-
centration after extraction. Diazomethane and dimethyl
sulfate proved to be the most effective of five
methylation reagents studied. Packed columns were
effective for gas chromatography of simple mixtures
and SCOT columns provided better overall performance
for complex mixtures. Computerized data reduction was
essential for practical use of GC-MS for samples
containing many compounds. A computerized spectra
matching program proved highly effective in identifying
compounds contained in the computer library. The system
was shown to be effective in solving problems related
to fishkills caused by pesticides, confirmation of
polychlorinated biphenyl residues in water and identi-
fication of compounds discharged by over a dozen indus-
tries. Over two hundred compounds were identified in
industrial effluents.
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CONTENTS
Section Page No.
I. RECOMMENDATIONS 1
II. INTRODUCTION 3
III. SAMPLE HANDLING 5
Collection 5
Extraction 6
Kuderna-Danish Evaporation 9
Clean-up Techniques 11
IV. DERIVATIVE FORMATION 15
Trimethylsilyl Derivatives 15
Methyl Derivatives 16
Diazomethane 16
Dimethyl sulfate 18
Others 18
Ozonolysis 20
V. GAS CHROMATOGRAPHY 21
Columns 21
Packed 21
Capillary and SCOT 21
Coatings 22
Flow Rate 22
Solvents 23
Temperature Programming 23
VI. MASS SPECTROMETRY 25
Instrumental 25
Operation 28
Specialized Techniques 31
LMRGC 31
Solids probe 34
VII. INTERPRETATION AND COMPUTER MATCHING 37
VIII. CONFIRMATORY TECHNIQUES 41
IX. CASE HISTORIES 43
Black Warrior River and Locust Fork 43
Branch Fish Kills
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Section Page No.
Polychlorinated Biphenyls (PCB's) 43
Industrial Effluent Characterization 45
Correlation of Compounds in Natural 48
Waters with Industrial Wastewater
Discharges
Waste Treatment 50
X. ACKNOWLEDGEMENTS 55
XI. BIBLIOGRAPHY 57
XII. APPENDICES 61
VI
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FIGURES
NO. PAGE
1 KUDERNA DANISH EVAPORATOR 10
2 OUTLINE OF FINNIGAN GC-MS-COMPUTER SYSTEM 27
3 RECONSTRUCTED GAS CHROMATOGRAM (RGC) OF A 30
PETROCHEMICAL PLANT EFFLUENT EXTRACT
4 MASS SPECTRUM OF FLUORENE 32
5 RECONSTRUCTED GAS CHROMATOGRAM AND LIMITED 33
MASS RECONSTRUCTED GAS CHROMATOGRAM
6 COMPLICATED RGC FROM AN INDUSTRIAL 35
EFFLUENT EXTRACT
7 LIMITED MASS RGC OF AN INDUSTRIAL EFFLUENT 36
EXTRACT
8 MASS SPECTRA OF EXTRACTS AND STANDARDS 44
CONTAINING MALATHION
9 FLAME IONIZATION DETECTOR GAS CHROMATOGRAMS 46
OF PCB'S AND KNOWNS
10 PARTIAL MASS SPECTRA OF PCB'S FROM 47
FIGURE 9
11 COMPUTER RECONSTRUCTED GAS CHROMATOGRAM OF 51
CHLOROFORM BLANK
12 RGC OF UPRIVER CONTROL SAMPLE EXTRACT 52
13 RGC OF PAPER MILL EFFLUENT EXTRACT 53
14 RGC OF DOWNSTREAM SAMPLE EXTRACT 54
15 APPARATUS FOR DIAZOMETHANE METHYLATION 89
16 APPARATUS FOR DIMETHYL SULFATE METHYLATION 91
VI l
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TABLES
No. Page
1 Methylation Efficiencies of Four Reagents 17
2 Qualitative Comparison of Methylation 19
Reagents
3 Minimum Acceptable Criteria for Mass 26
Spectrometer System
4 Steps in GC-MS-Computer Data Reduction 29
5 Comparison of Three Spectrum Matching 39
Programs
6 Compounds Identified in Wastewater of 49
Petrochemical Plant
Vlll
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SECTION I
RECOMMENDATIONS
Gas chromatography-mass spectrometry should be used as
the first approach to analysis of organic water pollu-
tants when the identities of the organic constituents
in the sample are in doubt. Computerized data
reduction and data interpretation are recommended to
improve efficiency and to eliminate the necessity for
an interpretation specialist at every laboratory.
The procedures described in this report are recommended
as a first step toward eventual development of a method
suitable for corroborative testing and consideration
as a standard method.
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SECTION II
INTRODUCTION
Currently, the most effective method for identification
of organic pollutants in water is gas chromatography-
mass spectrometry (GC-MS).
Any compound that can be gas chromatographed without
decomposition can be analyzed by GC-MS. Appendix 1,
containing over two hundred compounds identified in
industrial wastes, illustrates the wide applicability
of GC-MS. However, some classes of compounds are better
suited to other methods. Polymers, sugars, free amino
acids and many other biologically derived compounds are
not directly amenable to GC-MS because of their
non-volatility.
One evidence of the recent popularity of GC-MS is the
purchase of nineteen instruments within the last two
years by EPA laboratories throughout the country.
Significant time will be saved if these new users have
the benefit of five years experience at the Southeast
Environmental Research Laboratory in sample preparation,
GC conditions, data reduction, interpretation of spectra,
and all the other facets of this type of analysis.
This report describes the most successful current
practice in these areas and illustrates the power of
GC-MS in pollution analysis.
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SECTION III
SAMPLE HANDLING
Collection
Lake and river waters are not homogeneous. Industrial
effluents also change with time. Some investigators
prefer to take subsamples from various locations and
at different times and to combine them for analysis.
Many chemists avoid filtering the sample because the
colloidial and suspended particulates frequently have
organics absorbed on their surfaces.
Glass jars closed with Teflon-lined caps are preferred
for sample containers. The jars and caps should be
thoroughly cleaned with soap and water, rinsed with
distilled water, and rinsed with the solvent that will
later be used for extraction. After thorough drying,
the jars should be tightly capped until the sample is
taken. Additional jars should be prepared for
distilled water blanks.
For qualitative analysis, plastic bottles can be used
to collect samples that contain high concentrations of
organics. For these samples, e.g., paper mill efflu-
ents and municipal sewage, the adsorption of organics
on the walls of the bottle and the release of plasti-
cizers (phthaiate esters) into the sample are insigni-
ficant for short periods of storage.
The volume of sample collected is usually one or two
liters. Compounds present in a one-liter sample at
concentrations of 2 yg/fi, or greater will generally give
good quality spectra when processed by extraction,
concentration, and GC-MS techniques. For greater
sensitivity larger samples are required. For example,
twenty-liter samples of municipal sewage have been
processed with an apparent detection limit of 0.1 yg/Jl
based on an internal standard added to the extract
before concentration.
Because samples may have to be transported or stored
for considerable time before analysis, some means for
stopping biological action and preventing chemical
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changes is needed. Freezing is the preferred method.
The addition of strong acid as a preservative is not
recommended because it degrades some sensitive compounds
such as geosmin and 2-methyl isoborneol (taste and odor
causing compounds). Some space must be allowed in glass
containers for the expansion of water as it freezes.
Some workers find such a high proportion of breakage
on freezing that they prefer to pack the bottles in
ice for shipment and store them in a refrigerator at
about 4° C. Plastic containers, particularly the
popular cubitainers, do not usually rupture on freezing.
Samples are conveniently shipped by air freight. They
are usually frozen, placed in styrofoam containers for
insulation and protection against breakage, and
further packed in dry ice in an inexpensive styrofoam
chest.
Extraction
The first step in GC-MS analysis is extraction of the
organic compounds from the bulk of the water. Commonly,
a liter sample of water is extracted with two or three
50 to 100-ml portions of solvent. The combined
extracts are dried and the solvent is evaporated to 1
ml or less for GC analysis. Recovery studies on
compounds found in petrochemical refinery wastes and
paper mill effluents show the effectiveness of this
procedure. A liter of water, spiked with knowns, was
extracted with three portions (100/50/50 ml) of chloro-
form (duplicates were extracted with methylene chloride),
dried, and evaporated to 500 to 200 microliters for
injection and quantitation by flame ionizetion GC. The
recoveries were 50 to 100% at the 20 and 2 yg/& levels.
No significant differences were noted between methylene
chloride recovery and chloroform recovery; however,
methylene chloride is more likely to form emulsions.
Compounds with high vapor pressures (methyl styrene,
cymene, indene, methyl indene, terpinolene, camphor)
gave recoveries of 50-70%, whereas low vapor pressure
organics (indole, naphthalene, quinoline, biphenyl,
dimethyl naphthalene, diphenyl methane, acenaphthene,
bibenzyl, carbazole, benzophenone, phenanthrene,
fenchone, triphenylmethane, fenchyl alcohol,
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a-terpineol, and guaiacol) gave 72-100% recoveries.
Other solvents in common use are petroleum ether,
hexane (also 15% diethyl ether in hexane), diethyl
ether, and carbon tetrachloride. Petroleum ether and
hexane are so non-polar that they do not effectively
extract polar materials such as fatty acids and phenols.
Ether is not recommended (except in very small quanti-
ties for diazomethane methylations) because of its
flammability and inefficiency as a solvent, the danger
of explosion from peroxide impurities, and the
presence of additives. Various preservatives are added
to ether to inhibit peroxide formation but are not
usually listed on the container label. One peroxide
inhibitor identified by GC-MS and later confirmed by
the manufacturer was 2,6-ditertiarybutyl-p-cresol.
This compound was recognized as an artifact because
it also occurred in the blank. Finally, in solvent
comparison studies, ether was found to be only half as
effective as chloroform in extracting a paper mill
effluent.
Carbon tetrachloride is sometimes preferred because
it gives a narrow solvent peak on some GC columns;
however, it is less efficient than chloroform for
extraction of polar compounds. Carbon disulfide and
various freons have the advantage of very high vola-
tility but are difficuT'- 'zo purify and usually show
numerous impurity peaki,. .uditionally, carbon disul-
fide is very flammable.
Extraction is affected by the pH of the sample.
Organic acids are ionized salts in basic solution and
will not extract into organic solvercs. Therefore
samples containing fatty acids (municipal sewage)
or phenols (forest runoff) are best extracted at a
pH <5 to insure maximum covalent character.
Similarly, basic materials are best extracted at high
pH. A complete, but somewhat involved, procedure for
these "solubility class" separations was given by
Braus, Middleton and Walton (1). We recommend
extracting samples whose original pH is 5-7.5 with
solvent to isolate the neutral compounds. Then the
sample is acidified to pH 3 or less with hydrochloric
or sulfuric acid and extracted to isolate the acids
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and phenols. To isolate basic compounds/ the acid
solution is adjusted to pH 10 and extracted. Usually
very few basic compounds are found. Many variations
in procedure are possible and each problem requires an
individual judgment.
Several operational details help make extraction
simpler or more effective. Pesticide quality (dis-
tilled-in-glass) solvents are recommended. The
separatory funnel should have a Teflon stopcock and
the glass stopper should not be lubricated. If the
sample contains suspended matter, a representative
portion of it should be included in the sample for
extraction. The first portion of extracting solvent
should be used to rinse the sample container and then
added to the separatory funnel. Some samples, parti-
cularly those from anaerobic sites, will form an
emulsion or deposit a sludge in the solvent layer.
One of the main components of this material is elemental
sulfur. Some of the sulfur dissolves in the solvent
and precipitates as a whitish solid when the extract is
concentrated by evaporation. It usually chromatographs
as a single peak during GC-MS analysis and gives a
molecular ion at m/e 256 (Sg) and fragments from
successive losses of 32 mass units.
To separate the sludge from the solvent or to break an
emulsion, place a 2.5 cm ball of glass wool in a 2.5
cm diameter plain chromatography column—no frit—
and, after wetting the wool with fresh extraction
solvent, pour the emulsion through the glass wool.
Ignore the debris that remains. It may be necessary
to force the emulsion through with a little air
pressure.
After a sample is extracted, a little water may remain
dissolved in the solvent. The analyst can either dry
the extract before concentration or concentrate the
solvent directly. No studies have been done to show
which is best. Drying is usually accomplished by
filtering the extract through a column of anhydrous
sodium sulfate (200 ml of solvent through a 5 cm X 2.5
cm diameter column is common). The sodium sulfate
should first be heated to 500° C for 2 hours to remove
organic impurities, usually phthalate esters. Another
8
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method for drying is to pour the extract through a
pre-wet glass plug as described earlier.
Some analysts simply concentrate the extract and
inject it on the GC. With non-chlorinated solvents
even the ultra-water-sensitive electron capture
detector will accept this concentrate.
Numerous other methods have been tried to separate and
concentrate organic materials. In our experience
freeze concentration and steam distillation are vastly
inferior to extraction. Carbon adsorption has limited
use but the labor involved in processing makes it
unpopular. Polyurethane foams are now being used to
extract polychlorinated biphenyls directly from water.
These foams, coated lightly with DC-200, also extract
organochlorine pesticides. Our tests show that
neither material is effective for extracting dissolved
oil, phenols, or terpenes. Organic resins, in
particular Rhom and Haas' XAD series, show much more
promise but applications are still in the developmental
stages.
Kuderna-Danish Evaporation
The best method for concentrating the organic extract
is distillation with a Kuderna-Danish evaporator
(Figure 1), available from Kontes. The bottom and
middle flasks are filled to about half the nominal
flask capacity. Boiling chips are added and the whole
assembly is placed on a boiling water bath or a steam
bath in a hood. The water level should be maintained
just below the bottom joint and steam should bathe the
rounded bottom of the middle flask. Some people let
distillation become just vigorous enough to maintain
liquid in the upper bubble joint; others feel that it
is equally efficient, as well as quicker, to allow
moderate splashing out the top. Distillation is
usually continued until about one ml of liquid remains
in the concentrator tube; the whole assembly is then
removed and allowed to cool. As the liquid drains
from the condenser it rinses the residue from the walls
of the middle flask. The final volume is about five ml.
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FIGURE 1. KUDERNA-DANISH EVAPORATOR
10
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The calibrated concentrator tube is removed and placed
in a beaker of warm water (<70°) and the volume reduced
to any desired level, usually one ml, by directing a
stream of clean dry air or other gas over the surface.
A fresh glass disposable pipette is used as an air jet
for each sample. The pipettes are connected to the
air supply with Tygon tubing. Analysis of blanks so
treated have not indicated the presence of volatile
plasticizers from the tubing. If the extracting
solvent is not compatible with the GC system (if it
tails badly and obscures parts of the peaks), four or
five volumes of a more desirable solvent are added
and the volume is again reduced to the volume necessary
to give adequate GC sensitivity.
Several studies (2-4) have shown that considerable
loss of the extracted compounds takes place during
air evaporation. For very volatile compounds, 50%
loss on evaporation to about 1 ml is common; for less
volatile materials such as DDT-type pesticides, losses
are only a few percent. Fortunately the concentration
factor increases faster than the loss factor. There-
fore, enough compound for a good mass spectrum is
concentrated into a solvent volume acceptable to both
the GC and the MS. If the sample must be stored over-
night or longer the tube should be closed with a glass
stopper and placed in the refrigerator. Some prefer
to store extracts in Reacti-Vials or other screw-cap
vials fitted with a Teflon cap liner.
The Kuderna-Danish method is preferred to a rotary
evaporator because there is less danger of contamina-
tion in the all-glass apparatus, less danger of
bumping with loss of sample, and less loss due to
handling since transfers do not have to be made from
large round bottomed flasks to a calibrated container.
Clean-Up Techniques
When extracts of water are analyzed by GC for specific
organic compounds, e.g., pesticides, the extract
usually must be "cleaned-up" to remove interferences.
The most widely used technique is Florisil or silica gel
column chromatography, sometimes in combination with
acetonitrile partitioning. Thin-layer chromatography,
11
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preparative GC, and liquid chromatography are used less
frequently. Fortunately, since the mass spectrum of a
compound is much more definitive than its GC retention
time/ extracts for GC-MS analysis generally need not be
cleaned up, particularly when solubility class separa-
tion has been used during sample extraction.
If preliminary GC runs show that clean-up of the
extract is necessary, the concentrated extract is
chromatographed on activated florisil. In the original
technique (5), increments of solvent containing
increasing proportions of ethyl ether in petroleum ether
are used to elute fractions containing increasingly
polar organics. We use methylene chloride rather than
ethyl ether. This technique was used with a textile
plant effluent being analyzed for dieldrin by GC-MS (6).
As another approach, after solubility separation of
the crude extract, the neutral fraction may be further
separated on a silica gel column. Elution with
isooctane, benzene, 1:1 chloroform:methanol separates
organics into aliphatic, aromatic, and oxygenated
organic fractions, respectively (7).
Thin layer chromatography is sometimes used as a
preliminary separation technique for GC-MS analysis.
Samples of industrial and municipal effluents contain
so many components that direct TLC of extracts usually
gives only unresolved streaks. In a few case inter-
mediate clean-up by TLC is useful, resulting in the
separation of bands of compounds of the same type.
This type of sample clean-up has been used in the
identification of pesticide metabolites by GC-MS (8).
Preparative gas chromatography may be used as a
preliminary separation technique. In many cases
initial GC runs of crude sample extracts result in
several overlapping peaks, even under the best of
chromatographic conditions. These overlapping
portions of the effluent are collected in capillary
tubes, cooled if necessary by dry ice or a tissue
soaked in acetone, and rechromatographed on a high
resolution (usually capillary) column. With this
less complex mixture, GC parameters may be varied
more freely to obtain better separation. Preparative
12
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GC was used in the separation and identification of
polychlorinated biphenyl (PCB) isomers (9). In this
case, as with some water samples, several sharp pre-
parative chromatographic peaks that appeared to be one
component each were shown by capillary GC to consist of
two or more components.
Liquid chromatography (LC) seldom resolves sample
components as well as GC, but may be considered as
a preliminary separation tool in some cases. Volatile
compounds collected from a LC eluate may be analyzed
directly by GC-MS if the LC eluting solvent is
compatible with the GC-MS system.
One advantage of LC is its amenability to chromato-
graphy of aqueous samples. Furthermore, ion-exchange
LC columns provide for the separation of water soluble,
non-extractable organic compounds. One established
procedure (10) calls for concentration of the original
water sample by direct evaporation and/or freeze drying.
The concentrated sample is dissolved in a buffer solution
and chromatographed on a high pressure anion-exchange
column. Fractions, usually representing several over-
lapping peaks, are collected in their buffered eluting
solution and the solution is freeze-dried. The solid
residue is derivatized to make volatile components
that may be analyzed directly by GC-MS.
13
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SECTION IV
DERIVATIVE FORMATION
Many compounds are not volatile enough to be trans-
formed to the gas phase without decomposition. Such
polar compounds as carbohydrates, amino acids,
sulfonic acids, and nucleic acids are not directly
gas chromatographable. Some steroids, carboxylic
acids, and phenols can be directly chromatographed in
their free form, but require special columns and
techniques not readily available. Phenols "tail" on
most GC columns, steroids elute too slowly, and only
recently have columns become available for the
efficient separation of free long-chain carboxylic
acids.
Derivatization reagents are now available for all
these classes of compounds. To be useful for GC-MS
analysis, a derivative must (1) be formed quantita-
tively from the free precursor by a rapid reaction
with a readily obtainable reagent, (2) be volatile
enough for vaporization in the GC inlet, (3) be
thermally stable in the GC-MS system, and (4) be of
a chemical class that has been studied extensively by
mass spectrometry so that a spectral library is
available for matching. Methyl derivatives, in the
form of methyl esters of carboxylic acids or methyl
ethers of phenols, and trimethylsilyl (TMS) derivatives
of carbohydrates, steroids, phenols, amines, carboxylic
acids, and amino acids are the most common.
Trimethylsilyl Derivatives
A variety of TMS reagents are available, some of which
are tailored for specific functional groups. These
reagents generally react with groups containing active
hydrogen atoms (-PH, -SH, -NH, -COOH) to replace the
hydrogen with a trimethylsilyl [-Si(CH3)3] moiety.
Suggestions for the use of TMS reagents and information
on their chromatographic and mass spectrometric
properties have been collected by Pierce (11).
TMS derivatives of about fifty urinary constituents
separated by high-pressure anion-exchange liquid
15
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chromatography were prepared to increase their vola-
tility for identification by GC and MS (12). TMS
derivatives are also being used in the identification
of municipal waste components separated by liquid
chromatography (10).
Methyl Derivatives
Both diazomethane and dimethyl sulfate are used
routinely to prepare the methyl esters of carboxylic
acids and the methyl ethers of phenols.
Diazomethane. The diazomethane methylation method is
simple, fast, and almost quantitative for carboxylic
acids (Table 1). The procedure for diazomethane
methylation is given in Appendix 2. It is the method
of choice for extracts in which carboxylic acids are
the most important compounds to be studied. It has
been applied very successfully to the methylation of
acid fractions of municipal wastes. However, diazo-
methane has two important disadvantages as a methy-
lating reagent: it does not react quantitatively with
all phenols, and some undesirable side reactions
occur (13) .
Although pentachlorophenol reacts quantitatively and
other chlorinated phenols have been observed to
produce methyl ethers with diazomethane, guaiacol,
vanillin, and other phenols found in paper mill wastes
give poor yields (Table 1).
Samples that have been extracted with chloroform,
evaporated to nominal dryness and then redissolved in
ether and methylated, frequently contain half a dozen
or more di- and trichloroalkanes (C2 through Cy).
These are formed by the reaction of the residual
chloroform and diazomethane, a reaction enhanced by
exposure to uv light and excess diazomethane (14).
Since methylene chloride does not react to form
interfering peaks, it is now routinely used to extract
samples that will be methylated with diazomethane.
Another advantage of methylene chloride is that it does
not have to be completely evaporated before methylation;
loss of volatile phenols is thereby decreased.
16
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Table 1
Methylation Efficiencies of Four Reagents
Compound
Guaiacol
Vanillin
Palmitic acid
Dehydroabietic acid
% Yield*
Diazo-
me thane
15
40
80
95
Dimethyl
sulfate
90
70
60
60
HC1-CH3OH
0
0
90
10
"Me thy 1-8"
0
0
50
85
"MethElute"
85
55
70
70
*Yield of methyl ether or ester, based on GC peak heights. These yields
also represent losses occurring upon evaporation of solvents and other
procedural errors.
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Dimethyl Sulfate. In a comparison of methylation
techniques using the acid fractions of kraft paper
mill effluents, dimethyl sulfate gave much better
yields of phenolic methyl ethers than did diazomethane
(Tables 1 and 2). Dimethyl sulfate also methylates
the resin acids and fatty acids found in paper mill
wastes fairly well, but was not successful in methy-
lating the acid fraction of municipal wastes (Table 2).
Dimethyl sulfate is recommended for methylation of
extracts containing phenols. The procedure for
dimethyl sulfate methylation is given in Appendix 3.
An advantage of this method is that the aqueous sample
is used directly, after extracting bases and neutrals.
An important disadvantage is the time required—about
four hours.
Others. Three other methylation reagents tried are
compared with diazomethane and dimethyl sulfate in
Tables 1 and 2. Methanolic hydrochloric acid (6N)
reagent (15) (Table 2) was used with municipal waste
with results comparable to those obtained with diazo-
methane. However, it does not methylate pure vanillin
or guaiacol, and the esterification yield with
dehydroabietic acid was only 10% (Table 1). The
reaction is slower than diazomethane methylations, and
the solvent is restricted to methanol. There are also
problems with neutralization of the HCl.
"Methyl-8", dimethylformamide dimethyl acetal (16) was
found to methylate palmitic acid in fair yields and
dehydroabietic acid in good yields, but it did not
methylate the phenols (vanillin and guaiacol). It
methylated the municipal waste fraction nearly as well
as diazomethane and is therefore recommended for
samples containing only carboxylic acids.
The most convenient reagent tested is "MethElute", a
0.2 M solution of trimethyl anilinium hydroxide in
methanol (17). The sample extract is dissolved in
this reagent, and the solution is injected into the
GC inlet where the methylation reaction occurs.
By-products and excess starting materials appear near
the solvent peak, followed by the later-eluting
ethers and esters. Methylation of municipal waste and
paper mill acid fractions gave results comparable to
18
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Table 2
Qualitative Comparison of Methylation Reagents
Reagent
Diazo-
methane
Dimethyl
sulfate
"MethElute"
"Me thy 1-8"
HCl-MeOH
Phenols
Paper mill
waste
P
B
G
P
—
Municipal
waste
F*
P
—
—
—
Fatty Acids
Paper mill
waste
G
B
G
P
--
Municipal
waste
B
P
G**
G
E
Resin Acids
Paper mill
waste
G
G
G
B
—
Municipal
waste
__
--
--
—
—
B=Best, E=Excellent, G=Good, F=Fair, P=Poor
*0nly one sample tested; chlorinated phenol methyl ethers were detected
but yields are unknown. Cresol was not methylated.
**Two or three reagent peaks formed—they did not interfere in this
analysis.
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those using diazomethane. Because this reagent methy-
lates both carboxylic acids and phenols in fairly good
yields, as seen in Table 1, it has great potential but
should be tested on extracts from other sources.
Ozonolysis
Ozonolysis can be used as a derivatization technique.
Many unsaturated compounds can be converted to alde-
hydes or ketones through formation and cleavage of
ozonides. The aldehyde and ketone products may be
identified, leading to deduction of the original
position of unsaturation. The ozone for the reaction
can be generated using commonly available laboratory
apparatus (18). This technique was used to differen-
tiate the unsaturated fatty acids from their saturated
analogs in municipal sewage by comparison of a sample's
gas chromatogram before and after ozonolysis.
20
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SECTION V
GAS CHROMATOGRAPHY
Most samples require some gas chromatographic experi-
mentation to work out the optimum, or at least a
useful set of chromatographic conditions prior to GC-MS
analysis. Therefore, an additional gas chromatograph
is useful to avoid wasting MS instrument time while
gas chromatographic conditions are being determined.
Glass-lined injectors should be used in both GC's to
avoid decomposition of the sample by initial contact
with hot metal. The detector of the auxiliary GC
should be flame ionization to achieve sensitivity
similar to that of the MS. The additional gas chromato-
graph should be of the same type as the one interfaced
with the mass spectrometer because, after optimum
chromatographic conditions are determined, the smallest
change will affect the peak separation of a complex
mixture. Often the column from the auxiliary GC is
transferred to the GC-MS for the final analysis.
Columns
Packed Columns. The 1/8 inch diameter packed columns
commonly used in GC laboratories are inexpensive and
readily available. Most mass spectrometers will
accept them. The analyst must be careful not to
inject the large amounts of solvents (5-10 microliters)
traditionally used because this raises the gas pressure
in the MS past acceptable limits. For routine surveys
in which only the more concentrated pollutants are of
interest, packed columns are most often used.
Capillary Columns. Although capillary columns offer
the ultimate in peak resolution they are expensive.
They accept only small amounts of sample and require
long separation times.
Support-coated-open-tube (SCOT) columns 0.02 inches
in inside diameter, are a compromise between the tra-
ditional packed column and the true capillary column.
As such, they offer some advantages of both:
21
-------
• better peak resolution than packed columns
with complex mixtures
• moderate size sample injections (usually
1-2 y£)
• chromatographic times not much longer than
with packed columns (usually 20-40 minutes)
• broad choice of support-coating combinations
The technology involved in preparing them is so complex
and involved that it is cheaper to buy them commercially
than to develop the expertise and equipment to make
them. Perkin-Elmer SCOT columns are normally terminated
with a male nut that fits the PE 900 series gas
chromatographs. To render these columns useful with
other gas chromatographs, these male nuts need only be
pushed back and replaced with 1/16-inch female slotted
nuts in which the slot is made just large enough to
accept the capillary tubing. Although these columns
are relatively expensive, they can be returned to the
factory and recoated when column deterioration becomes
evident (broadened and/or tailing peaks). Some of our
columns have been recoated 4 or 5 times without
apparent effect on their separation efficiencies.
Column Coatings. Many liquid phases and supports are
on the market. The two we have found most useful are
SE-30 (non-polar) and Carbowax 20M terminated with
terphthalic acid (polar). We also keep on hand DECS,
OV-17, OV-101, OV-225, DC-200, DX-300, QF-1, LB-550X,
and Apiezon-L for special needs.
Flow Rate
Helium is the usual carrier gas for GC-MS systems and
should also be used as the carrier gas in auxiliary gas
chromatographs. The optimum carrier gas flow for GC-MS
systems operating under vacuum and using a Gohlke
separator is 16-18 ml/min. If about half this helium
flow is used for optimizing conditions with the auxiliary
GC (operating under atmospheric pressure), the other
chromatographic conditions (program rate, initial
temperature, initial hold) will hold when the column is
22
-------
transferred to the GC-MS. The resulting chromatogram
(or computer-reconstructed chromatogram) will closely
approximate the chromatogram obtained on the auxiliary
GC.
Solvents
Chloroform and methylene chloride are excellent
solvents for extraction. They give smaller and
narrower solvent peaks than hydrocarbon solvents in
GC using flame ionization detection. Unfortunately in
the inlet of a MS their ionization cross-sections are
such that they easily trip the electronic protection
circuit and cause automatic machine shutdown. As a
result, no more than 1.8 yJl of chloroform or methylene
chloride solutions can be injected into our GC-MS
system without taking special precautions. With
solvents such as toluene, hexane, and other hydro-
carbons , which do not cause this problem, up to 3 y£
samples can be injected.
Temperature Programming
Temperature-programmed GC is nearly always used for
GC-MS analysis. A medium program rate of 4° per
minute is common. Frequently the program is extended
to the upper limit recommended for the liquid phase.
At the resulting high temperatures, considerable bleed
occurs with many columns. For example, MS fragments
at m/e 73, 147, and 221 resulting from dimethyl
silicones appear when the usually stable silicone
columns are overheated. The program should therefore
be terminated short of these high temperatures.
When the initial temperature is too high, many
components of interest elute with or close to the
solvent. This may explain the absence of low
molecular weight compounds from Appendix I.
Limited experience with sub-ambient temperature
programming indicates that this approach is feasible
for the analysis of such compounds.
23
-------
SECTION VI
MASS SPECTROMETRY
Instrumental
The first GC-MS system used at SERL in 1968 was a
Perkin-Elmer/Hitachi RMU-7 double-focusing mass spectro-
meter connected to a Perkin-Elmer 900 gas chromatograph
through a Watson-Biemann separator. The data output
was to be collected and processed by computer but this
portion of the system was never fully developed.
Without a workable computer system, a typical twenty
peak chromatogram required approximately twelve hours
of manual data handling before interpretation could
begin. Despite these limitations, the first system
demonstrated the power of GC-MS by providing answers
to problems considered too complex for other types of
instrumental analysis (6, 19). Based on almost three
years'experience with this system, a set of MS system
criteria was prepared for use in testing and evaluating
the GC-MS-computer systems available in 1971. These
criteria, modified slightly (Table 3), are still valid.
Two GC-MS-computer systems (the Varian CH-7 and the
Finnigan 1015) were shown to satisfy these criteria
(20). Later, during 1972, an improved version of the
DuPont 21-490 system was also shown to satisfy the
criteria. The less expensive Finnigan system, outlined
in Figure 2, was chosen and installed during 1971.
The GC, a vendor-modified Varian Model 1400 with no
independent detector, serves as a specialized inlet to
the mass spectrometer.
The Gohlke separator is an all-glass jet separator
that separates organic samples from the helium carrier
gas based on differences in the diffusion rates of the
gases in a turbulent jet. Its small surface area
eliminates chemisorption and catalytic degradation and
thus overcomes the chief disadvantage of the Watson-
Biemann fritted glass separator.
25
-------
Table 3
Minimum Acceptable Criteria for
Mass Spectrometer System
Mass Spectrometer
Scan rate
Mass range
Resolution
Minimum identifiable
level of pesticide
Ionizing voltage range
2 sec/decade or 230 amu/sec
12-650 amu
1/400 or unit throughout range
10 ng in GC inlet
10-100 volts
Data System
Field experience
High speed storage
and retrieval
Data acquisition mode
Calibration method
Minimal software
Both software and hardware
proven in the field
Magnetic tape
Continuous cyclic scan
Semi-automatic
Computer reconstructed
chromatogram, background
correction, and spectrum
plotting routines
26
-------
ro
Vac ion
mnn
Chromatograph
Digital Equip.
Dec Tapes ""
-^
Diablo Disk
Gohlke
Separator
^>^
^s^*~- PDP fi/« —
p 1
^^-^^, vOmpUlvr -^
" |
Reduced- Data
Interpretation
B nf MantaH 7j\nae
Finnigan :-*i — Direct Probe Inlet
Moss Spectrometer -^ Liquid Inlet •
•
\
System
* maUSUIGS Plnttnr
ADC a DAC ^ I
Reconstructed
Chromotogroms
ond Moss Spectra
FIGURE 2. OUTLINE OF FINNIGAN GC-MS-COMPUTER SYSTEM
-------
The Finnigan 1015 mass spectrometer is a quadrupole
instrument having a range of 1-750 amu. Resolution is
one mass unit throughout the range (e.g., 1/20 at mass
20, but 1/625 at mass 625). As a consequence, instru-
mental sensitivity at low mass is much higher than in
a magnetic instrument. At a scan speed of 120 amu/sec
the sensitivity is adequate to give identifiable
spectra from 20 ng of material introduced into the GC
inlet.
The liquid inlet is used for introduction of calibrating
compounds, the direct probe for solid materials.
The System Industries interface and the analog-to-
digital and digital-to-analog converters are used to
permit the Digital Equipment Corporation (DEC) computer
to control the mass spectrometer during calibration,
data acquisition, and checking; to accept data from the
mass spectrometer; and to control the Houston plotter
during data reduction.
The DEC PDF 8/e computer, the heart of the data system,
has a 4096 word core and an ASR33 teletypewriter.
Programs, raw data, and reduced data are stored on
either the two DECtape units or the Diablo disk.
Output of reduced data is achieved under computer
control via the plotter, the teletypewriter, or a
coupling device. The coupling device connects the
POP8 via telephone lines to a large computer and permits
semi-automatic spectrum identification by a matching
procedure described in Section VII.
Operation
The basic steps in processing a sample by GC-MS computer
are listed in Table 4 and can best be illustrated by
reference to an example.
The mass spectrometer is calibrated with perfluoro-
n-tributyl amine, which gives a number of characteristic
fragments in the mass range 50-614. The computer uses
the signals from these fragments to establish the
relationship between the digital-to-analog converter
values and the m/e values.
28
-------
Table 4
Steps in GC-MS-Computer Data Reduction
1. Formation of calibration reference file
2. Data acquisition
3. Reconstructed gas chromatogram plot
4. Manual selection of GC peak and background spectra
5. Creation of background corrected spectra files
6. Output of plotted spectra
7. Interpretation of spectra
After the sample is injected into the GC, the mass
spectrometer automatically scans its pre-set mass
range every five seconds (or other pre-set interval).
As it does so, the plotter draws a trace that is equi-
valent to a GC signal (lower trace in Figure 3). If
the sample was pre-analyzed on an auxiliary GC, this
trace can be used to determine when to terminate the
run.
After the run is complete, the computer plots a recon-
structed gas chromatogram (RGC) like that shown in the
upper trace in Figure 3. The largest peak is automa-
tically plotted at amplitude 100 and the other peaks
are shown proportional to it. Each point on the
spectrum number scale under the RGC represents a
complete mass spectrum collected on magnetic tape.
These spectra can be displayed individually and thus
the analyst can see the composition of each peak.
Consider the peak at spectrum number 287 (marked with
an arrow in Figure 3). The spectrum stored on the tape
will be that of one of the sample compounds plus
various fragments from column bleed, traces of air, oil,
moisture, and perhaps a small residual amount of material
from the peak that eluted just prior to the one at 287.
These background fragments may mask some of the peaks of
29
-------
U)
O
109 118 129 130 1W ISO ISO 170 ISO IS! 289 210 220 230 2*1 2SI 200 2TO 280 JSO 300 410
FIGURE 3.
RECONSTRUCTED GAS CHROMATOGRAM (RGC) OF A PETROCHEMICAL PLANT
EFFLUENT EXTRACT
-------
the sample. To overcome this problem, the operator
selects a spectrum that contains nearly the same
background and none of the compound fragments and has
the computer subtract the background from the sample.
In this example a logical choice is the chromato-
graphic valley at spectrum number 284. The resulting
mass spectrum plotted by the computer is shown in
Figure 4 (the compound structure and M"1" were added
manually).
The processing of the MS data that formerly required
many hours of hand calculations and graph plotting
now takes less than three hours, and most of this time
does not require operator attention. Only 30 minutes
of operator time are required to enter into the
computer the instructions to output the reduced data
for a 20 peak chromatogram. Elapsed instrument time
to reduce the data ranges from slightly more than one
hour for the disk system to more than two-and-a-
quarter hours for the tape system. The time difference
is due to the time lost by the computer while searching
the tapes for the appropriate data.
The overall GC-MS-computer system as outlined in Table
4 works well; however, two obvious improvements can
be made in the computer programs. The first is faster
data output utilizing a cathode ray tube with a hard
copy device and the second, a modification to permit
time-shared acquisition and processing of data. With
these modifications overall data reduction time should
be reduced by half.
Specialized Techniques
LMRGC. Specialized techniques of MS or data reduction
can be used to detect a specific material or class of
materials in a mixture. The most common technique is
the generation of the limited mass reconstructed gas
chromatogram (LMRGC). For example, in Figure 5, the
RGC shows as peaks those spectra that contain signifi-
cant numbers of any ion fragments of m/e 35 to 250.
Above the RGC is the LMRGC in which the computer was
instructed to respond only to those spectra that
contain the m/e 149 fragment. This fragment is often
31
-------
SPETTHJ1
207
carntf
M
tu
MODE! OUTP
PMNT71
PLOT?: r
FILE! AGO
SPEC NO I £87
AMP EXP7I Y
THRESHOLD t: I
EXPAND BY: 5
HIM Xl
SUBT BKGD?: r
BKGD FILEI AGO
SPEC NO I 284
NOHM ON:
SAVE RESULT7I Y
170 ISO 190 200 2VO 228 230 210 235
FIGURE 4. MASS SPECTRUM OF FLUORENE
-------
100
80
§60
20
LMRGC m/e!49
3561
0
(Phthalate)
292
A
I I I I I I i i i i i i i i i i
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380
SPECTRUM NUMBER
CO
u>
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 • 300 320 349 360 380
SPECTRUM NUMBER
FIGURE 5. LOWER: RECONSTRUCTED GAS CHROMATOGRAM OF A PETROCHEMICAL COMPANY
EFFLUENT. UPPER: LIMITED MASS RECONSTRUCTED GAS CHROMATOGRAM
INDICATES ONLY SPECTRA 292 AND 356 CONTAIN SIGNIFICANT FRAGMENTS
OF M/E 149, TYPICAL OF PHTHALATE ESTERS
-------
found in the spectra of phthalate esters. The results
indicate that two of the sample peaks may be phthalates.
Similar characteristic fragments exist for several other
classes of compounds.
Limited mass monitoring is also possible in the data
acquisition step. In this mode, the mass spectrometer
is stepped sequentially through a series of limited
mass ranges. Longer detection times can be spent on
each individual mass than in normal runs; hence, the
sensitivity for a given peak can be enhanced. The
technique can also be used to eliminate peaks that
might otherwise interfere with the spectrum of
interest. Figure 6 shows a complicated RGC from an
industrial effluent. In Figure 7, the sample was
rerun using the limited mass range technique. Based
on the peak patterns and the particular masses
monitored, the impurity was identified as a PCB
mixture.
Solids Probe. Samples not amenable to GC can sometimes
be introduced without decomposition into the MS through
the solid probe inlet. Most GC-MS instruments have
this accessary. In one example, a blue material
isolated from a municipal sewage plant would not
chromatograph. When introduced into the MS by the
solids probe, it was identified as copper phthalocyanine,
Since the uses of this compound are limited, the source
was soon identified as a paint factory.
34
-------
00
8
«— »
5L
s..
ftl
I*.,
o
'? )0 20 33 40 SO 60 73 83 93 133 110 120 133 140 150 163 173 18C
SPECTRUM MJMBER
FIGURE 6. COMPLICATED RGC FROM AN INDUSTRIAL EFFLUENT EXTRACT
-------
OJ
e
8.
Rj
iM|fiM|Ti?i|Miim
10 20 30 1C
SPECTHLH NU«R
70
>m^^
80 90 100 110 120 130
FIGURE 7. RGC OF EFFLUENT EXTRACT FROM FIGURE 6 USING LIMITED MASS TECHNIQUE
TO ENHANCE SENSITIVITY AND REDUCE INTERFERENCES
-------
SECTION VII
INTERPRETATION AND COMPUTER MATCHING
The mass spectrum provides a chemical fingerprint that
is characteristic of a material and can be interpreted
to give the structure. The majority of organic spectra
can be interpreted through a combination of experience
and a knowledge of chemistry and mass spectral fragmen-
tation theory. Depending on the complexity and unique-
ness of the spectrum and the analyst's familiarity with
the compound or class of compounds, interpretation can
be made in a matter of seconds or it can require hours
or even days. Tables that list the eight or ten most
intense peaks (21-23) enable the analyst to search for
compounds at a rate of about 10 minutes per search.
This becomes tedious if more than one or two spectra
are analyzed. Computerized empirical spectra matching
program can identify spectra at a much faster rate.
Many matching schemes using computers have been
described in the literature. After a thorough survey
of the literature and of EPA's needs, a research grant
was awarded to Battelle Columbus Laboratories to
modify the spectra matching system of Hertz, Hites,
and Biemann (24). The modifications included
increasing the speed of matching and constructing a
data base suitable for pollutant identification.
Matching, taking advantage of the information
redundancy of mass spectra, is based only on the two
most intense peaks in every 14 amu slot. There are
four main steps in the matching process:
1) Screening based on molecular weight range
2) Screening based on most intense peak of the
unknown spectrum
3) Pre-searching based on spectrum family
4) Calculation of the similarity index and
ordering of best matches based on peak-by-
peak comparison of unknown to those reference
spectra that passed the pre-search.
37
-------
This modified program, SEWL3P, has been evaluated and
improved during the past year. Matching the spectrum
of an unknown against the present data base of 11,000
spectra (10,600 general spectra from the Aldermaston
collection and 400 pollutant spectra from Southeast
Environmental Research Laboratory and Battelle)
requires approximately 45 seconds. The similarity
index gives the user an immediate indication of whether
the "best hit" is a poor match (<0.2 if no closely
related compound is in data base), one of several fair
matches (0.2-0.35 if the correct compound is not in
data base) , or a good match (>0.35 where the second
best hit is significantly lower).
In cases where the correct answer is not included in
the data base, related compounds are usually found as
the best hits. This can lead to a rapid identification
by other means. In one study made at the Southeast
Environmental Research Laboratory, 50% of the unknowns
present in an environmental sample were found correctly
as the best hit; 8%, as the second best; and 2% as the
third best.
Addition of new pollutant spectra to the library takes
place continuously. As the data base grows, the success
of the system is expected to improve.
To reduce the operator time involved in utilizing this
system, PDP8 utility programs were developed with
Battelle that enable a laboratory to transfer all of
the necessary data from the laboratory computer through
a telephone coupling device to the central matching
computer. These programs also eliminate human errors
and prejudices in selection and transmission of data.
At the time the National Water Contaminants Characteri-
zation Research Program and Battelle were developing
this system, a somewhat less sophisticated matching
program was developed at the National Institutes of
Health (25) and a simple one at Finnigan Corporation
and System Industries (26). A comparison of these
programs is given in Table 5. Each has certain
strengths that adapt it to different phases of pollu-
tant identification.
38
-------
Table S
Comparison of Three Spectra Matching Programs
Par JUTE ter
System
EPA/Battelle NIH
Finnigan/
System Industries
Basis for match
Average number of
input peaks used
Data selection
and entry
Average natch
time for best
fit (based on
present library
size)
Output ordered
and ranked by
Effect of an
extraneous peak
Number of spectra
in data base
Expansion of data
base
Present availa-
bility to Agency
users
Minimum hardware
required
Masses and intensi-
ties of the two
most intense peaks
in all 14-amu slots
-25
Semi-automatic or
manual
45 seconds
Similarity index
(high-low)
Slight reduction in
similarity index
11.000
Masses and inten-
sities selected
from the two most
intense peaks in
14-amu slots
Manual
Screens only.
Does not choose a
•best fit-
Screening tine
(including dialog)
approximately 2
minutes for 4
mass peaks.
File number
(low-high)
Eliminates the
correct match
8,800
EPA program manager User
Nationwide on
Battello time-
shared computer
Acousticoupler
S. TTY
Preferred hardware* Coupler, KLBE/c,
and CRT
Estimated cost
per match (exclu-
ding library.
storage costs)
$1.50
Nationwide on
NIH tine-shared
computer
Acousticoupler
& TTY
Coupler t CRT
$1.00
Masses of the
most intense peak
in all 14-amu
slots
-15
Manual (semi-
automatic version
has been written)
<15 seconds for
System 250 disk
<3 minutes for
System 150
DECtape
Number of mis-
matched peaks
(low-high)
Increases the
mismatch index
by 1
300
User
System Industries
PDP8 routine—
also available on
GE time-shared
computer
TTY
CRT
Free
•Comparisons of match time and costs made on this basis.
39
-------
The EPA/Battelle program is the most useful because it
quickly provides ordered listings of the best matches
with minimal operator handling. These listings
are based on detailed comparison of both masses and
intensities for all peaks in the reduced unknown
spectrum with those of the 11/000 reduced spectra of
the library. When the correct compound is not in the
data base, the best matches have low similarity indices
but generally are related to the correct compound.
The NIH system provides sequential screenings of the
data base through comparison of the intensities of
user-selected masses from the unknown with those of
the corresponding masses in reference spectra. This
system has been useful in suggesting possible
structural fragments associated with the selected
masses.
The Finnigan/System Industries program appears well
suited for low-cost, rapid, in-house matching of
spectra against small data bases. The library
furnished with the system was developed for drug
enforcement use (26) and is not applicable to
environmental pollutants. Work is underway to develop
a similar library of the 240 industrial organic
pollutants listed in Appendix 1. When this is
completed, a better assessment of the Finnigan/System
Industries program can be made.
40
-------
SECTION VIII
CONFIRMATORY TECHNIQUES
Although the mass spectrum of a compound can be
described as a fingerprint, it is not always completely
unique. The spectra of different members of homologous
series (hydrocarbons and fatty acids) and of isomers
(polychlorobiphenyls and alkanes) are usually indistin-
guishable. In the case of m- and p-cresol, not only
are the spectra indistinguishable, but on many GC
columns the retention times are the same.
A broad range of confidence levels exist for an iden-
tification by GC-MS. The lowest level is identifica-
tion based entirely on a chemist's interpretation of
the spectrum without any reference to a standard or
known spectrum. Next is a poor match with a standard
spectrum or a low similarity index (<0.2) from a
machine search. Computer matches with similarity
indices above 0.4 or close resemblance with spectra
from the literature are termed tentative identifications.
An even higher level of confidence is placed in a good
computer or literature MS match combined with a GC
retention time match with a known measured in the
laboratory. This level is accepted as a confirmed match
in our laboratory, particularly when the compound has
been run here earlier and its spectrum placed in our
files and in the computer library. The best identifi-
cation based solely on GC-MS is a match of both the GC
and MS data with a known run under the same conditions.
The ultimate in identification is agreement between
GC-MS and other independent methods, e.g., NMR or IR.
Unfortunately, the amounts of sample required usually
preclude the use of conventional NMR and IR in water
analysis.
41
-------
SECTION IX
CASE HISTORIES
Black Warrior River and Locust Fork Branch Fish Kills
Fish kills in the Black Warrior River occurred at the
Locust Fork Branch, near Birmingport, Alabama/ in
October, 1969, and again near Demopolis, Alabama, in
September, 1970. The 1969 kill involved 750 thousand
fish and the 1970 incident killed 8 thousand fish.
Both kills were suspected to have been caused by the
spraying of malathion in conjunction with a U. S.
Corps of Engineers mosquito control program in the
area. The presence of malathion was confirmed by GC-MS
in extracts of both the Locust Fork Branch (Figure 8-A)
and the Black Warrior River area near Demopolis
(Figure 8-B).
The molecular ion (m/e 330) was not observed in spectra
of either of the samples or of the standard when it was
introduced from the gas chromatographic column (Figure
8-C). Instead, the fragment (m/e 173) resulting from
cleavage of (CH-jO^PSo* was the largest significant
ion. A small parent ion was found in a sample intro-
duced by the direct probe (Figure 8-D). The fragmen-
tation pattern of 8-D is significantly different from
8-C, primarily because of different sample pressures
in the ion source, illustrating the necessity of
comparing mass spectra of samples with standards under
identical conditions. The major fragmentations of
malathion are indicated in Figure 8-D.
The results of these analyses were used in a brief
prepared by the Alabama Department of Conservation and
submitted to the Alabama State Attorney.
Polychlorinated Biphenyls (PCB's)
Aroclors, manufactured by the chlorination of biphenyl,
are distillation fractions containing 20 or more PCB
isomers. They are used widely in industry and have
become ubiquitous pollutants of the aquatic environment.
We have used mass spectrometry several times to confirm
the presence of PCB's, identified tentatively by
43
-------
B
»3
MAIATHION - LOCUST CREEK
« m
173
US
'ar"'''te • 'ao'" v>a—ws > •
MALATHION - (LACK WARRIOR
CREiK
MMATHtON STANDARD- GC/MS
''
0 " l*f BS^^^S 30iS 7!B SB"
MALATHION STANDARD • DIRECT PROBE
,*$ \°
330
"To ; « w-^Tir—KB ' of—as—to—BS—SB—ao • a«i—ato acr so j» s
FIGURE 8. MASS SPECTRA OF EXTRACTS AND STANDARDS
CONTAINING MALATHION
44
-------
electron capture GC, in water and mud samples. The
FID chromatograms of the extracts are invariably
complicated, but the specificity of the mass spectro-
meter allows unequivocable identifications of submicro-
gram amounts of PCB's,
In one case, a Florida Bay sediment extract, cleaned
on a florisil column, was analyzed for Aroclor 1254.
The GC effluent was split 1:1 between the flame detec-
tor and the MS. The GC flame detector pattern is shown
in Figure 9. Eleven of these peaks were shown by MS to
be PCB's. Their retention times and chlorine numbers
(number of chlorine atoms per PCB molecule, as
determined by MS) correspond with those of an Aroclor
1254 standard, also shown in Figure 9.
Mass spectra showing several chlorine isotope clusters
for some of the PCB's from the sediment are compared
with corresponding spectra from an Aroclor 1254
standard in Figure 10. The parent ion was observed in
all the PCB's. The major fragmentation path is loss
of successive chlorine atoms from the parent molecule.
Industrial Effluent Characterization
In April, 1971, EPA Division of Field Investigations,
Denver, Colorado, requested chemical characterization
of the wastewaters from seven different companies in
an industrial area of western Louisiana.
One liter of the effluent from a petrochemical company
was extracted with chloroform; the organic layer was
dried over anhydrous sodium sulfate and concentrated
in a Kuderna-Danish apparatus to about 1 ml. Further
concentration (to 0.25 ml) was effected by carefully
blowing dry nitrogen over the sample, which was warmed
only by body heat from the fingers. The concentrated
sample was chromatographed on a 50 ft SCOT column
coated with carbowax 20M-TPA.
Figure 3 shows the ion current summation (ICS) and,
plotted above it, the computer-reconstructed gas
chromatogram (RGC). Although many of the RGC peaks
exhibited a variation in height or area relative to
the FID chromatogram obtained prior to the GC-MS run,
45
-------
PCB's- SEDIMENT EXTRACT
6 CHLORINE
0 NUMBERS
1
•50
40
20
WTO
m
JO
Q
0 3
14
12
10
864
-TIME,min.
FIGURE 9. FLAME IONIZATION DETECTOR GAS CHROMATOGRAMS
OF PCB'S FROM THE ENVIRONMENT AND IN AN
AROCLOR STANDARD. THE NUMBERS ARE CHLORINE
ATOMS PER PCS MOLECULE DETERMINED BY MS
46
-------
PCB's-SEDIMENT EXTRACT
. . M*,4CI
*«' too
. SO
SCI
(M*-CI>
— ~TOJ 370 --V/B
BO 580
£
ftt
z
1 «
i
Jo
300 -J
4Cl M*«
100
3CI 3CI
M',3CI go
(
ji
L~4r^r4^-V
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s
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Z
C 4O
4
1 S
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'
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3CI ;; i
!
' i i
!<
C" TZ60 28B 300 3JO HO X
PCB's-AROCLOR 1254
wo
DO
60
40
20
M**CI
?CI
(M*->CI)
~H» f.
3CI
D SO """
•ins — 280
no
80
g
| 60
: z
2 40
2
20
Too
4C1
2CI
SCI
|
!
KX)
M»,5CI j. K
j
60
4C
M
! [l
. 550 240 KO 280 J ! 300 320' 340
M»
4CI
5CI
3CI ,!|
,i
~54ff 2«) 280^ 500 320 W J(
FIGURE 10. PARTIAL MASS SPECTRA OF SOME OF THE PCB'S SHOWN IN FIGURE 9
-------
the resemblance was sufficient to relate the FID GC
peaks to their RGC counterparts.
After the RGC and desired LMRGC's were plotted,
requests for spectra, with background or interfering
peak subtraction, were logged into the computer. When
the spectra of the individual peaks were produced by
the computer, they were identified by the spectra
matching program through the central data bank located
at the Battelle Research Institute, Columbus, Ohio.
Table 6 shows the list of compounds identified in the
plant wastewater.
Correlation of Compounds in Natural Waters
with Industrial Wastewater Discharges
In the summer of 1972 the Organic Analysis Unit of the
Lower Mississippi River Field Facility requested our
assistance in determining whether paper mills in that
region contributed measurably to organic pollution of
the Mississippi River. Only one mill will be discussed
as an example.
The mill wastewater (1.8 Si) was extracted with two
500 ml portions of chloroform. Combined extracts were
dried over sodium sulfate and concentrated to 1 ml in
a Kuderna-Danish apparatus. The extract was further
concentrated to 0.3 ml by blowing dry nitrogen over
the sample.
Six liters of Mississippi River water sampled 0.25 mi
above the mill (control sample) and 6 Jl sampled 0.25
mi below the mill were each extracted with two 1 &
portions of chloroform. Combined extracts of each
sample were dried over sodixun sulfate, concentrated to
1 ml in a Kuderna-Danish apparatus, and further concen-
trated to 0.25 ml with dry nitrogen, in addition to
the upriver control sample, a solvent blank was
necessary because of the very large concentration
factors (8,OOOX) required. Two liters of the same lot
of chloroform were therefore treated similarly.
48
-------
Table 6
Compounds Identified in Wastewater
of Petrochemical Company
RGC Spectrum #
(from Figure 3) Compound Name
2 m-xylene*
4 p-xylene*
10 1,5-cyclooctadiene
16 o-xylene*
29 isopropylbenzene (cumene)
36 styrene*
47 o-ethy1toluene
65 o-methylstyrene*
70 diacetone alcohol
75 indan*
86 2-butoxyethanol
89 3-methylstyrene
109 indene*
121 dimethylfuran isomer
129 n-pentadecane
140 1-methylindene*
145 3-methylindene
156 acetophenone
160 n-hexadecane
168 a-terpineol
177 naphthalene*
193 a-methylbenzyl alcohol
202 2-methylnaphthalene*
206 benzyl alcohol
2io l-methylnaphthalene*
221 ethylnaphthalene isomer
233 2,6-dimethylnaphthalene*
233 phenol*
244 methyl ethyl naphthalene isomer
249 cresol isomer
256 acenaphthene
265 acenaphthalene
278 methylbiphenyl isomer
287 fluorene
292 phthalate diester
296 3,3-diphenylpropanol
356 phthalate diester
identification was confirmed with a standard.
49
-------
The results of the GC-MS analysis are summarized in
Figures 11-14. The solvent contained the impurities
shown in Figure 11. Figure 12 shows that the upriver
control sample contains only one compound (guaiacol)
that is commonly found in paper mill effluents. The
paper mill effluent contained eleven compounds (Figure
13). Five of these compounds were confirmed in the
downriver sample (camphor, fenchyl alcohol, terpinene-
4-ol, alpha-terpineol and anethole isomer "C") as shown
in Figure 14.
Waste Treatment
The cases already discussed are examples of typical
short-term problems that confront most service labora-
tories . They usually require a few man-days to
several man-months effort. In addition to these,
several extensive projects based on GC-MS analysis are
in progress at SERL. GC-MS is invaluable in studying
the effects of waste treatment practices in areas such
as municipal sewage, paper mills, textile mills, and
refineries. In these studies, GC-MS determines
• the chemicals present in the raw wastewaters,
• which chemicals are reduced or removed by the
treatment,
• which chemicals resist the treatment and are
discharged,
• which new chemicals are produced by the
treatment, and
• which treatment is effective with each
compound.
Each of these studies is a major effort that requires
development of specific techniques for sampling,
extraction, cleanup, concentration, and GC-MS. In
addition to providing answers to specific pollution
problems, these studies are also demonstrating that
GC-MS is an indispensable tool in water pollution
studies.
50
-------
Ui
FIGURE 11. COMPUTER RECONSTRUCTED CHROMATOGRAM OF CHLOROFORM BLANK
-------
(Ji
ao '» JT15 ab 'M jaTSe 120 aw
FIGURE 12. RGC OF UPRIVER CONTROL SAMPLE EXTRACT
-------
o-t
B.
B
U.
I.
U)
8.
JL
S.
to * w « SB
to » wo iia UB ue i-w ias iw lie
to VB «B to
FIGURE 13. RGC OP PAPER MILL EFFLUENT EXTRACT
-------
2- PHOPIWIYLTHIOIMENE
R.
9.
V
HEXACHLOflOCtHANE
N-NONYL ALDEHYW
2-AcET¥LTHIOPHEHE
ALPHA-TERPINEOL
I I I I I I I '" P ' I " I' I'
e ID 28 an « a so TB
BPETTHJI MMEH
tee UB 'i2B lac 110 'IBB 'i» tie ri
aob
zib
ZIB
» »(i MB »• W W 3" '"> •"
FIGURE 14. RGC OF DOWNSTREAM SAMPLE EXTRACT
-------
SECTION X
ACKNOWLEDGEMENTS
Mary Walker, Ann Alford and Mike Carter, mass spectro-
metrists at the Southeast Environmental Research
Laboratory, have made substantial contributions to
this report and to the application of GC-MS to water
pollution problems.
The data on extraction efficiency were contributed by
A. D. Thruston, Jr.
William Loy, Surveillance and Analysis Division,
Region IV, has made extensive application of GC-MS
and his contribution to Appendix One is appreciated.
55
-------
SECTION XI
BIBLIOGRAPHY
1. Brans, H., Middleton, F. M., and Walton, W.,
"Organic Chemical Compounds in Raw and Filtered
Surface Waters/1 Anal. Chem., 23, 1160 (1951).
2. Burke, J. A., Mills, P. A., and Bostwick, D. C. ,
"Experiments with Evaporation of Solutions of
Chlorinated Pesticides," J. Assn. of Official
Anal. Chemists, 49, 999 (1966).
3. Chiba, M., and Morley, H. V., "Studies of Losses of
Pesticides During Sample Preparation," Ibid., 51,
56 (1968).
4. Goldberg, M. C., DeLong, L., and Sinclair, M.,
"Extraction and Concentration of Organic Solutes
from Water," Anal. Chem., 45, 89 (1973).
5. Mills, P. A., Onley, J. H., and Gaither, R. A.,
"Rapid Method for Chlorinated Pesticide Residues
in Nonfatty Foods," J^ Assn. Official Anal. Chem.,
46., 186 (1963).
6. Garrison, A. W., and Hill, D. H., "Organic Pollu-
tants from Mill Persist in Downstream Waters,"
American Dyestuff Reporter, 61, 21 (1972).
7. Rosen, A. A., and Middleton, P. M. , "Identification
of Petroleum Refinery Wastes in Surface Waters,"
Anal. Chem. , 27., 790 (1955) .
8. Paris, D., Personal Communication, 1972.
9. Webb, R. G., and McCall, A. C., "Identities of
Polychlorinated Biphenyl Isomers in Aroclors,
J._ Assn. Official Anal. Chem. , 55, 746 (1972) .
10. Katz, S., Pitt, W. W., Jr., Scott, C- D. and Rosen,
A A., "The Determination of Stable Organic Com-
pounds in Waste Effluents at Microgram/Liter Levels
by Automatic High-Resolution Ion Exchange Chromato-
graphy," Water Research, 6_, 1029 (1972).
57
-------
11. Pierce, A. E. , Silylatign of Organic Compounds,
Pierce Chemical Company, Rockford, 111. (1968).
12. Mrochek, J. E., Butts, W. C., Rainey, W. T., Jr.,
and Burtis, C. A., "Separation and Identification
of Urinary Constituents by Use of Multiple-
Analytical Techniques," Clinical Chemistry, 17,
72 (1971).
13. Hopps, H. B., "Preparation and Reactions of Diazo-
methane," Aldrichimica Acta, 3_, 9 (1970), published
by Aldrich Chemical Company, Milwaukee, Wisconsin.
14. Green, C. R., "The Reaction of Diazomethane with
Polyhalomethanes," University Microfilms, Incor-
porated, Ann Arbor, Michigan (1970).
15. Crowell, E. P., Arnovic, S. M., and Burnett, B. B.,
"Gas Chromatographic Determination of Mono- and
Dibasic Acids," £._ Chromatog. Sci. , 9_, 296 (1971).
16. "Alkyl-8 Reagents" from Pierce Previews, November,
1972, published by Pierce Chemical Co., Rockford,
Illinois.
17. "MethElute," Product Bulletin No. 49300 (1971) by
Pierce Chemical Co., Rockford, Illinois.
18. Beroza, M. and Bierl, B. A., "Rapid Determination
of Olefin Position in Organic Compounds in Micro-
gram Range by Ozonolysis and Gas Chromatography,"
Anal. Chem. , 39^ 1131 (1967) .
19. Garrison, A. W., Keith, L. H. , and Alford, A. L.,
"Confirmation of Pesticide Residues by Mass
Spectrometry and NMR Techniques," Advances in.
Chemistry Series, No. Ill, 26-54 (1972).
20. Neher, Maynard B., "Evaluation of Gas Chromatograph/
Mass Spectrometer/Computer Systems," Battelle
Columbus Laboratories Research Report to the Water
Quality Office of the Environmental Protection
Agency (1971), Battelle Laboratories, Columbus,
Ohio.
21. Cornu, A., and Massat, R., Compilation of Mass
Spectral Data, Heyden & Son Limited, London (1966).
58
-------
22. Atlas of Mass Spectral Data, Vols. 1-3, Edited by
Stenhagen, E., Abrahamsson, S., and McLafferty,
F. W., Interscience, New York (1969).
23. Eight Peak Index of Mass Spectra, Vols. 1&2, Mass
Spectrometry Data Centre, AWRE, Aldermaston,
England (1970).
24. Hertz/ H. S., Kites, R. A., and Biemann, K.,
"Identification of Mass Spectra by Computer-
Searching a File of Known Spectra," Anal. Chem.,
4_3, 681 (1971) .
25. Heller, S. R. , "Conversational Mass Spectral
Retrieval System and Its Use as an Aid in Struc-
ture Determination," Anal. Chem., 44, 1951 (1972).
26. Finkle, B. S., Taylor, D. M. , and Bonelli, E. J. ,
"A GC/MS Reference Data System for the Identifi-
cation of Drugs of Abuse," J. Chromatog. Sci. ,
10, 312 (1972).
59
-------
SECTION XII
APPENDICES
No. Page
1 Organic Compounds Identified in Water by 62
Gas Chromatography-Mass Spectrometry
2 Procedure for Diazomethane Methylation 88
3 Procedure for Dimethyl Sulfate Methylation 90
61
-------
APPENDIX ONE
ORGANIC COMPOUNDS IDENTIFIED IN WATER BY
GAS CHROMATOGRAPHY-MASS SPECTROMETRY
Compound (1)
Sample source
Concentrations, toxicities (4),
comments, analyst
cr\
6,8 ,11,13-Abietatetraen-18-
oic acid (2,C) (3)
13~Abieten-18-oic acid (3)
Abietic acid (C) (3)
Acenaphthalene
Acenaphthene
(C)
(C)
Acetophenone (C)
Paper mill's raw waste and trick-
ling filter effluent
Paper mill's raw waste and trick-
ling filter effluent
Paper mill's raw waste and lagoon
Petrochemical plant's five-day
lagoon effluent
Petrochemical plant's five-day
lagoon effluent
Wood preserving plant's lagoon
effluent
Wood preserving plant's settling
pond
Pesticide plant's raw effluent
Chlorinated paraffin plant's
lagoon
Petrochemical plant's five-day
lagoon effluent
Toxic to salmon at 2-5 mg/1 (10).
Keith.
Toxic to salmon at 2-5 mg/1 (10).
Keith.
Toxic to salmon at 5 mg/1 (10).
Keith, Loy.
Keith.
Caused skin tumors in mice (9)
Keith.
Loy.
0.2 mg/1. Loy.
McGuire.
0.29 mg/1; LD50 in rats is 3g/kg
(7). Loy.
Keith.
-------
a\
Acetosyringone (C)
Acetovanillone (C)
(C)
2-Ac e t yl th io phe ne
Acrylonitrile (C)
Adiplc acid (C)
Adiponitrile (C)
Aldrin
m-Anethole
o-Anethole
p-Anethole
Anthraquinone (C)
Anteisomargaric acid (C)
(3)
Gulf coast paper mill's settling
pond
Gulf coast paper mill's settling
pond
Paper mill's raw waste and lagoon
Paper mill's raw waste
Acrylic fiber plant's settling
pond
Nylon plant's raw waste
Nylon plant's raw waste
Pesticide plant's raw effluent
Paper mill's raw waste
Paper mill's raw waste
Paper mill's raw waste
Wood preserving plant's settling
pond
Paper mill's raw waste and five-
day lagoon
0.14 mg/1. Loy.
Estimated at 0.025 mg/1. Loy.
Keith, Loy.
Keith.
100 mg/1; 24 hr. TLm to bluegllls
is 22.5 mg/1 arid 30 mg/1 caused
100% mortality to pin perch (8).
Garrison.
3.7 mg/1; 24 hr. TLm for bluegills
is 330 mg/1 (11). Loy.
320 mg/1; 24 hr. TLm for bluegills
is 815 mg/1 (8). Loy.
96 hr. TLm at 25° C is 0.01 mg/1
for bluegill (8). McGuire.
Keith.
Keith.
Keith.
0.05 mg/1. Loy.
Keith.
-------
Anteisopentadecanoic acid
(C) (3)
Arachidic acid (3)
Arachidonic acid (C) (3)
Behenic acid (C) (3)
Benzaldehyde (C)
Benzyl alcohol
2-Benzothiazole (C)
(C)
Biphenyl
Borneo1
1-Butanol (C)
2-Butoxyethanol
n-Butylisothiocyanate (C)
Paper mill's five-day lagoon
Paper mill's raw vaste
Paper mill's five-day lagoon
Paper mill's raw effluent and
five-day lagoon
Paper mill's raw waste
Petrochemical plant's five-day
lagoon effluent
Latex accelerators and thickeners
plant's holding pond
Synthetic rubber plant's aerated
lagoon
River below textile finishing
plant
Paper mill's raw waste and trick-
ling filter effluent
Petrochemical (alcohols) plant's
raw effluent
Petrochemical plant's five-day
lagoon effluent
Latex accelerators and thickeners
plant's holding pond
Keith
MLD for fish is 5 mg/1 of the
sodium salt (8). Garrison.
Keith.
Keith.
Keith.
Keith.
0.16 mg/1; LD50 for mice is 100
mg/kg (7). Loy.
Very disagreeable odor. Loy.
ID50 in rats is 2.2 g/kg (7). Loy.
Toxic—probable human lethal dose
is 50-500 mg (5). Keith, Loy.
16.0 mg/1; 90 Ib/day discharge.
Keith.
Keith.
Raw effluent 0.5 mg/1 and holding
pond 0.1 mg/1. Loy.
-------
a\
ui
Camphor (C)
(C)
Caproic acid (C)
Carbazole (C)
Chlordane
Chlordene
o-Chlorobenzoic acid (C)
(3)
bis-(2-Chloroethoxy)
methane (C)
bis-2-Chloroethyl ether
(C)
bis-2-Chloro isopropyl
ether (C)
trans-Communic acid (3)
o-Cresol (C)
Paper mill's raw waste and trick-
ling filter effluent
Gulf coast paper mill's settling
pond
Nylon plant's raw waste
Wood preserving plant's settling
pond
Pesticide plant's raw effluent
Pesticide plant's raw waste
Chlorinated paraffin plant's
lagoon
Synthetic rubber plant's treated
waste
Synthetic rubber plant's treated
waste
Glycol plant's thickening and
sedimentation pond
Paper mill's raw waste and
trickling filter effluent
Wood preserving plant's settling
pond
Minimum detectable taste is 2 mg/1
(8). Keith, Loy.
0.031 mg/1. Loy.
220 mg/1; 24 hr. TLm for bluegills
is 200 mg/1 (11). Loy.
0.27 mg/1; intraperitoneal LD50 for
rats is 200 mg/kg (9). Loy.
96 hr. TLM at 25° C is 0.02 mg/1
for bluegill (8). McGuire.
McGuire.
0.24 mg/1. Loy.
140 mg/1. Loy.
0.16 mg/1. Loy
Loy.
Toxic to salmon at 2-5 mg/1 (10),
Keith.
1.4 mg/1; 48 hr. TLm for fathead
minnows is 24 mg/1; odor thres-
hold is 0.07 mg/1 at 30° C (8).
Loy.
-------
o-Cresol (C)
m-Cresol (C)
p-Cresol (C)
Cumene (isopropylbenzene)
Cyclohexano1 (C)
1,5-Cyclooc tad iene
o\ p-Cymene (C)
it
Decane (C)
1-Decanol (C)
Dehydroabietic acid (C) (3)
(C) (3)
Petroreflnery's eight-hour
lagoon effluent
Wood preserving plant's settling
pond
Paper mill's raw waste and lagoon
Petrochemical plant's five-day
lagoon effluent
Nylon plant's raw waste
Petrochemical plant's five-day
lagoon effluent
Paper mill's raw waste and trick-
ling filter effluent
Pesticide plant's raw waste
Polyolefin plant's lagoon
Petrochemical (alcohols) plant's
raw effluent
Wood preserving plant's settling
pond
Paper mill's raw waste and trick-
ling filter effluent
0.120 mg/1; 300 Ib/day discharge.
Keith.
2.5 mg/1; 24 hr. TLm for carp is
24 mg/1; odor threshold is 0.33
mg/1 at 30° C (8). Loy.
More toxic than phenol (5); odor
threshold 0.05 mg/1 (8). Loy.
Keith.
LD50 to rats is 1-10 g/kg (12).
Loy.
Keith.
Keith.
McGuire.
0.03 mg/1. Keith.
2.5 mg/1; 15 Ib/day discharge.
Keith.
0.02 mg/1; the sodium salt is toxic
to salmon at 5 mg/1 (10); LD50
in rats is Ig/kg (7). Loy.
Keith.
-------
Dehydroabietic acid (C) (3)
Gulf coast paper mill's settling
pond
0.47 mg/1. Loy.
(C) (3) Tall oil refinery's settling pond Loy.
Diacetone alcohol
4,4'-Diamino-dicyclohexyl
methane
Dibenzofuran (C)
(C)
(C)
2,3-Dibromo-l-propanol (C)
Dibromopropene isomer
Dibutylamine (C)
Dieldrin (C)
Petrochemical plant's five-day
lagoon effluent
Nylon and polyester plant's
effluent after neutralization
and sedimentation
Wood preserving plant's settling
pond
Wood preserving plant's lagoon
effluent
Nylon plant's settling pond
Acrylic fibers plant's settling
pond
Acrylic fibers plant's settling
pond
Latex accelerators and thickeners
plant's raw effluent
Anaerobic lagoon of yarn finish-
ing mill
Causes liver damage and anemia in
animals (5). Keith.
0.4 mg/1. Loy.
0.12 mg/1. Loy.
Loy.
Garrison.
0.5 mg/1. Loy.
Garr ison.
Less than 1 mg/1; LD50 in rats is
550 mg/kg (7). Loy.
48 hr. TLm for bluegill is 3.4 ug/1
and 0.3 yg/1 for marine shrimp;
lethal to rainbow trout after
three months' exposure to 1 yg/1
(5). Garrison.
-------
CP>
00
Dieldrin
N,N-Diethylformamide (C)
Diethy1 phthalate (C)
3,4-D ihydroxyacetophenone
(pungenin) (3)
3,5-Dimethoxy-4-hydroxy-
acetophenone (C) (3)
Pesticide plant's raw effluent
96 hr. TLm values for several fish
species are 0.005-0.05 mg/1 (8).
McGuire.
Latex accelerators and thickeners Less than 1 mg/1. Loy.
plant's raw effluent
Synthetic rubber plant's settling Loy.
pond
Paper mill's trickling filter
effluent
Paper mill's raw effluent and
five-day lagoon
2,4-Dimethyldiphenylsulfone Nylon plant's settling pond
Dimethyl furan isomer
2,6-Dimethyl naphthalene
(C)
Dimethyl naphthalene isomer
Dimethyl phthalate (C)
(C)
Acrylic fibers plant's settling
pond
Petrochemical plant's five-day
lagoon effluent
Petrochemical plant's five-day
lagoon effluent
Pesticide plant's raw effluent
Plastic (PVA) plant's settling
pond
Probably low T&O threshold. Keith,
Keith.
Garrison.
Garr ison.
Keith.
0.015 mg/1. Keith
McGuire.
LD50 to rats is 8400 mg/kg (7).
Loy.
Synthetic rubber plant's settling Loy.
pond
-------
VD
Dimethyl pyridine isomer
Dimethyl quinolirie isomers
Dimethyl sulfone (C)
Dimethyl sulfoxide (C)
10,12-Dimethyl tridecanoic
acid (3)
4,6-Dinitro-o-cresol (C)
(2-methyl-4,6-dinitro-
phenol)
2,4-Dinitrotoluene (C)
2,6-Dinitrotoluene (C)
(C)
3,4-Dinitrotoluene (C)
Diphenylene sulfide (C)
Wood preserving plant's settling
pond
Wood preserving plant's settling
pond
Paper mill's raw waste and trick-
ling filter effluent
Paper mill's raw waste and trick-
ling filter effluent
Paper mill's five-day lagoon
Specialty chemical plant's
effluent
Explosives (DNT) plant's raw
waste and settling pond
effluent
Explosives (DNT) plant's raw
waste and settling pond
effluent
TNT plant's raw effluent
Explosives (DNT) plant's raw
waste and settling pond
effluent
Wood preserving plant's settling
pond
0.1-0.2 mg/1. Loy.
0.1 mg/1. Loy.
Keith.
Keith.
Keith.
18 mg/1; minimum lethal dose at
6 hr. was 3-4 mg/1 for minnows
(8). Loy.
190 mg/1 in raw waste. Loy.
150 mg/1 in raw waste and 0.02 mg/1
in pond effluent. Loy.
0.68 mg/1. Loy.
40 mg/1 in raw waste. Loy, Garrison.
0.1 mg/1. Loy.
-------
Diphenyl ether
3,3-Diphenylpropanol
2,6-Di-t-butyl-p-benzo-
qu inone (C)
p-Dlthlane (C)
Dodecane (C)
(C)
Eicosane (C20) (c)
End r in
Ethyl carbamate (C)
2-Ethyl-l-hexanol (C)
Pesticide plant's raw effluent
Petrochemical plant's five-day
lagoon effluent
Surface drainage from closed
waste treatment system of
particle board plant
Synthetic rubber plant's treated
waste
Petrorefinery's lagoon effluent
after activated sludge treat-
ment
Petrorefinery's eight-hour
lagoon effluent
Paper mill's raw effluent
Petrorefinery's lagoon effluent
after activated sludge treat-
ment
Pesticide plant's raw effluent
Paper mill's trickling filter and
aerated lagoon
Gulf coast paper mill's settling
pond
Threshold odor and taste level is
0.013 mg/1 (8). McGuire.
Keith.
Estimated at 0.01 mg/1. Loy.
0.12 mg/1; strong offensive odor.
Loy.
0.22 mg/1; 0.4 Ib/day discharge.
Keith.
0.031 mg/1; 79 Ib/day discharge.
Keith.
Kerosene based defearner. Keith.
0.30 mg/1; 2.9 Ib/day discharge.
Keith.
TLm values are less than O.OOS mg/1
for six fish species (8).
McGuire.
Keith.
0.019 mg/1. LD50 to rats is 3200
mg/kg (7). Loy.
-------
2-Ethyl-l-hexanol (C)
(C)
Ethylidenecyclopentane
Ethyl isothiocyanate (C)
Ethyl naphthalene isomer
Ethyl naphthalene isomer
m-Ethyl phenol (C)
Ethyl phenylacetate (C)
o-Ethyl toluene
Eugenol
Fenchyl alcohol (C)
Fenchone (C)
Fluoranthene (C)
Laboratory sewage
Plastic (PVA) plant's settling
pond
River below textile finishing
plant
Paper mill's raw waste
Latex accelerators & thickeners
plant's raw effluent
Petrochemical plant's five-day
lagoon effluent
Pesticide plant's raw effluent
Paper mill's raw waste and lagoon
Resin plant's lime treated hold-
ing pond effluent
Petrochemical plant's five-day
lagoon effluent
Webb.
Loy.
Loy.
Keith.
Less than 1.5 mg/1; used as a
military poison gas (7). Loy.
Keith.
McGuire.
Loy.
Loy.
Keith.
Paper mill's raw waste and lagoon LD50 in rats is 2 g/kg (7). Keith.
Paper mill's raw waste and trick-
ling filter effluent
Paper mill's raw waste and trick-
ling filter effluent
Wood preserving plant's settling
pond
Taste threshold 2 mg/1 (5). Keith,
Loy.
Keith.
0.6 mg/1; oral LD50 for rats is
2000 mg/kg (9). Loy.
-------
Fluorene (C)
to
2-Formy 1 1 hio phene
Furfural (3)
(C)
Guaiacol (C)
CC) (3)
Heneicosane (C2l) (C)
Heptachlor
Heptachloronorbornene
isomers
Heptadecane
Wood preserving plant's settling
pond
Petrochemical plant's five-day
lagoon effluent
Paper mill's raw waste
Paper mill's raw waste
Synthetic rubber plant's settling
pond
Gulf coast paper mill's settling
pond
Paper mill's raw waste and trick-
ling filter effluent
Petrorefinery's lagoon effluent
after activated sludge treat-
ment
Pesticide plant's raw waste
Pesticide plant's raw effluent
Nylon plant's settling pond
0.17 mg/1. Loy.
Keith.
Keith.
Ingestion of 0.06g produces per-
sistent headache (7). Keith.
0.002 mg/1. Keith.
0.43 mg/1; toxic to perch at 70-80
mg/1; odor threshold is 0.002
mg/1 (8). Loy.
Keith, Loy.
0.19 mg/1; 1.8 Ib/day discharge.
Keith.
McGuire.
McGuire.
Garrison.
(C)
Petrorefinery's eight-hour lagoon 0.022 mg/1; 53 Ib/day discharge.
effluent Keith.
-------
U)
Heptadecane (C)
Hexachlor epoxide
Hexachlorobenzene (C)
Hexachlorobutadiene
Hexachlorocyclopentadiene
Hexachloronorbornadiene
isomers
Hexadecane (C)
(C)
(C)
Hexadieneal
1-Hexanol (C)
Petrorefinery's lagoon effluent
after activated sludge treat-
ment
Pesticide plant's raw waste
Chlorinated solvents plant's raw
effluent
Pesticide plant's raw effluent
Pesticide plant's raw waste
Pesticide plant's raw effluent
Nylon plant's settling pond
Petrorefinery's eight-hour
lagoon effluent
Petrorefinery's lagoon effluent
after activated sludge treat-
ment
Paper mill's raw waste
Petrochemical plant's five-day
lagoon effluent
Pesticide plant's raw effluent
Petrochemical (alcohols) plant's
raw effluent
0.34 mg/1; 3.3 Ib/day discharge.
Keith.
McGuire.
Loy.
McGuire.
McGuire.
McGuire.
Garrison.
0.026 mg/1; 66 Ib/day discharge.
Keith.
0.42 mg/1; 4.0 Ib/day discharge.
Keith.
Kerosene based defoamer. Keith.
Keith.
McGuire.
65.0 mg/1; 375 Ib/day discharge.
Keith.
-------
Homovanlllic acid (3)
p-Hydroxyacetophenone (C)
p-Hydroxybenzaldehyde (C)
(3)
o-Hydroxybenzoic acid (3)
Hydroxybiphenyl isomer
4~Hydroxy-3 methoxypropio-
phenone (C) (3)
p-Hydroxythiophenol (3)
Indan (C)
Indene (C)
Isodrin
Isoeugenol
Isopalmitic acid (C) (3)
Isopentyl alcohol (C)
Isooctyl phthalate (C)
Isopimaric acid (C) (3)
Paper mill's raw waste and five-
day lagoon
Paper mill's raw waste and lagoon
Paper mill's raw waste and lagoon
Paper mill's raw waste
Pesticide plant's raw effluent
Paper mill's raw effluent
Paper mill's raw waste
Petrochemical plant's five-day
lagoon effluent
Petrochemical plant's five-day
lagoon effluent
Pesticide plant's raw effluent
Paper mill's raw waste and lagoon
Paper mill's five-day lagoon
Laboratory sewage
Nylon plant's raw waste
Paper mill's raw waste and trick-
ling filter effluent
Keith.
Keith.
Keith.
Keith,
McGuire.
Keith.
Keith.
0.007 mg/1. Keith.
0.026 mg/1. Keith.
TLm for bluegill is 0.006 mg/1.
(8). McGuire.
Keith.
Keith.
0.17 mg/1. Webb.
Loy.
Toxic to salmon at 2-5 mg/1 (10).
Keith.
-------
Jasmone
Lignoceric acid (3)
Limonene (C)
Linoleic acid (C) (3)
Handelic acid (3)
Margaric acid (C) (3)
2-Mercaptobenzothiazole (C)
ui
alpha-Methylbenzyl alcohol
Methyl biphenyl isomer
Methyl 3,4-Dimethoxybenzyl
ether (3)
2-Methyl-4-ethyl dioxolane
(C)
Methyl ethyl naphthalene
isomer
Pesticide plant's raw effluent
Paper mill's raw waste
Paper mill's raw waste and trick-
ling filter effluent
Paper mill's raw waste and lagoon
Paper mill's raw waste
Paper mill's raw waste
Synthetic rubber plant's aerated
lagoon
(C) Paper mill's raw waste and lagoon
Petrochemical plant's five-day
lagoon effluent
Petrochemical plant's five-day
lagoon effluent
Paper mill's raw waste
Fiberglass plant's effluent
Petrochemical plant's five-day
lagoon effluent
McGuire.
MID for fish is 5 mg/1 of the
sodium salt (8). Garrison
Keith.
MID for fish is 10 mg/1 of the
sodium salt (8). Keith.
Keith.
MLD for fish is 5 mg/1 of the
sodium salt (8). Keith.
Very disagreeable odor. Loy.
Probably highly toxic. Keith.
Keith.
Keith.
Keith.
Distinct odor of black walnuts.
Loy.
Keith.
-------
1-Methyl indene (C)
3-Methyl indene
1-Methyl naphthalene
(C)
(C)
(C)
2-Methyl naphthalene (C)
(C)
Methyl naphthalene isomer
Methyl naphthalene isomers
13-Methyl pentadecanoic
acid (3)
Petrochemical plant's five-day
lagoon effluent
Petrochemical plant's five-day
lagoon effluent
River below textile finishing
plant
Petrorefinery'a eight-hour
lagoon effluent
Petrochemical plant's five-day
lagoon effluent
0.002 mg/1. Keith.
0.003 mg/1. Keith.
Probable lethal human dose 500-5000
mg/kg (5). Loy.
0.005 mg/1; 12 Ib/day discharge.
Keith.
0.02 mg/1. Keith.
Synthetic rubber plant's settling 0.002 mg/1. Keith.
pond
Petrorefinery's eight-hour lagoon 0.013 mg/1; 33 Ib/day discharge.
effluent Keith.
Petrochemical plant's five-day
lagoon effluent
Wood preserving plant's lagoon
effluent
Pesticide plant's raw effluent
Paper mill's five-day lagoon
0.03 mg/1. Keith.
Loy.
McGuire.
Keith.
Methyl phenanthrene
Wood preserving plant's lagoon Loy,
effluent
-------
-J
-J
Methyl quinoline isomers
o-Methylstyrene (C)
beta-Methylstyrene
Methyl trisulfide
Myristic acid (C) (3)
Naphthalene (C)
(C)
(C)
2-Naphthoic acid (C)
Neoabietic acid (C) (3)
Nitrobenzene (C)
Wood preserving plant's settling
pond
Petrochemical plant's five-day
effluent
Petrochemical plant's five-day
lagoon effluent
Paper mill's raw waste
Paper mill's raw waste
Nylon plant's settling pond
Surface drainage from closed
treatment of system of
particle board plant
Petrochemical plant's five-day
lagoon effluent
Pesticide plant's raw waste
Wood preserving plant's settling
pond
Paper mill's raw waste
Chemical company's lagoon after
steam stripping
0.5 mg/1. Loy.
0.001 mg/1. Keith.
Keith.
Keith.
MLD for fish is 5 mg/1 of the
sodium salt (8). Keith.
MLD to minnows for 6 hrs. is 15
mg/1 (8). Garrison.
Less than 0.01 mg/1. Loy.
0.05 mg/1. Keith.
McGuire.
0.16 mg/1. Loy.
Toxic to salmon at 2-5 mg/1 (10).
Loy, Keith.
0.11 mg/1; minimum lethal dose at
6 hr. was 90-100 mg/1 for minnows
(8); approximate cone, in water
causing faint odor is 0.03 mg/1
(8). Loy.
-------
-J
00
2-Nitro-p-cresol (C)
o-Nitrophenol (C)
o-Nitrotoluene (C)
(C)
(C)
m-Nitrotoluene
p-Nitrotoluene (C)
(C)
Nonachlor
Nonadecane (C)
(C)
Nonylphenol (C)
Chemical company's lagoon after
steam stripping
Chemical company's lagoon after
steam stripping
Paper mill's five-day lagoon
TNT plant's raw effluent
DNT plant's raw effluent
DNT plant's raw effluent
Chemical company's lagoon after
steam stripping
DNT plant's raw effluent
Pesticide plant's raw effluent
Petrorefinery's lagoon effluent
after activated sludge treat-
ment
Petrorefinery's eight-hour lagoon
effluent
Anaerobic lagoon of yarn finishing
mill
9.3 mg/1. Loy.
1.4 mg/1; minimum lethal dose at
6 hr. was 125-130 mg/1 for
minnows (8). Loy.
Keith.
0.15 mg/1. Loy.
7.8 mg/1 in raw waste and 0.012 in
pond effluent. Loy.
Garrison.
0.04 mg/1; minimum lethal dose at
6 hr. was 45-50 mg/1 for minnows
(8). Loy.
8.8 mg/1 in raw waste. Loy,
Garrison.
McGuire
0.31 mg/1; 3.0 Ib/day discharge.
Keith.
0.013 mg/1; 33 Ib/day discharge.
Keith.
LD50 for rats (orally) is 400-1400
mg/fcg (6); estimated human lethal
dose 500-5000 mg/kg (5). Garrison.
-------
Nonylphenol
Norcamphor
beta-Oclmene (C)
1-Octanol (C)
Octachlorocyclopentene
Octadecane (C)
(C)
Oleic acid (C)
(C) (3)
Octylphenol
Palmitic acid (3)
(C)
(C) (3)
(C) (3)
River below textile finishing
plant
Paper mill's raw waste
Paper mill's raw waste
Petrochemical (alcohols) plant's
raw effluent
Pesticide plant's raw effluent
Petrorefinery's eight-hour lagoon
effluent
Nylon plant's settling pond
Tall oil refinery's settling pond
Paper mill's raw waste and trick-
ling filter effluent
River below textile finishing
plant
Textile chemical plant's raw
effluent
Tall oil refinery's settling pond
Paper mill's raw waste and trick-
ling filter effluent
Gulf coast paper mill's settling
pond
Loy.
Keith.
Loy.
19.0 mg/1; 110 Ib/day discharge.
Keith.
McGuire.
0.017 mg/1; 43 Ib/day discharge.
Keith.
Garrison.
MLD for fish is 5 mg/1 of the
sodium salt (8). Loy.
Keith, Loy.
LD50 to mice is 25-50 mg/kg (6).
Loy.
MLB for fish is 5 mg/1 of the
sodium salt (8). Garrison.
Loy.
Keith, Loy.
0.013 mg/1. Loy.
-------
Palmitoleic acid (C) (3)
Pentachlorocyclopentadiene
isomers
Pentachloronorbornadiene
isomer
Pentachloronorbornene
Isomer
Pentachloronorbornene
isomer
Pentachloronorbornadiene
epoxide isomer
Pentachlorophenol (C)
oo
o
(C) (3)
(C)
(C)
(C)
Paper mill's five-day lagoon Keith.
Pesticide plant's raw effluent McGuire.
Pesticide plant's raw effluent McGuire.
Pesticide plant's raw effluent McGuire.
Pentadecane (C)
Pesticide plant's raw waste
Pesticide plant's raw waste
Latex accelerators and thickeners
plant's holding pond
Wood preserving plant's raw
effluent
Resin plant's lime treated
holding pond effluent
Synthetic rubber plant's aerated
lagoon
Wood preserving plant's lagoon
effluent
Petrorefineryfs eight-hour
lagoon effluent
McGuire.
McGuire.
0.4 mg/1; 0.2 to 0.6 mg/1 toxic to
19 varieties of fish; odor thres-
hold of 0.86 mg/1 at 30° C (8).
Loy.
Garrison.
Loy.
Loy.
Loy.
0.030 mg/1; 76 Ib/day discharge.
Keith.
-------
Pentadecane (C)
oo
Petrorefineryls lagoon effluent
after activated sludge treat-
ment
Paper mill's raw waste
Petrochemical plant's five-day
lagoon effluent
Pentadecanoic acid (C) (3) Paper mill's lagoon
Phenanthrene (C)
(C)
Phenol (C)
" (C)
11 (C)
11 (C)
" (C) (3)
Phenyl ether (C)
Wood preserving plant's lagoon
effluent
Wood preserving plant's settling
pond
Laboratory sewage
Petrorefinery's eight-hour
lagoon effluent
Wood preserving plant's settling
pond
Petrochemical plant's five-day
lagoon effluent
Paper mill's raw waste
Nylon plant's settling pond
0.49 mg/1; 4.8 Ib/day discharge.
Keith.
Kerosene based defoamer. Keith.
Keith.
MLD for fish is 5 mg/1 of the
sodium salt (8). Keith.
5 mg/1 killed rainbow trout and
bluegills in 24 hrs. C8). Loy.
1.4 mg/1. Loy.
96 hr. TLm for bluegills is 13.5
mg/1 and odor threshold is
0.02-0.03 mg/1 (8). Webb.
0.2 mg/1; 510 Ib/day discharge.
Keith.
0.66 mg/1. Loy.
0.06 mg/1. Keith.
Keith.
0.05 mg/1; odor threshold is 0.013
mg/1 (8). Garrison.
-------
00
o-Phenylphenol
Pimaric acid (C) (3)
(C) (3)
beta-Pinene (C)
Pinene isomer
Polychlorinated biphenyls
(Arochlor 1254) (C)
2-Propionylthiophene
4-n-Propylphenol (C)
Pyrene (C)
Quinoline (C)
Sandaracopimeric acid
(C) (3)
Stearic acid (3)
River below textile finishing
plant
Paper mill's raw waste and trick-
ling filter effluent
Gulf coast paper mill's settling
pond
Paper mill's raw waste
Gulf coast paper mill's settling
pond
Nylon plant's raw waste
Paper mill's raw waste
Paper mill's raw waste and lagoon
Wood preserving plant's settling
pond
Wood preserving plant's settling
pond
Paper mill's raw waste and lagoon
Textile chemical plant's raw
effluent
Probable lethal human dose 500-
5000 mg/kg (5). Loy.
Toxic to salmon at 2-5 mg/1 (10).
Keith, Loy.
0.12 mg/1. Loy.
Loy.
0.008 mg/1. Loy.
0.2 vig/1. Loy.
Keith.
Loy.
0.33 mg/1. Loy.
1.5 mg/1; oral LD50 for rats is
460 mg/kg (9); 5 mg/1 was lethal
to bluegills in 4 hrs. at 13° C
(8). Loy.
Toxic to salmon at 2-5 mg/1 (10).
Keith.
MLD for fish is 5 mg/1 of the
sodium salt (8). Garrison.
-------
00
CO
Stearic acid (C) (3)
Styrene (C)
(C)
Syringaldehyde (C)
(C) (3)
Terpinene-4-ol
alpha-Terpineol (C)
(C)
Terpineol isomer
Terpinolene
1,1,2,2-Tetrachloroethane
(C)
Tetrachlorophenol isomer
(3)
Gulf coast paper mill's settling
pond
Petrochemical plant's five-day
lagoon effluent
Synthetic rubber plant's settling
pond
Gulf coast paper mill's settling
pond
Paper mill's lagoon
Paper mill's raw waste
Nylon plant's settling pond
Paper mill's raw waste and trick-
ling filter effluent
Petrochemical plant's five-day
lagoon effluent
Gulf coast paper mill's settling
pond
Paper mill's raw waste
Chlorinated solvents plant's
raw effluent
Wood preserving plant's raw
effluent
0.02 mg/1. Loy.
0.03 mg/1; ID50 in rats is 5 g/kg
(5). Keith.
0.003 mg/1. Keith.
Estimated at 0.01 mg/1. Loy.
Keith, Loy.
Keith.
Garrison.
Keith, Loy.
Keith.
0.200 mg/1. Loy.
Kerosene based defoamer. Keith.
2.2 mg/1; LD50 intravenous in
rabbits is 80 mg/kg (7). Keith.
Odor threshold of 0.9 mg/1 at 30° C
(8); probable lethal human dose
is 50-500 mg/kg (5). Garrison.
-------
Tetradecane (C)
(C)
Petrorefinery's lagoon effluent
after activated sludge treat-
ment
Petrorefinery's eight-hour lagoon
effluent
Tetratuethylbenzene isomer Pesticide plant's raw waste
oo
2,2'-Thiodiethanol (C)
(Thiodiglycol)
Toluic acid (C) (3)
Trichlorobenzene isomer
Trichlorobenzene isomer
Trichlorocyclopentene
isomers
1,1,2-Trichloroethane (C)
Synthetic rubber plant's treated
waste
Chlorinated paraffin plant's
lagoon
River below textile finishing
plant
Textile chemical plant's raw
effluent
Chlorinated solvents plant's
raw effluent
Trichloroguaiacol (C) (3) Paper mill's raw waste
n-Tridecane (C)
(C)
Petrorefinery's eight-hour
lagoon effluent
Petrorefinery's lagoon effluent
after activated sludge treat-
ment.
0.58 mg/1; 5.6 Ib/day discharge.
Keith.
0.039 mg/1; 99 Ib/day discharge.
Keith.
McGuire.
Estimated at 2 mg/1. Loy.
0.24 mg/1. Loy.
May cause liver damage; estimated
human lethal dose is 50-500
mg/kg (5). Loy.
Estimated lethal human dose is
50-500 mg/kg (5). Garrison.
Pesticide plant's raw effluent McGuire.
5.4 mg/1; TLm for marine pinperch
is 150-175 mg/1 (5). Loy.
Present in sample toxic to salmon
(10). Keith.
0.042 mg/1; 107 Ib/day discharge.
Keith.
0.39 mg/1; 3.8 Ib/day discharge.
Keith.
-------
oo
n-Tridecane (C)
Triethylurea (C)
3,4,5-Trimethoxyaceto-
phenone (C) (3)
2,4,6-Trimethylpyridine
(C)
2,4,6-Trinitrotoluene (C)
n-Undecane
(C)
(C)
(C)
Valeric acid (C)
Vanillin (C)
(O
Veratraldehyde (C)
Paper mill's raw waste
Latex accelerators & thickeners
plant's raw effluent
Paper mill's raw waste and trick-
ling filter effluent
Wood preserving plant's settling
pond
TNT plant's raw effluent
Paper mill's raw waste
Petroref iriery 's eight-hour
lagoon effluent
Polyolefin plant's lagoon
Petrorefinery's lagoon effluent
after activated sludge treat-
ment
Nylon plant's raw waste
Kerosene based defoamer. Keith.
6.4 mg/1. Loy.
Keith.
0.3 mg/1. Loy.
0.7 mg/1; MLD for minnows over 6
hrs. is 4 mg/1 (8). Loy.
Keith.
0.027 mg/1; 69 Ib/day discharge.
Keith.
0.02 mg/1. Keith.
0.05 mg/1; 0.4 Ib/day discharge.
Keith.
Paper mill's raw waste and trick-
ling filter effluent
Gulf coast paper mill's settling
pond
Paper mill's raw waste & lagoon Keith.
500 mg/1; 48 hr. TLm for daphnia
magna is 4-5 mg/1 (11). Loy.
Odor threshold 0.15 mg/1 (8).
Keith, Loy.
Estimated at 0.02 mg/1. Loy.
-------
o-Xylene (C)
(C)
m-Xylene (C)
p-Xylene (C)
2,5-Xylenol (C)
3,4-Xylenol (C)
3,5-Xylenol (C)
Synthetic resin plant's settling
pond
Petrochemical plant's five-day
lagoon effluent
Petrochemical plant's five-day
lagoon effluent
Petrochemical plant's five-day
lagoon effluent
Wood preserving plant's settling
pond
Wood preserving plant's settling
pond
Wood preserving plant's settling
pond
Loy.
0.006 mg/1. Keith.
0.008 mg/1; MLD is 10-90 mg/1;
taste and odor threshold 0.3-1.0
mg/1 (8). Keith.
0.002 mg/1. Keith.
0.82 mg/1; behavior similar to
3,4-xylenol (8). Loy.
0.5 mg/1; 24 hr. TLm for carp is
30 mg/1; odor threshold similar
to phenol (8). Loy.
1.5 mg/1; 24 hr. TLm for carp is
53 mg/1; odor threshold similar
to phenol (8). Loy.
(1) Arranged alphabetically. Prefixes showing position (e.g. : p-, alpha-, bis-, trans-) are not
considered part of the name for this purpose.
(2) Confirmed identifications are marked (C). All others are to be regarded as tentative. Using
only GC-MS data, identifications can be confirmed by concurrent examination of the sample and
a known compound for duplication of GC retention time and MS fragmentation pattern. As a
substitute for the concurrent mass spectrum, the MS comparison can be with data from the
literature, from computer files, or from other reference collections.
(3) Converted to the methyl ester or ether (usually by dlazomethane or dimethylsulfate) before analysis.
-------
(4) Brief discussions of how toxicity data are obtained for water pollutants are found in
references (8) and (11). LD50 (lethal dose, 50%) is the amount of compound administered
by direct feeding or injection that kills 50 percent of the test animals. The term TLm
(tolerance limit, median) designates the exposure concentration required to kill 50 percent
of the test organisms within a specified time period, e.g. 96 hrs. MID (minimum lethal dose)
is the minimum concentration required to kill one or more of the test species.
(5) Gleason, M. N., Gassel, R. E., Hodge, H. C., and Smith, R. R., Chemical Toxicology of
Commercial Products, The Williams & Wilkens Co., Baltimore, Md., 3rd Edition (1969).
(6) Garrison, A. W. and Hill, D. H. , American Dyestuff Reporter, 61^, 21 (1972).
(7) The Merck Index of Chemicals and Drugs, Merck & Co., Inc., Rahway, N.J., 7th Ed (1960).
<8) Water Quality Criteria, Edited by McKee, J. E. and Wolf, H. W., California State Water
Resources Control Board, 2nd Edition (1963).
(9) Survey of_ Compounds Which Have Been Tested for Carcinogenic Activity, Supplement 2_, USDHEW,
Public Health Service, Bethesda, MD. (1969).
CO
-J (10) Rogers, I. H., "Isolation and Chemical Identification of Toxic Components of Kraft Mill Wastes.
presented at the joint annual meeting of the Pacific Coast and Western Technical Sections of
the Canadian Pulp and Paper Association, Jasper, Alberta, May 25-27, 1972,
(11) Water Quality Criteria Data Book, Volume 3 , "Effects of Chemicals on Aquatic Life," by
Battelle's Columbus Laboratories for the Environmental Protection Agency, Project No.
18050 GWV (1971).
(12) Handbook of Analytical Toxicology, The Chemical Rubber Co., Cleveland, Ohio (1969).
-------
APPENDIX TWO
PROCEDURE FOR DIAZOMETHANE METHYLATION
The apparatus is shown in Figure 15.
1. Evaporate the sample extract just to dryness with a
stream of nitrogen in a centrifuge tube, the bottom
tube of a Kuderna-Danish apparatus/ or the sample
storage vial. A small amount of methylene chloride may
be retained, but the presence of chloroform may produce
artifacts. Dissolve the extract in one-half to one
milliliter of distilled-in-glass ethyl ether.
2. Add about 5 ml of distilled-in-glass ether to the
first tube of the apparatus to saturate the nitrogen
carrier gas with ether. Add 0.7 ml of ether, 0.7 ml of
carbitol, 2-(2-ethoxyethoxy)ethanol, 1.0 ml of 37%
aqueous KOH (not over 2 days old), and 0.1-0.2 g of
N-methyl-N-nitros o - p-toluenesulfonamide ("Diazald,"
Aldrich Chemical Co.) to the second tube. The base
immediately begins to release diazomethane from the
sulfonamide.
3. Immediately position the second test tube and adjust
the nitrogen flow to about 10 ml per minute. Caution I
Diazomethane is an extremely toxic and explosive gas.
A good fume hood and safety glasses are mandatory. No
chipped glassware should be used, as rough glass
surfaces catalyze decomposition of diazomethane.
4. Position the third tube (a safety trap to prevent
reagent carry-over) and the sample tube to bubble the
nitrogen and diazomethane gas mixture through the
sample. Continue the reaction until the slight yellow
color of diazomethane persists in the sample solution
(from a few seconds to 30 minutes, depending upon the
sample concentration). In the case of dark colored
extracts in which the diazomethane is not visible, a
reaction time of 30 minutes is recommended.
5. Allow the esterified sample to stand unstoppered in
the hood for 15 to 30 minutes to allow excess diazo-
methane to escape from the ether solution. Discard all
waste from the reaction with care and rinse the apparatus
with acetone. Evaporate the sample to the volume
necessary for gas chromatography.
88
-------
REDUCTION VALVE
METERING
VALVE
NITROGEN TANK
GLASS OR
STAINLESS STEEL-
0.7mm I D
RUBBER STOPPER
0.7cm 00
TUBE I
(I6x 150mm)
RUBBER STOPPER
0.7cm 0 D
O.I cm 0 0
TUBE 2
(13 x 85mm)
GENERATOR
RUBBER STOPPER
0.7cm 0 0
O.I cm 00
TUBE 3
(15 x 85mm)
TRAP
0.1 cm 0 0
TUBE 4
(I5x 85mm) or
KD CONCENTRATOR TUBE
SAMPLE
FIGURE 15. APPARATUS FOR DIAZOMETHANE METHYLATION
89
-------
APPENDIX THREE
PROCEDURE FOR DIMETHYL SULFATE METHYLATION
The apparatus is shown in Figure 16.
1. Bring the original sample (300 ml) to pH 11 and
extract with chloroform to remove neutral and basic
compounds.
2. A 500-ml 3-neck (standard taper 24/40) round
bottom flask, equipped with a fourth neck for a ther-
mometer, is fitted with two pressure-equalizing
addition funnels, the probe of a single-probe pH meter,
and a magnetic stirrer.
3. Nitrogen is introduced into the top of the first
addition funnel and exits from the top of the second
one. Place forty ml of Eastman reagent grade dimethyl
sulfate into the first addition funnel and a 50%
solution of sodium hydroxide (80 ml) into the second.
4. Pour the sample into the flask and flush the system
with nitrogen.
5. After raising the temperature to 85° C, begin
dropwise addition of both the dimethylsulfate and
the sodium hydroxide solution. Maintain temperature
between 80-90° (Caution—exothermic reaction. Have
ice available to add to water bath.) and the pH
between 10.5-11. Since diraethylsulfate is not
readily soluble in water vigorous stirring must be
used. The addition time is about 1 hour.
6. After all the dimethylsulfate is added, maintain
the reaction vessel at 85-90° for an additional 15-20
minutes and then cool to room temperature.
7. Add 5 ml concentrated ammonium hydroxide to destroy
excess dimethylsulfate and re-extract the reaction
mixture with chloroform to remove the methyl esters of
acids and the methyl ethers of phenols.
8. Dry the chloroform extract and evaporate to the
appropriate volume for GC analysis.
90
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Nitrogen Out
50% Sodium Hydroxide
FIGURE 16. APPARATUS FOR DIMETHYL SULFATE METHYLATION
91
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
w
4. Title CURRENT PRACTICE IN GC-MS ANALYSIS OF
ORGANICS IN WATER
;, Authors) Webb, R.G., Garrison, A.W., Keith, L.H.,
and McGuire, J.M.
9. Organization Environmental Protection Agency
National Environmental Research Center—
Corvallis
Southeast Environmental Research Lab.
Athens, Georgia 30601
1? Sponsoring O
3. R
6.
8. Pc.-'ormit- Qrgar at/on
Rt ortffu.
10.
16020 GHP
11, Contract/Z.-dfrt sir).
]13. Type- ' Repe. "
Perron Covered
is. supplementary Notes Environmental Protection Agency Report No.
EPA-R2-73-277 , August 1973.
16. Abstract Experiences during five years of evaluating the application of
gas chromatography-mass spectrometry (GC-MS) to wastewater analysis at the
Southeast Environmental Research Laboratory have resulted in the selection
of recommended practices for such applications. Liquid-liquid extraction
with solvents such as methylene ch^ride and chloroform removed greater
than 50 percent of compounds found in pulp mill and petrochemical waste
at concentrations of 2 yg/& to 20 jag A. The Kuderna-Danish evaporator was
the most effective means of concentration after extraction. Diazomethane
and dimethyl sulfate proved to be the most effective of five methylation
reagents studied. Packed columns were effective for gas chromatography of
simple mixtures and SCOT columns provided better overall performance for
complex mixtures. Computerized data reduction was essential for practical
use of GC-MS for samples containing many compounds. A computerized spec-
tra matching program proved highly effective in identifying compounds
contained in the computer library. The system was shown to be effective
in solving problems related to fishkills caused by pesticides, confirma-
tion of polychlorinated biphenyl residues in water and identification of
compounds discharged by over a dozen industries. Over two hundred
compounds were identified in industrial effluents.
i?a. Descriptors *pollutant Identification, *Water Sampling, *Solvent
Extractions, *Gas Chromatography, *Mass Spectrometry, Industrial
Wastes, Organic Wastes, Data Storage and Retrieval, Water Pollution
Sources, Water Chemistry
ub. identifiers *GC~MS, GC/MS, *Computer-aided Organic Compound Identifica-
tion, derivative Formation, Clean-up, Case Histories, Kuderna-Danish
Evaporator
17c. COWRR Field & Group 05A
18. Availability
! 19. St -taity C-ass,
, (Report)
, 20. Sfcur, t.y Class.
21. £j:of
Pages
22. Price
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, O. C. 2O24O
Abstractor R« G. Webb
| institution S.E. Environmental Research Lab,
\VRS1C IO2 (RE.V JUNK l«7i'.
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