PUBLICATIONS AND ARTICLES RELATING
TO THE
CHEMICAL ANALYSIS OF OIL POLLUTION
EMISSION
WAVELENGTH
FLUORESCENCE
INTENSITY
EXCITATION
WAVELENGTH
U.S. ENVIRONMENTAL PROTECTION AGENCY
INDUSTRIAL ENVIRONMENTAL RESEARCH LABORATORY-Cl
OIL AND HAZARDOUS MATERIALS SPILLS BRANCH
EDISON, NEW JERSEY 08817
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COVER ILLUSTRATION is A THREE- DIMENSIONAL REPRESENTATION OF A TOTAL
FLUORESCENCE SPECTRUM, FOR FURTHER INFORMATION SEE SECTION I:
" DETERMINATION OF PETROLEUM OILS IN SEDIMENTS BY FLUORESCENCE
SPECTROSCOPY AND NMR " ,
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SECTION I
FLUORESCENCE PUBLICATIONS
AQC Newsletter Articles
ANALYSIS FOR CRANKCASE OIL IN WATER BY FLUORESCENCE SPECTROPHOTOMETRY
U. Frank
PASSIVE TAGGING OF OILS BY FLUORESCENCE SPECTROPHOTOMETRY
U. Frank
A METHOD FOR QUANTITATING OIL DIRECTLY IN
WATER BY FLUORESCENCE SPECTROPHOTOMETRY
U. Frank
SOLVENT IMPURITIES AND FLUORESCENCE SPECTROPHOTOMETRY
U. Frank, H. Jeleniewski
PASSIVE TAGGING OILS BY FLUORESCENCE SPECTROPHOTOMETRY
U. Frank
AN IMPROVED SOLVENT FOR FLUORESCENCE ANALYSES OF OILS
U. Frank
RECLAIMING A WASTE SOLVENT
U. Frank
EFFECT OF FLUORESCENCE QUENCHING ON OIL IDENTIFICATION
U. Frank
IDENTIFICATION OF PETROLEUM OILS BY FLUORESCENCE SPECTROSCOPY
U. Frank
SYNCHRONOUS EXCITATION FLUORESCENCE SPECTROSCOPY
U. Frank, L. Pernell
SYNCHRONOUS EXCITATION FLUORESCENCE SPECTROSCOPY
1 U. Frank, M. Gruenfeld
Research Papers
IDENTIFICATION OF PETROLEUM OILS BY FLUORESCENCE SPECTROSCOPY
U. Frank
DETERMINATION OF PETROLEUM OILS IN SEDIMENTS
BY FLUORESCENCE SPECTROSCOPY AND NMR
U. Frank, M. Gruenfeld
A REVIEW OF SOME COMMONLY USED PARAMETERS
FOR THE DETERMINATION OF OIL POLLUTION
M. Gruenfeld, U. Frank
PETROLEUM HYDROCARBONS FROM EFFLUENTS: DETECTION
IN MARINE ENVIRONMENT
J. T. Tanacredi
Bibliography
BIBLIOGRAPHY OF RECENT METHODS FOR THE
FLUORESCENCE ANALYSIS OF PETROLEUM OILS
U. Frank
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NEWSL ETTER
£01 TON
Analytical
U S ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
CINCINNATI , OHIO 43268
PHONf : 513-684- 7301
SELECTED REPRINTS
FLUORESCENCE
These selected reprints describe work that was performed at the
Industrial Environmental Research Laboratory - Ci., Oil and Hazardous
Materials Spills Branch, Edison, New Jersey 08817.
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Analytical Quality Control Newsletter (U.S. EPA), No. 13, April 1972
Analysis for Crankcase Oil in Water by Fluorescence Spectrophotometry
Potential environmental damage from repeated applications of spent
crankcase oil onto unpaved roadways, for dust control, is under investi-
gation at Edison. Our laboratory recently examined a suspected "run-off"
water sample, collected in the vicinity of such a road, for traces of
crankcase oil. After the usual extraction and concentration steps,
analysis was attempted by gas chromatography (GC), using a flame ioni-
zation detector. An unresolved chromatogram was obtained, in which the
presence of crankcase oil could not be established. This difficulty
was attributed to excessive interfering contaminants in the sample ex-
tract, that masked any GC profile resulting from traces of crankcase
oil. This analysis was successfully performed, however, by using a
modified version of the fluorescence spectrophotometric method previously
reported by Thruston and Knight (Env. Science and Tech., 5_, 64-69, 1971)
We excited the sample extract at 290 my instead of the recommended
340 my, because we had previously established that this change considerably
improved differentiation between the spectrum of a known crankcase oil
and the spectra of ten other common oils that were examined (crudes and
crude oil fractions). The entire sample extract was dissolved in the
minimum cyclohexane volume needed to fill a 1 cm rectangular cell. A
Perkin Elmer MPF-3 fluorescence spectrophotometer was used for this
analysis. The water sample was found to contain traces of crankcase oil.
The pollutant was identified through the shape of its emission spectrum,
and the wavelength of its maximum emission (329 my). It matched in these
spectral properties a known crankcase oil, but differed considerably from
all the other oils examined. Fluorescence spectrophotometry therefore
shows unique promise as a method for characterizing oils in situations
that are not amenable to analysis by gas chromatography. fU. Frank)
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Analytical Quality Control Newsletter (U.S. EPA), No. 15, October 1972
PASSIVE TAGGING OF OILS BY FLUORESCENCE SPECTROPHOTOMETRY
Fluorescence spectrophotometry is currently under evaluation as
a method for passive tagging of oils. The potential usefulness
of this technique was previously reported by Thruston and
Knight (Env. Science and Tech., 5_, 64-69, 1971), but diffi-
culties were encountered while applying some of their recommenda-
tions. Misleading emission spectra were obtained from dilute
solutions of crude and processed oils in cyclohexane (0.5
mg/liter) when these were excited at the recommended wavelength
of 340 my. All the resulting spectra were essentially identical,
consisting of a pronounced maximum at 380 my surrounded by a
variable background envelope. These spectra were more fully
resolved by examining several solutions containing different
oil concentrations, at various excitation wavelengths in the
range 290 my - 400 my. Two distinct emission spectra re-
sulted; the maximum that was previously noted at 380 my, was
due to the cyclohexane solvent Raman C-H stretch band, while
the background envelope was due to oil. These conclusions
were confirmed by separately exciting at 340 my pure cyclo-
hexane and the oils as tnin solvent-free films. A decrease
in dissolved oil concentration increased interference by the
Raman band. Several other solvents were also excited at
340 my and yielded interferring Raman bands: hexane, pentane
and isooctane were tested. In this study 290 my was selected
as the excitation wavelength of choice. It yielded improved
spectral separation, with the emission maxima of the oils
occurring between 330 my - 360 my and the Raman band of cyclo-
hexane occurring at 320 my. (U. Frank, 201-548-3347)
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Analytical Quality Control Newsletter, (U.S. EPA), No. 18, July 1973
A METHOD FOR QUANTITATING OIL DIRECTLY IN WATER
BY FLUORESCENCE SPECTROPHOTOMETRY
A preliminary evaluation of fluorescence spectrophotometry for the
quantitation of oil directly in water has been completed. Since
in this approach the oil is not extracted from water, two big advan-
tages over other methods are realized: shorter analysis time and
small sample volume requirements. As little as 3 ml suffices for
analysis; the usual methods require 1000 ml sample volumes. In
the present study, standard solutions of oils were prepared for
fluorescence measurement by vigorously mixing 2 yl - 60 yl portions
of the oils with 100 ml portions of distilled water. Ten ml
aliquots of the emulsions were then mixed with 5.0 ml of 2-
Propanol. This solvent acts in an intermediary capacity to solu-
bilize oil in water. A weathered West Texas Sour Crude Oil, an
unweathered No. 4 fuel oil, and an unweathered South Louisiana
Crude Oil were used to prepare the standard solutions. The fluo-
rescence intensities of the solutions were measured at 340 my in
10 mm path length cells, by exciting at 290 my. The resulting in-
tensity vs. concentration plots were linear, and passed through
the origin. This confirms the usefulness of the method for single
point analysis. The influence of oil weathering on the method was
evaluated by exposing portions of the South Louisiana Crude Oil and
No. 4 fuel oil, as thin films, to environmental conditions for 30
days. The fluorescence intensities of these oils in the water-
alcohol solvent system remained unchanged. Quantitative results
obtained with this method were also compared to those obtained with
the IR extraction method described in the AQCL Newsletter #15,
October 1972. Both methods were found to yield identical re-
sults. (U. Frank, FTS 201-548-3510, Coml. 201-548-3347)
SOLVENT IMPURITIES AND FLUORESCENCE SPECTROFHOTOMETRY
Solvents that are used for the analysis of oils by fluorescence
spectrophotometry often contain impurities which give rise to
interfering emission spectra. "Spectroanalyzed" and "99 Mol%
Pure" cyclohexane (Fisher Scientific Co.) were tested at room
temperature by exciting from 200 - 400 nm in 10 mm path length
cells. The "Spectroanalyzed" cyclohexane was found to contain
impurities which cause a significant contribution to typical oil
emission profiles. "99 Mol% Pure" cyclohexane displayed only the
characteristic Raman band (described in the AQCL Newsletter #15),
and is therefore the solvent of choice. Similarly, methylcyclo-
hexane obtained from two different sources, Fisher Scientific Co.,
and Eastman Kodak Co., was examined. This solvent is commonly
used for fluorescence studies of oils at cryogenic temperatures
(77°K), where it forms a transparent glass. Both the Fisher and
Eastman Kodak methylcyclohexane yielded emission spectra of im-
purities when excited at this temperature. The Eastman Kodak
product was found to be a more suitable solvent for excitations
at low wavelengths, while the impurities in the Fisher product
contributed less to emission spectra when excited at wavelengths
abouve 290 nm. (U. Frank/H. Jeleniewski, FTS 201-548-3510, Coml. 201-548-3347)
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Analytical Quality Control Newsletter (U.S. EPA), No. 20, January 1974
PASSIVE TAGGING OILS_BY_FLUORF.SCENCE SPECTROMETRY
A question has been raised by several investigators about the
stability of fluorescing components in oils exposed to environ-
mental conditions, especially sunlight. A preliminary evaluation
of the reliability of fluorescence spectra for passive tagging
oils was carried out by performing a limited weathering study.
Six oils (No. 2, No. 4 and No. 6 Fuel Oils; and South Louisiana,
W. Texas Sour, and Bachaquero Crude oils) were placed as surface
slicks on ocean water, contained in wide mouth jars, and exposed
on a building roof for 30 days. Similarly, four other oils (one
light Arabian ar^ four Persian Gulf Crude oils) were subjected
to outdoor weathering for 50 to 300 hours in 500 gallon tanks
provided with constantly circulating salt water drawn from Casco
Bay, Maine. After the indicated weathering, the oils were re-
covered from the water surface and solutions were prepared
having concentrations of 1 and 10 mg/1 oil in solvent, using
99 mol % rjure cyclohexane. Solutions of the corresponding un-
weathered oils were prepared in a similar manner. Solvent
selection was in accordance with AQC Newsletter #18, "Solvent
Impurities and Fluorescence Spectrometry". Analyses were per-
formed by exciting the solutions at 290, 320, 340 and 360 nm
and scanning each emission spectrum in the range of 220 to 600
rim. The resulting spectral envelopes, which are used for passive
tagging oils, were found to retain their characteristic shapes
despite extensive weathering, although their intensities were
slightly reduced.
(U. Frank; FTS 202-548-3510, Coml. 202-548-S347)
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Analytical Quality Control Newsletter (U.S. EPA), No. 21, April 1974
AN IMPROVED SOLVENT FOR FLUORESCENCE ANALYSES OFViOILS
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Analytical Quality Control Newsletter (U.S. EPA), No.22, July 1974
Effect of Fluorescence Quenching on DM I dent If I .cat Ipn
Questions have been raised by several Investigators about the
effects of quenching on fluorescence spectra when used for
passively tagging oils. Quenching as defined for the
fluorescence analysis of oil, occurs at high concentrations and
Is characterized by supresslon of the oil's emission radiation.
The exact concentrations at which quenching becomes apparent was
determined for four oils. Solutions having known concentrations
of the oils In 99 Mol % pure cyclohexane were prepared. Emission
spectra were obtained In the range 220-600 nm, by exciting at 290
and 31*0 nm. Graphs were then constructed by plotting
fluorescence Intensities versus concentrations. The points at
which the plots began to deviate from linearity marked the "onset
of quenching." Those points corresponded to the following
concentrations:
01 L CONCENTRATION OF "ONSET OF QUENCHING"
mg/1
Ex 290 nm E.x 340 nm
#2 Fuel Oil 30 No Emission
#6 Fuel Oil 6 7
Bachaquero Crude 6 18
Iran - Gach Crude 16 25
By Inspection of the spectral envelopes It was found that above
the onset of quenching the shapes of the envelopes varied with
concentratIon/ while below the onset (region of llnarlty) the
shapes remained constant. !n addition, at concentrations above
100 mg/1 the envelopes were sometimes distorted beyond
recognition. It Is therefore vital to prepare oil solutions with
concentrations below the onset of quenching when passively
tagging oils by the fluorescence technique. (U, Frank. FTS 201-
5U8-3510, Coml . 201-5U-33U7)
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Analytical Quality Control Newsletter (U.S. EPA), No.24, January 1975
IDENTIFICATION OF PETROLEUM OILS BY FLUORESCENCE SPECTROSCDPY
A simple and rapid method for fingerprinting petroleum oils by
taking advantage of the three dimensional character of their
fluorescence spectra has been developed. Our approach
involves excitation of the oils at 15 wavelengths, in the
range of 220-500 nanometers (nm), at 20 nm intervals. The
emission monochromator is rapidly scanned at each excitation
wavelength to obtain the emission maximum. These maxima are
then plotted versus the excitation wavelengths to derive
"silhouette profiles", which are used to fingerprint the oils.
We tested our method by matching weathered with unweathered
portions of nine petroleum oils, and discriminating among
them. Each oil yielded a unique and different profile that
remained substantially unchanged despite weathering. (U
Frank, FTS 201-5U8-3510, Coml. 201-51*8-33^7)
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Analytical Quality Control Newsletter (U.S. EPA), No. 31, October 1976
Synchronous Excitation Fluorescence Spectroscopy
A preliminary evaluation of the utility of synchronous excitation
fluorescence spectroscopy for the quantitative determination of
petroleuai oil and phenol has demonstrated that by the use of
this technique Rayleigh-Tyndall and Raman scatter interferences
are totally eliminated. These scatter interferences, which
are usually encountered in emission spectra obtained by con-
ventional constant wavelength excitation, techniques, were pre-
viously described in AQC Newsletter No. 13. Since the scatter
interfer^nces are avoided by synchronous excitation, this tech-
nique offers two advantages over constant wavelength excitation:
(1) shorter analysis time because solvent blank determinations
and corrections are not necessary, and (2) increased sensitivity
because spectral interferences are avoided. In our study we
compared synchronous excitation spectra to emission spectra of
oil and phenol solutions, and of appropriate solvent blanks.
Synchronous excitation spectra were obtained by simultaneously
scanning the excitation and emission monochromators with the
emission monochromator leading by a 20 nm interval. Emission
spectra were obtained at 290 nm for oil and 270 nm for phenol.
(U. Frank, FTS 342-7510, Coml 201-548-3347/L. Perneli, FTS
342-7517 Coml 201-548-3347)
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Analytical Quality Control Newsletter (U.S. EPA), No.32, January 1977
Synchronous Excitation Fluoregcence Spectroscopy
Use o t" synchronous excitation fluorescence spectroscopy for the
quantification of petroleum oils and phenol was previously de-
scribed in AQC Newsletter No. 31, October 1976. Use of this tech-
nique has now been extended to aniline, benze.ne, benzolic acid,
cresol, ethyl benzene, naphthalene, phenol, toluene, and xylene.
These materials are part of more than 300 toxicants that may be
designated "hazardous" by anticipated legislation (40 CFR Part 116)
Detection limits obtained by the synchronous excitation tech-
nique varied between 0.5 racg/1 (ppb) - 0.1 mg/1 (ppm), using
100 ml cyclohexane for extracting 1 liter water. A preliminary
evaluation of interference by commonly present materials in
brackish lake and marsh waters, was also performed. No inter-
ference was found. (U. frank, FTS 34G-6626, Coml. 201-321-6626/
M. Gruenfeld, FTS 340-6625, Coml. 201-321-6G25)
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Reprinted from: Proceedings 1975 Conference on Prevention and Control
of Oil Pollution, March 25 - 27, 1975, San Francisco, API, Wash., DC
IDENTIFICATION OF PETROLEUM OILS
BY FLUORESCENCE SPECTROSCOPY
Uwe Frank
Industrial Waste Treatment Research Laboratory
U.S. Environmental Protection Agency
Edison, New Jersey
ABSTRACT
A simple and rapid method for the identification of weathered
petroleum oils (passive tagging) by fluorescence spectroscopy is
described. The approach used takes advantage of the three-
dimensional character of the oil fluorescence spectra. Oil identifica-
tion methods of other investigators that use fluorescence spectro-
scopy are also reviewed within the context of the three-dimensional
system. Our method involves excitation of the oils at 15 wave-
lengths, between 220-500 nanometers (nm), at 20-nm intervals. The
emission monochromator is rapidly scanned at each excitation
wavelength to obtain an emission spectrum. The maximum emission
intensities are then plotted versus the^ excitation wavelengths to
derive silhouette profiles. These are used as fingerprints for passive
tagging petroleum oils. The influence of weathering, quenching, and
solvent effects on our method are also examined.
INTRODUCTION
The discharge of petroleum products into the marine environ-
ment has caused extensive environmental damage in the past. The
current increasing transportation and offshore production of petro-
leum is, therefore, of vital concern to the U.S. Environmental
Protection Agency. Effective legislation, with adequate analytical
support for enforcement, should reduce this damage, and method-
ology for the identification of the source of discharged oil is needed.
Fluorescence spectroscopy is a rapid and promising tool for the
source identification of weathered oil (passive tagging). While in the
past few years many laboratories have used this tool for passive
tagging both crude and refined petroleum products, few innovations
in the technique have come about. Although three parameters are
inherent to the fluorescence technique, only two of them have been
commonly used. This can be explained more clearly within the
context of a three-dimensional system. Because fluorescence spec-
troscopy entails three parameters (excitation wavelength, emission
wavelength, and fluorescence intensity), the total spectrum of an oil
can be presented as a topographical map of a mountainous region.
Figure 1 depicts the total fluorescence spectrum of an oil with three
fluorescence maxima; the excitation wavelength, emission wave-
length, and fluorescence intensity represent the x, y, and z axes,
respectively.
Most publications on the identification of oils by fluorescence
spectroscopy do not mention the three-dimensional character of oil
fluorescence spectra. Their authors limit themselves by using only
two of the three parameters. Within the context of the three-
dimensional system, the approach of these investigators can be
described as taking "cuts" through figure 1 in planes that are
parallel to the y axis and at specific points along the x axis. Figure 2
illustrates this; each cut is an emission spectrum at one excitation
wavelength.
Figure 1. Three-dimensional presentation of the total fluorescence
spectrum of an oil
Thus, Thruston and Knight [1] utilize 340 nanometers (nm) as
the excitation wavelength for oil solutions in cyclohexane at three
concentration levels. They then ratio the 386-nm and 440-nm
emission maxima to one another. A similar approach is taken by
Coakley [2] who excites each oil at a discrete wavelength. He
selects for each oil the excitation wavelength that yields the
maximum emission for that oil. His wavelengths are usually in the
range 290-320 nm. Jadamec [3], in a more recent method, uses 254
nm as the excitation wavelength. He generates emission spectra for
passive tagging 8 oil spill samples.
All of these methods utilize the same basic approach. The oils
are excited at specific wavelengths and their emission spectra are
obtained. The methods differ only in their wavelength of excitation.
Other fluorescence methods are available or have been proposed
for passive tagging oils, but their utility for this purpose has not
been established. Lloyd [4,5] describes a method whereby cross-
sectional cuts are taken at 45° angular planes to the x and y axes.
This is accomplished by simultaneously scanning the excitation and
emission monochromators of the fluorescence instrument. Another
method by Freegarde et al. [6] proposes the analysis of oils by
constructing contour maps. While this approach somewhat simulates
a three-dimensional system, it is extremely time consuming and
requires computer services for data manipulation.
87
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88
CONFERENCE ON PREVENTION AND CONTROL OF OIL POLLUTION
EMISSION
Z INTENSITY
EXCITATION
WAVELENGTHS
EMISSION
WAVELENGTHS
Figure 2. Three-dimensional presentation of the emission spectra of
the oil in figure 1 excited at three wavelengths
Our method circumvents these limitations; sample analysis and
data handling are rapid and commensurate with the other com-
monly used fluorescence techniques [1,2,3,]. We use all three
fluorescence parameters, and thereby exploit the advantages that
derive from the three-dimensional system that is illustrated in figure
1. We obtain in our method a silhouette profile of an oil's total
three-dimensional spectrum as it is observed in a plane that parallels
the x axis. Figure 3 illustrates how a simplified silhouette profile of
an oil is obtained from the three-dimensional spectrum that is
illustrated in figure 1. In actual practice, we excite each oil at 20-nm
wavelength intervals between 220 nm and 500 nm. The emission
monochromator is rapidly scanned at each excitation wavelength,
and the maximum emission intensity of each scan is recorded. These
maximum intensities are then plotted manually versus the excitation
wavelengths, and the silhouette profiles of the oils are obtained by
connecting the points with straight lines.
We evaluated the ability of the method to identify weathered
oils, i.e., to correlate weathered oils with unweathered portions of
the same oils (passive tagging). Pertinent environmental factors such
as photodecomposition, evaporation, dissolution, and biodegrada-
tion were considered separately and in combination. Quenching and
solvent effects that may influence the accuracy of our method were
also examined.
Weathering effects
Previous investigators indicate, that weathering may drastically
degrade petroleum oils. Thiuston and Knight [1], Coakley [2], and
Freegarde et al. [6] surmised that significant changes in the
intensity and shape of the emission spectrum can occur when oils
aie exposed to sunlight. Two studies were therefore conducted in
order to establish our method's ability to cope with weathering
effects. The first study dealt primarily with photodecomposition
effects, and the second study examined the combined effects of
water, radiation, heat, and bacteria.
Quenching effects
Thniston and Knight [1] found that the intensity and shape of
oil fluorescence spectra substantially depend on the concentrations
of the solutions that are measured. Fluorescence quenching is a
phenomenon that occurs at high solution concentrations and is
^— SILHOUETTE PtOMLE
EMISSION SPECTRA
EXCITATION
WAVELENGTHS
EMISSION
WAVELENGTHS
Figure 3. Three-dimensional presentation of the derivation of the
silhouette profile from the oil :n figure 1
characterized by the formation of excimers. These are combinations
of excited molecules. Excimer formation and their effect on
fluorescence spectra was first noted by Forster and Kasper [7], who
demonstrated that while a polynuclear aromatic compound (PNA)
yielded a fluorescence emission maximum at 390 nm, a hundredfold
increase in the concentration of this compound shifted its fluores-
cence maximum to 480 nm. Other PNAs, exhibit similar behavior.
Because other investigators [8,9] have demonstrated that petroleum
oil fluorescence is primarily attributable to PNAs, we maintain that
the distortion of high concentration oil fluorescence spectra is due
to excimer formation.
Excimer formation is considered important to our method
because its effects can combine with or can be confused with the
effects of weathering. Also, losses of volatiles by spilled oils
invariably result in increased concentrations of PNAs in the
weathered residues; the PNAs are higher boiling materials that do
not readily volatilize. As a consequence, excimer formation can
become quite prominent and can cause ambiguities during passive
tagging analyses. Therefore, the oil solution concentrations at which
substantial quenching appears must be known before our method
can be successfully used.
Solvent effects
Cyclohexane, our solvent of choice, presents two problems: (1)
interference by Raman Scatter, and (2) interfering emissions by sol-
vent impurities. Raman scatter is caused by the interaction of ex-
citation radiation with the bonds of the cyclohexane molecules. It
reveals itself as an emission band that appears 0.3 microns""1 from
the Rayleigh band in cyclohexane [10]. Also, impurities in cyclo-
hexane yield spectra that can coincide with those of oils. The effects
of these interferences on our method are also examined in this
paper.
Experimental
Apparatus. A Perkin-Elmer model MPF-3 Fluorescence Spectre-
photometer,1 with a constant temperature cell bath (P.E. Part No.
220-1419) was used. The cell (10 mm pathlength, quartz; P.E. Part
No. 990-2711) was maintained at 20° ± 0.5°C.
'Mention of trade names or commercial products does not
constitute endorsement by the U.S. Government.
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MONITORING
89
Procedure. The silhouette profile of a typical oil was obtained in
the following manner:
A stock solution with an accurately known concentration
approximating 1000 mg/1 oil in cyclohexane was prepared. An
aliquot of this solution was then diluted to obtain a final
concentration of 5 mg/1. This was measured in a cell which was
placed into the constant temperature cell holder for a five-minute
equilibration period. The analysis was performed at a constant
temperature of 20° C.
In order to prevent the recorder pen from going off scale during
a run, the excitation wavelength that yielded the maximum emission
resonse was determined. This was performed by manually scanning
the excitation and emission monochromators until a maximum pen
deflection was obtained. Next, the instrument settings were adjusted
to yield ca. 90% recorder scale deflection at this excitation
wavelength.
The oil was then excited at 15 wavelengths, 20 nm apart, in the
range 220 to 500 nm (i.e. 220, 240, 260, •••, 480, 500 nm), to
obtain the maximum emission at each wavelength. This was
accomplished by setting the excitation monochromator at each
excitation wavelength and rapidly scanning the emission mon-
ochiomator over the entire fluorescence spectral region of the oil.
Fifteen compressed emission spectra were thereby obtained. A
cyclohexane "blank" was similarly analyzed by exciting at the 15
wavelengths to determine its contributions to the oil spectra. None
were found at the instrument settings used.
The silhouette profiles were prepared by manually plotting the
maximum emission intensity of each spectrum versus its excitation
wavelength and then connecting these points with straight lines.
Two criteria were used to compare one profile with another: (1)
shape, and (2) the excitation wavelength of the maximum emission
intensity.
Weathering effects. Photodecomposition effects were examined
by placing four oils (No. 2 and No. 6 fuel oil; South Louisiana and
Bachaquero crude oil) as surface slicks in wide-mouth, quart-size
jars, containing ocean water. The jars were exposed to the elements
on a building roof during the entire month of August.
The other environmental effects were examined by subjecting
five other crude oils to weathering conditions which simulate typical
oil spill incidents. The weathering was performed by the Depart-
ment of Environmental Protection, State of Maine, at the facilities
of the TRIGOM laboratory, South Portland, Maine, as part of a U.S.
Environmental Protection Agency (EPA) sponsored grant. The oils
were weathered by spilling one pint of oil on salt water in
500-gallon fiberglass tanks. Seawater drawn from Casco Bay, Maine,
was constantly circulated through the tanks whose geometry was
such that the outlets were located at the bottom and the inlets at
the top. All weathering was performed under ambient outdoor
conditions, during the summer months, and for periods of 7-14
days. The weathered oils were skimmed from the water surface,
dissolved in cyclohexane, and centrifuged in order to separate the
oil from paiticulate matter and residual water. The solutions were
then decanted, and the solvent stripped off with the aid of an air
stream at room temperature. Silhouette profiles of the weathered
and unweathered oils were then prepared and compared.
Quenching effects The concentration at which quenching phe-
nomena became apparent was determined for four representative
crude and refined oils. A No. 2 fuel oil (a low-viscosity distillate), a
No. 6 fuel oil (a high-viscosity residual), a South Louisiana crude oil
(a low-viscosity crude), and a Bachaquero crude oil (a medium-
viscosity crude) were examined. Solutions having accurately-known
concentrations of these oils in cyclohexane were prepared. Emission
spectra in the range 220-600 nm were obtained by exciting at 290
nm and 340 nm, and fluorescence intensity versus concentration
plots were prepared. The maximum concentration within the linear
region of each plot was designated at the onset of quenching (figure
4).
Solvent effects. The emission spectra of ten oils were examined
in order to demonstrate the effects of solvent Raman scatter on the
fluorescence spectra of the oils. Solutions having concentrations of
1 mg/1 and 10 mg/1 oil in cyclohexane were excited at 290 and 340
nm. A cyclohexane blank measurement was obtained concurrently
z
o
70
60
SO
40
30
20
10
I
\_
ONSET OF
QUENCHING
•
10 20 30 40 SO 60 70
CONCENTRATION (ng/1)
Figure 4. Emission intensity versus concentration plot of No. 2 fuel
oil in cyclohexane demonstrating the onset of quenching. The
maximum concentration within the linear region is 30 mg/liter
with each oil measurement using the same instrument settings. The
emission spectra of two solvents (Fisher Scientific Co., Spectro-
analyzed and 99 Mol % Pure cyclohexanes) were also checked for
the presence of fluorescing impurities.
Results and discussion
Weathering effects. The criteria that are used to judge the
efficacy of a method for passive tagging oils are: (1) whether the
method can correctly correlate weathered and unweathered portions
of the same oils, and (2) whether it can distinguish between oils.
Our first weathering study, i.e., the study of photodecomposition
effects, demonstrates that the profiles of four oils remain relatively
unchanged despite weathering (figure 5) and that the method
distinguishes between the oils (figure 6). Four discrete silhouette
shapes and excitation wavelength maxima were obtained, yet the
profiles of the weathered and unweathered portions of each oil were
quite similar. We therefore conclude that our method not only
discriminates between oils but also that exposure of the oils to
sunlight did not adversely affect our method.
The results of our second weathering study, i.e., the study of
other environmental effects, also support the contention that our
method is adequate for passive tagging oils. The silhouette profiles
of the weathered and unweathered portions of five crude oils are
shown in figure 7. Each weathered oil is again uniquely matched
with its unweathered counterpart.
Quenching effects. The optimum solution concentration for
fluorescence measurement was found to be 5 mg/1 oil in cyclo-
hexane. Table 1 illustrates our reasons for selecting this concentra-
tion by listing the concentrations of the oils at which the onset of
quenching occurs (figure 4). Oil solutions that exceeded this
-------
90 CONFERENCE ON PREVENTION AND CONTROL OF OIL POLLUTION
100
90
80
f 7°
l/l
| 60
2 50
C/)
to
S 40
30
20
10
NUMBER 2 FUEL
UNWEATHERED OIL
WEATHERED OIL
NUMBER 6 FUEL
S. LOUISIANA CRUDE
BACHAQUERO CRUDE
_lJ I
240 320 400 480 240 320 400 480 240 320 400 480 240 320 400 480
EXCITATION WAVELENGTH (rim)
Figure 5. Silhouette profiles of weathered and unweathered portions of four crude and processed oils
NUMBER 2 FUEL OIL
S.LOUISIANA CRUDE
NUMBER 6 FUEL OIL
BACHAQUERO CRUDE
240 260 320 360 400 440 480 520
EXCITATION WAVELENGTH (nm)
Figure 6. Overlay of the silhouette profiles of the unweathered oils
in figure 5
concentration yielded fluorescence spectral envelopes that changed
with solution concentration. The spectral envelopes of less concen-
trated solutions remained constant despite concentration changes.
We, therefore, suggest that it is vital to use solution concentrations
that are within the linear portion of figure 4 to avoid spectral
distortion by quenching effects.
Solvent effects. Raman scatter can be quite prominent in
cyclohexane at concentrations below 10 mg/1 oil in solvent. Its
impact depends on the excitation wavelength that is used and on
the fluorescence efficiency (emission intensity) of the oil. Typical
Raman band interference with a spectrum of oil is illustrated in
figure 8. Two excitation wavelengths are used, and they produce
different effects. This figure demonstrates the importance of
measuring neat solvent prior to oil analysis. Fortunately, most
petroleum oils have such a high fluorescence efficiency that this
solvent Raman band overlap does not handicap our ability to
achieve oil identification.
The presence of fluorescing impurities in the solvent is a factor
that must also be taken into account. Two grades of cyclohexane
were examined: Spectroanalyzed and 99 Mol % Pure cyclohexane
(Fisher Scientific Company). Impurities in the Spectroanalyzed
grade solvent yielded substantial interfering fluorescence envelopes,
but the 99 Mol % Pure solvent was essentially free of these
interferences. We successfully purified the Spectroanalyzed cyclo-
hexane, however, by simple distillation [11].
Summary
We have described the three-dimensional character of oil
fluorescence spectra and reviewed within this context the pertinent
work of previous investigators. We have also presented a straight-
forward method for identifying the source of discharged petroleum
oil, i.e., correlating a weathered oil correctly with an unweathered
portion of the same oil (passive tagging). Our method utilizes the
three-dimensional aspect of fluorescence spectra, and requires only
simple data manipulation. In order to validate our method for
passive tagging oils, we tested its ability to match weathered with
unweathered portions of nine petroleum oils and to discriminate
among them. Each oil yielded a unique and different profile in these
tests, and the profiles remained substantially unchanged despite
weathering. We also demonstrate that some common phenomena
such as fluorescence quenching, Raman scatter, and solvent impuri-
ties do not handicap our method.
-------
MONITORING
91
UWEATHERED OIL
WEATHERED OIL
USD TRECO CRUDE
LIGHT ARABIAN CRUDE
HEAVY I HAH I AH CRUDE
240 32° 4°0 480 240 320 400 480 Z40 320 400
240 320 «00 480 240 320 400
EXCITATION WAVELENGTH nm
Figure 7. Silhouette profiles of weathered and unweathered protions of five crude oils
Table 1. Concentrations of oil in cyclohexane
at which the onset of quenching appears for 4 crude and
refined oils
OIL
Number 2 Fuel Oil
Number 6 Fuel Oil
Bachaquero Crude
Iran Gach Crude
CONCENTRATION (mg/1)
Ex 290 nm Ex 340 nm
30
6
6
16
No Emission
7
18
25
ACKNOWLEDGMENTS
The author is grateful to Mi. Michael Gruenfeld, Supervisory
Chemist of this laboratory, for many helpful discussions and for his
assistance in preparing this manuscript, and to Mr. Henry Jeleniew-
ski for the data acquisition that has made these findings possible.
REFERENCES
1. Thruston, A.D., and Knight, R.W. 1971. Environmental science
& technology, 5:64.
2. Coakley, W.A. 1973. Proceedings of Joint Conference on the
Prevention and Control of Oil Spills, p. 215. Washington,
D.C.: American Petroleum Institute.
3. Jadamec, J.R. 1974. Abstracts of Pittsburgh Conference on
Analytical Chemistry and Applied Spectroscopy. Cleveland,
Ohio.
4. Lloyd, J.B.F. 1971. / Forens. Sci. Soc. 11:83.
5. Ibid., p. 153.
6. Freegarde, M.; Hatchard, C.G.; and Parker, C.A. 1971. Lab.
Practice, 20(1):35.
7. Forster, T., and Kasper, K. 1955. Z. Elektrochem. 59:976.
8. McKay, J.F., and Latham, D.R. 1972. Analytical Chemistry
13:2132.
EXCITATION AT 340nm
EXCITATION AT 290nm
OIL
340
380
290 320
EMISSION WAVELENGTH
350
Figure 8. Raman band interference with the emission spectra of a
La Rosa crude oil. The concentration 1 mg/l oil in 99 Mol % Pure
cyclohexane is used
9. McKay, J.F., and Latham, D.R. 1973. Analytical Chemistry
7:1050.
10. Parker, C.A. 1959. Analyst 83:446.
11. Frank, U. 1974. EPA Analytical Quality Control Newsletter
21:11.
-------
NO. 400
Presented at the
1977 Pittsburgh Conference On
Analytical Chemistry And
Applied Spectroscopy
February 28 - March 4, 1977
Cleveland, Ohio
DETERMINATION OP PETROLEUM OILS IN SEDIMENTS
BY FLUORESCENCE SPECTROSCOPY AND NMR
U. FRANK AND M. CRUENFELD, Oil 8 Hazardous Material! Spills Br«nch, Ind. Env.
Res. I.ab.-Ci, U.S. Environmental t'otection Agency, Edison, N. J. 08817
The use of several fluorescence methods and one NMR procedure were evaluated for monitor-
ing the presence of petroleum oils in sediments. Analyses were performed on sediments
from a mangrove swamp in Puerto Rico that was impacted by a major oil spill in 1973, and
on sediments from a proximate area that had no known history of petroleum oil pollution.
The latter sediments served as control samples.
The following fluorescence techniques, that are believed to be most cCHiunonly used for
oil spill source identification, were examined: (a) Single Wavelength Excitation^•^>^»^;
(b) Synchronous Excitation^; and (c) Derived Silhouette Profiles". These techniques
are compared and contrasted within the context of a three dimensional system, using the
three interdependent variables that are inherent to fluorescence Spectroscopy, i.e., ex-
citation wavelength (x), emission wavelength (y) , and fluorescence intensity (z). With-
in this context, the fluorescence characteristics of petroleum oils are presented as
"total fluorescence spectra" and the spectral information that is obtained by each tech-
nique is discussed as an appropriate portion of such spectra. Figure 1 shows a hypo-
thetical three dimensional total oil spectrum (illustrated as mountains by solid lines),
and the spectral information that Is available. This figure also highlights the rather
limited information used by techniques (a) and (b). Technique (a) measures only the
slice of the total spectrum that parallels the y axis; technique (b) measures only the
slice of the total spectrum that lies at a 45 angle to the x and y axes. Technique (c)
differs, however. This method measures the total spectrum's silhouette profile as pro-
jected onto the plane bordered by the x and z axes. Unlike methods (a) and (b), silhou-
ette profiles of method (c) incorporate the essence of three dimensional fluorescence
spectra, within a two dimensional format. A comprehensive discussion of method (c) is
available in Reference 6. In addition to Its greater informational content, this method
is rapid and easily used. It was therefore selected for the mangrove sediment analyses.
Use of NMR spectroscopy for confirming the presence of petroleums in the mangrove sedi-
ments was also evaluated. The NMR procedure is based on the absorption of aromatic
compounds in the 6.5-8.0 ppm spectral region, relative to tetramethylsilane at 0 ppm;
substantially elevated levels of aromatics in sediments are thought to indicate the
presence of petroleums. These absorptions were observed in the spill impacted sediments
but not in the'control sediments. Figure 2 illustrates a composite section of the NMR
spectra of the contaminated sediment (upper), and the control sediment (lower). The
fluorescence and the NMR data agree on the presence of petroleums in the spill impacted
sediments.
FIGURE I
Method (a)1'2'3'4
Method (b)S
. Method (c)6
FIGURE 2
10
References:
1.
8
P-P-
Hatchard, and
Practice, 20 (1)
4. J.R.
5. P J
6. II. F
CA. ,
Jndamec
ohn, and
rank, Pr
A.P.I. ,
M. Freegard, C.G.
C.A. Parker, Lab.
35-40 (1971)
A.D. Thruston and R.W. Knight, Env.
Sci . and Tech., 5_, 64 (1971)
W.A. Coakley, Proc. of Joint Conf.
on Prevent. 6 Contr. of Oil Spills,
Nash., D.C., A.P.I., 215 (1973)
and T.J. Porro, Abstract Pittsburgh Conference (1974)
I. Soutar, Anal. Chem. , 4^, 520 (1976)
ic. of Joint Conf. on Prevent. S Contr. of Oil Spills, San Pranclsco,
87 (1975)
-------
Reprinted from: Proceedings 1977 Oil Spill Conference, March 8-10
1977, New Orleans, API, Wash., DC
A REVIEW OF SOME COMMONLY USED PARAMETERS
FOR THE DETERMINATION OF OIL POLLUTION
Michael Gruenfeld and Uwe Frank
Oil and Hazardous Materials Spills Branch
Industrial Environmental Research Laboratory-Cincinnati
U.S. Environmental Protection Agency
Edison, New Jersey 08817
ABSTRACT
A state-of-the-art review is provided describing specific parameters of
petroleum oils that are used b\ various investigators to demonstrate the
presence of oil pollution in water, sediments, and biological tissues. Several
representative publications are discussed with regard to the techniques used
for distinguishing between petroleum h\drocarbons and organics that are of
recent biological origin. The techniques include chromatographic proce-
dures using alumina and silica gel for separating hydrocarbons from other
organics, followed by instrumental methods such as gas chromatography,
fluorescence spectroscopy. ultraviolet absorption specfroscopy, et al. The
various oil parameters thai are used to demonstrate the presence of petro-
leum oils are discussed, and the most effective ones are recommended. In
addition, a recent studv is also described in which several of the parameters
were used to demonstrate the presence of oil pollution in sediments from a
mangrove swamp In Puerto Rico.
INTRODUCTION
Petroleum oils that are spilled or otherwise discharged into the aqueous
environment migrate in all directions. Spilled oil floats on water and is
carried onto shorelines. Oil also migrates downward through the water
column and contacts fish, benthic sediments, animals and plants. Several
kinds of chemical analyses are commonly performed to monitor the presence
of oil pollution. "Fingerprinting" methods are used to locate the source of
oil discharge. Methods of quantitation are used to measure dispersed oil
levels in water, and oil levels that are incorporated by benthic sediments and
tissues of animals and plants. While most fingerprinting methods require
gram quantities of oil and are therefore restricted to oil rich environmental
samples such as surface slicks and shoreline residues, the quantitation
methods are used to measure ppm (mg/kg) or smaller amounts of oil. These
methods are emphasized in the following discussion. Descriptions of highly
satisfactory fingerprinting methods are available, however.2
Although hydrocarbons are the major components of petroleum oils, they
are also produced by living marine organisms. Methods for measuring
petroleums in sediments, plants and animals, and methods for measuring
sub-ppm levels of oils in water consequently select characteristic petroleum
oil parameters that differentiate between recent biologically produced hy-
drocarbons and petroleum derived hydrocarbons. The following discussion
reviews some of these parameters and illustrates their use for confirming the
presence of petroleum oil in samples.
Unresolved complex mixture
Petroleum is an extremely complex mixture of thousands of different
hydrocarbons and related compounds. Hydrocarbons, according to Ander-
son, et at.1, are the most numerous and abundant organic compounds
comprising crude oils and refined petroleum products. Comprehensive dis-
cussions of petroleum oil composition are available in the preceding refer-
ence, and in reports by Zafiriou, el al..20 Bieri, et al.,3 and the U.S.
Department of Commerce."
When injected into a gas chromatograph (GC), petroleum oils exhibit an
inverted "cup and saucer effect" (Figure 1). This description of a rather
characteristic feature of GC profiles of petroleum oils was obtained from the
Department of Commerce report. This report also describes this configura-
tion as a "smear of unresolved hydrocarbons", Zafiriou, et al.'l° call it an
"unresolved envelope", while Farrington and Medeiros" describe it as due
to an "unresolved complex mixture (UCM)." The presence of this configu-
ration results from the inability of gas chromatographic methods to separate
all petroleum oil components from one another. According to Blumer and
Sass,J this unresolved envelope is characteristic of the homologous and
isomeric hydrocarbons in fossil fuels. According to Clark and Finley,"'8 the
presence of this large unresolved envelope below discrete n-paraffin peaks
(discussed below) strongly suggests the presence of petroleum oils in en-
vironmental samples.
Environmental samples that contain only recently biologically produced
hydrocarbons exhibit less complex chromatograms. According to Ander-
son, et al.', living organisms use rather specific biosynthetic pathways
which favor the production of hydrocarbons in preferred size ranges. Such
chromatograms exhibit a few exceptionally large peaks and often show some
degree of baseline resolution.
n-alkane homologs
Crude oils and most refined petroleum products contain a homologous
series of n-alkanes. These appear as discrete peaks above the unresolved
envelope of the GC profiles of oils (Figure 2). According to Zafiriou, etal*°
n-alkanes from Ci-Ceo are present in petroleum oils with adjacent members
occurring in similar quantities. Zafiriou contrasts this with the distribution of
n-alkanes in petroleum-free plants, organisms, recent sediments, and shales,
all of which yield GC profiles showing only a limited range of normal
BACHAQUERO CRUDE OIL
-INCREASING TEMPERATURE
BEGIN TEMP.
BASELINE
Figure 1. Gas chromatogram of a Bachaquero crude oil exhibiting the
presence of an unresolved complex mixture
487
-------
488
1977 OIL SPILL CONFERENCE
DEFINITION OF TERMS
PROGRAM START
INCREASING TEMPERATURE
INJECTION
Figure 2. Gas chromatogram of a No. 2 fuel oil exhibiting the presence of n-alkane homologs, and the isoprenoids
pristane and phytane
alkanes (mostly C-is-C-js), and a strong predominance of odd-numbered
over even-numbered compounds. The exceptionally broad distribution of
n-alkanes (Ci-Cso) in crude oils and most refined products is also em-
phasized by Anderson, el at.' These authors find a distinct predominance of
odd carbon number versus even carbon number n-alkanes in petroleum-free
environmental samples (i.e., samples containing only recent biologically
produced hydrocarbons). While odd-to-even n-alkane carbon number ratios
in petroleum oils are approximately I.O, GC profiles of environmental
samples containing only recently produced hydrocarbons often show a
strong predominance of one or two n-alkanes over all others. Use of GC
profiles of environmental samples to demonstrate petroleum incorporation is
also suggested by Clark and Fmley.' According to these authors, the first
indication of petroleum uptake is often seen by the presence of a large
unresolved envelope below the n-alkane peaks, and by the presence of
n-alkane peaks in the range Cr2-C22 which have the same order of mag-
nitude. The n-alkanes are quite susceptible to microbial catabolism. and may
not appear in GC profiles of substantially weathered oils. Such chromato-
grams should retain their characteristic inverted cup and saucer appearance.
however.
Isoprenoids
Gas chromatograms of petroleum oils often contain major peaks that
appear in addition to those of n-alkanes. Two isoprenoids. pristane (2. 6. 10,
14-tetrame'hylpentadecane) and phytane (2. 6, 10, 14-tetramethylhexa-
decane) are of particular interest in evaluating the presence of petroleums in
samples. When relatively non-polar stationary phases are used (e g..
Apiezon L. OV-1, O V- IOI, or SE-30), pristane and phytane yield GC peaks
that adjoin and are usually only partially separated from the Ci? and Ci«
u-alkane peaks (Figure 2) This characteristic four peak configuration is
common to many oils and indicates the likely incorporation of petroleums by
marine environmental samples. Pristane is commonly produced by marine
organisms, however, and consequently does not confirm petroleum incorpo-
ration. But. according to Anderson. I'lul.' and Ehrhardt and Hememann."1
phytane has not been found as a natural component of marine organisms. Its
presence in samples therefore suggests oil incorporation.
Fluorescing components
Aromatic hydrocarbons are abundant in most crude and refined petroleum
products, and their presence in marine samples is often thought to indicate
petroleum incorporation. For example. Margrave and Phillips15 use the
presence of triaromatic and greater substituted aromatic compounds as a
useful indication of petroleum pollution. The U.S. Department of Com-
merce19 report emphasizes that aromatic hydrocarbons in marine waters are
currently believed to be from petroleum sources. Similarly, Brown and
Huffman7 cite evidence suggesting that marine organisms do not produce
aromatic hydrocarbon mixtures. Conversely, Blumer and Youngblood6
describe forest fires and prairie fires as a likely source of many polynuclear
aromatic hydrocarbons in marine sediments. Similarly, Anderson, ci a/.'
indicate that although aromatic hydrocarbons have not been isolated from
plankton, they may be produced by some marine organisms. Despite these
possibilities, elevated levels of polynuclear aromatic hydrocarbons in
marine samples are widely thought to indicate the presence of petroleums.
Several publications describe methods for measuring aromatic hydrocar-
bons in marine samples Fluorescence spectroscopy is the most highly
recommended technique. According to Gordon. <•; «/.'' fluorescence
methods are rapid, sensitive, and simple. While these authors believe
fluorescing materials to be aromatics. they describe the uncertainty of actual
compound identity as a major drawback. Gordon and Keizer1'1 describe
several fluorescence methods for measuring aromatics in water, while
Zitko,21 and Hargrave and Phillips1'' provide some useful fluorescence data
describing petroleum incorporation by animals and sediments. Several
fluorescence spectra of oils are illustrated in Figure 3.
FOSTERTON CRUDE
VENEZUELAN BUNKER C U0.33)
.REDWATER CRUDE
KUWAIT CRUDE
GUANIPA CRUDE
PEMBINA AND
LEDUC CRUDE
340 380 420
EMISSSION WAVELENGTH (nm)
Figure 3. Fluorescence spectra of several petroleum oils13
-------
OIL SPILL BEHAVIOR AND EFFECTS
489
Naphthalenes and substituted naphthalenes
The toxicity of higher boiling aromatics such as napthalenes, to fish, is
well established according to Anderson, el al.' In fact, these authors feel
that the toxicity of naphthalenes to fish may even exceed that of lower boiling
aromatics such as benzene, toluene, and xylene. Naphthalenes are more
soluble in water than most other high boiling aromatic hydrocarbons, and
consequently have a greater tendency to migrate from petroleum oils into
marine waters, sediments, and animals. Discussions of these properties and
of the unique uptake and depuration characteristics of naphthalenes, and
their known toxicity to marine organisms are presented by Rossi, el ill.. "
and also in several other papers by Anderson that are cited by these authors
Naphthalene, methyl naphthalene, and dimethyl naphthalene, according
to the preceding authors, are the major high-boiling aromatic hydrocarbons
that transfer from oils into the water column and consequently contact
marine organisms. In addition, napthalenes are readily absorbed by the
organisms, but only slowly depurated. As a consequence of their enhanced
availability, high toxicity, rapid uptake and slow depuration, napthalenes
are often viewed with special interest during oil pollution studies. These
aromatics are apparently not produced by marine organisms, and con-
sequently serve to indicate petroleum incorporation. Naphthalenes are read-
ily measurable, using the ultraviolet spectroscopic method in Neff and
Anderson."
Use of parameters
The preceding discussion addressed several readily measurable and com-
monly used parameters for monitoring and confirming the presence of
petroleum oils in waters, sediments and animals of the marine environment.
Oil pollution is indicated whenever samples from oil spill or discharge
impacted areas are found to contain petroleums, while samples from proxi-
mate non-impacted areas do not contain oil. Several parameters are
suggested for monitoring the presence of petroleum oils in marine environ-
mental samples:
I. gas chromatograms exhibiting the presence of an inverted "cup and
saucer eff'ect"-unresolved complex mixture
2. gas chromatograms exhibiting the presence of n-alkane homologs
covering a broad range of molecular weights, and with adjoining
n-alkane peaks of essentially equal size
3. gas chromatograms exhibiting the presence of the isoprenoid phytane,
most often as a partially resolved peak adjoining the n-Ci« alkanepeak
4. the occurrence of substantial fluorescence spectra, following excita-
tion at specified wavelengths; aromatic components are thereby indi-
cated
5. the presence of napthalene, methyl naphthalene, and dimethyl
naphthalene; measurement can be accomplished by ultraviolet absorp-
tion spectroscopy
Measurement of these parameters is usually accomplished after
chromatographic separation of hydrocarbons from other organics, using
silica gel, alumina, or florisil chromatographic techniques. The occurrence
of any one of these parameters may suffice to establish the presence of
petroleum oils. Of course if more than one is found, then petroleum is shown
to be present with a higher degree of certainty.
Sample analysis
Application of these parameters is illustrated by discussing a recent study
in which some of the parameters were used to establish the presence of oil
pollution in aquatic sediments from a mangrove swamp in Puerto Rico. The
area was impacted by a major oil spill in 1973. General ecological aspects of
this project are discussed in greater detail by Nadeau and Bergquist."
Gas chromatography (GLC), fluorescence spectroscopy, and nuclear
magnetic resonance (NMR) spectroscopy were used to analyze extracts of
sediments from the spill site, and from an area having no known history of
petroleum oil pollution. Extractions were performed by triturating 20 g
portions of air dried sediment with four 50 ml portions of carbon tet-
rachloride. Separation of hydrocarbons from other organics was ac-
complished as in Blumer, el al.,5 using silica gel and alumina packed
columns. The eluates were evaporated to dryness, and the residues analyzed
by the following procedures.
GLC. The analysis was performed with a 50ft OV-101 support-coated
open tubular(SCOT) column, using a flame ionizationdetector(FID) and
a temperature program of 75°-275°C at 6°/minute. Program activation was
initiated to coincide with solvent peak elution.
NMR. Spectra were obtained using a 60 Megahertz (MHz) instrument.
All peak field positions were referred to tetramethylsilane (TMS) at 0
ppm, using the delta scale.
Fluorescence. Measurements were made according to Frank,12 using
15 different excitation wavelengths between 220-500 nanometers (nm) at
20 nm intervals. This procedure differs somewhat from that illustrated in
Figure 3.
Analysis results are summarized in Figures 4-6. GC profiles of the
contaminated sediments exhibit the typical inverted "cup and saucer ef-
fect" This unresolved complex pattern does not appear in GC profiles of the
clean sediments, however (Figure 4). The n-alkane homologs and the
40
30 20
RETENTION TIME (min.)
10
Figure 4. Gas chromatograms of sediment sample extracts: (upper line) sediment from an oil spill impacted area; (lower line)
sediment from a non-impacted area
-------
490
1977 OIL SPILL CONFERENCE
80 -
m
i-
60
HI
O
—r
IMS
*^u/K^y^i/^
10
1 0
p.p.m.(5)
Figure 6. NMR spectra of sediment sample extracts: (upper) sediment from an oil spill impacted area; (lower) sediment from
a non-impacted area; peaks between 0.5-2.0 ppm and 6.5-8.0 ppm are in absorption regions characteristic of aliphatic and aromatic
hydrocarbons, respectively; the aromatic absorption region is emphasized by increasing instrument sensitivity
-------
OIL SPILL BEHAVIOR AND EFFECTS
491
in aquatic organisms. Proceedings of Join! Conference on Preven-
tion and Control of Oil Spills. American Petroleum Institute,
Washington, D.C.
9 Clark, R. C. and J. S. Finley, [974, Analytical techniques for isolating
and quantifying petroleum paraffin hydrocarbons in marine or-
ganisms. Marine Pollution Monitoring (Petroleum). NBS Special
Publication 409. Proceedings of a Symposium and Workshop held at
National Bureau of Standards, Gaithersburg, Maryland, May 13-17
10. Ehrhardt, M. and J. Heinemann. I974. Hydrocarbons in blue mussels
from the Kiel bight Marine Pollution Monitoring (Petroleum) NBS
Special Publication 409. Proceedings of a Symposium and Work-
shop held at National Bureau of Standards, Gaithersburg, Maryland.
May 13-17
II. Farrington, J. W. and G C. Medeiros, 1975. Evaluation of some
methods ot analysis for petroleum hydrocarbons in marine or-
ganisms. Proceedings of Joint Conference on Prevention und Con-
trol of Oil Spills. American Petroleum Institute, Washington, D.C.
12. Frank, U., 1975. Identification of petroleum oils by fluorescence spec-
troscopy Proceedings of Joint Conference on Prevention and Con-
trol of Oil Spills. American Petroleum Institute, Washington, D.C.
13. Gordon, D. C. and P. D. Keizer, 1974. Estimation of Petroleum
Hydrocarbons in Seawaterby Fluorescence Spectroscopy: Improved
Sampling and Analytical Methods. Technical Report No 481. Envi-
ronment Canada, Fisheries and Marine Service
14. Gordon, D C., P. D. Keizer, and J. Dale, 1974. Estimates using
fluorescence spectroscopy of the present'state of petroleum hydro-
carbon contamination in the water column of the northwest Atlantic
Ocean. Marine Chemistn-. v2, pp251-261
15. Hargrave, B. T. and G. A. Phillips, 1975. Estimates of oil in aquatic
sediments by fluorescence spectroscopy. Environmental Pollution.
v8. pp193-21 I
16. Rossi, S. S., J. W. Anderson. andG. S. Ward, 1976. Toxicity of water
soluble fractions of four test oils for the polychaetous annelids,
Meanthes arenaceod?ntala and Capttella capitata. Environmental
Pollution. vlO, pp9-18
17. Nadeau, R. J. and E. T. Bergquist, 1977. Effects of a Major Oil Spill
Near Cabo Rojo, Puerto Rico on Tropical Marine Communities
Proceedings, 1977 Oil Spill Conference. American Petroleum Insti-
tute, Washington, D.C.
18. Neff, J. M. and J. W. Anderson, 1975. An ultraviolet spectrophotomet-
nc method for the determination of naphthalene and alkyl-
naphthalenes in the tissues of oil-contaminated marine animals.
Bull, of Environ. Contain. To.iicol v!4, pp!22-128
19. U.S. Department of Commerce, 1976. Measurement and Interpretation
of Hydrocarbons in the Pacific Ocean. Dept. of Commerce Report
AID.6BA. 76/EPR.3EX.76. Final report on Contract No. 4-35266.
April 1976
20. Zafiriou, O., M. Blumer, and J. Myers, 1972. Correlation of Oils and
Oil Products by Gas Chromatography. Woods Hole Oceanographic
Institution. WHOI-72-55
21. Zitko, V., 1971. Determination of residual fuel oil contamination of
aquatic animals. Bull. of Environ. Contain. Toxicol. v5, pp 559-564
-------
Petroleum hydrocarbons
from effluents:
detection in marine environment
John T. Tanacredi
Hunter College, New York, N. Y.
The marine environment has become the
primary disposal ground for an increasing
quantity of petroleum wastes. Mushrooming
demands for petroleum products and the lack
of economic incentive to recycle waste oil will
increase the concentrations of detrimental
petroleum hydrocarbons in the marine en-
vironment.
Although a continuous, low-level discharge
of waste petroleum hydrocarbons into the
marine environment may not be as dramatic
as a major oil spill, the consequences could be
more devastating over an extended period. As
noted by Blumer,1 earlier interpretations of the
environmental effects of oil must now be re-
evaluated in the light of recent evidence of its
effects on marine organisms 2~5 and its environ-
mental persistence, which resembles that of
DDT, PCBs, and other synthetic materials.6-9
Of the 4.73 mil m3 (1.25 bil gal) of new
oils purchased annually by the automotive in-
dustry,10 an estimated 68 percent will leave
automobile engines as waste. Of this 3.22 mil
m3 (850 mil gal), it is estimated11 that 0.7 to
1.5 mil m3 (200 to 400 mil gal )are recycled
annually, with only 0.4 mil m3 (100 mil gal)
actually being refined. In the New York
metropolitan area, of the estimated 0.3 mil m3
(94 mil gal) of automotive and industrial waste
petroleum recorded annually only 40 percent
is being reprocessed.12 The sources of waste
petroleum in municipal wastewater systems
and their receiving waters range from indi-
viduals who change the oil in their automobiles
and indiscriminately dump the wasted crank-
case oil into a nearby sewer to large establish-
ments that sporadically discard accumulated
stocks of waste oils. On many occasions,
especially during periods of plant by-pass,
large "oil slicks" pass through water pollution
control facilities undetected and untreated.13
A major portion of this waste petroleum will
eventually be discharged to a receiving body
of water because most hydrocarbons are more
resistant to degradative processes than other
compounds commonly found in wastewater,14
and because skimming and setting operations
fail to remove a major portion of petroleum
hydrocarbons which are finely dispersed.15
The current energy dilemma makes it im-
perative to consider this waste not simply as a
pollutant but also as a potentially re-usable
source of energy. This paper will attempt to
determine:
1. Whether waste automotive petroleum hy-
drocarbons are present in the treated effluents
discharged by water pollution control facilities
into Jamaica Bay,
2. Whether there is a sufficient quantity of
petroleum-derived hydrocarbons present in the
Jamaica Bay waters to warrant immediate at-
tention,
3. Whether a significant portion of this
waste petroleum persists in the surface waters
of the Bay, and causes a chronic exposure of
this ecosystem to petroleum-derived hydro-
carbons.
4. Whether petroleum hydrocarbons are be-
ing incorporated in biological tissue of a Bay
marine organism, Mya arenaria.
ANALYTICAL APPROACH
Adlard 16 showed that a variety of analytical
techniques should be used in a multi-parameter
approach to oil analysis. Because of the com-
plex chemistry of the oil, each oil sample lends
itself to differentiation from others. This
passive-tagging approach establishes specific
qualitative parameters for oil samples, in the
form of "profiles" or "fingerprints" to be com-
pared to a "standard profile." Thus, positive
correlations for environmental samples are
either established or not established with stan-
dards depending upon those portions of the
petrochemical waste that exhibit themselves in
fingerprints and remain stable under environ-
mental conditions.
Jamaica Bay, one of the few viable wetland
ecosystems in the New York Metropolitan area,
216 Journal WPCF
The author conducted the chemical analyses for this report at
a facility of the U.S. Environmental Protection Agency, Industrial
Environmental Research Laboratory-Ci, Edison, New Jersey. Use of
these laboratory facilities is provided in order to encourage grad-
uate level environmental research.
-------
Petroleum Hydrocarbons
was chosen as the study site because its unique
hydrological characteristics afford a long resi-
dence time for treated or untreated effluents
(Figure 1). The sampling scheme involved
collections of weekly (September 7, 1973
through November 5, 1973) final effluent sam-
ples from the. four major water pollution con-
trol facilities emptying into the Bay, surface
water samples of the Bay (0.61 m (2 ft) below
surface by Kemmerer water sampler), and
marine organism samples. My a arenaria were
collected from an area considered to be of high
pollution potential (HPP) (Mya II) and an
area of low pollution potential (LPP) (Mya
III).
METHODOLOGY
Water samples were collected in 980 ml
wide-mouth, glass Mason jars with Teflon-lined
caps. Five ml of 1:1 sulfuric acid and 20 ml
of carbon tetrachloride (CC14) were added to
all water samples to retard bacterial action and
for extraction purposes. All samples were re-
frigerated after collection. Glassware was
detergent washed and oven heated at 120°C
to ensure removal of possible contaminants.17
The quantity of total extractable hydrocarbons
in water sample extracts was determined by
use on an infra-red (IB) method tentatively
accepted for oil in water analysis by the
EPA's Industrial Waste Treatment Research
Laboratory, Edison, N. J., with some slight
modifications that were necessary because of
sample concentration factors.18 Carbon tetra-
chloride extracts were jet-air evaporated, con-
centrated, and residues weighed and brought
to volume in hexanes for ultraviolet (uv)-
fluorescence and gas chromatographic (GC)
analysis. Organism extraction required shucked
clams to be homogenized in a blender with 50
ml of n-hexane, mixed with three times wet
tissue weight of anhydrous Na2So,j, and re-
frigerated for 24 h. The tissue extract mix was
Soxhlet extracted in n-hexane for 6 h. These
extracts were divided into subfractions by
hexane/benzene/methanol elution through a
3 percent water deactivated silica gel/alumina
column at a flow rate of 120 ml/s. The three
analytical methods used for sample analyses
were gas chromatography, uv-fluorescence
spectroscopy and cc-mass spectroscopy.
B R o
FIGURE 1. Tri-phase sampling scheme for Jamaica Bay, New York. Organism
samples were designated Mya II and Mya III representative of high
pollution potential (HPP) and low pollution potential (LPP),
respectively.
February 1977 217
-------
Tanacredi
INCREASING TIME AND TEMPERATURE
FIGURE 2. Gas chromatographic profiles of
"standard" petroleum entities:
(A) waste automobile crank-
case oil, (B) 10W/30 virgin
motor oil, (C) transmission
fluid. (D) Arabian light crude
oil. (E) No. 2 fuel oil, (F)
standard Ce-C36 hydrocarbon
"spike" used for retention time
and relative peak height cor-
relations.
Gas chromatographic method. Hydrogen-
flame gas chromatography with flame ioniza-
tion detector and a 15.24 m X 0.51 mm (50
ft X 0.02 in.) ID, stainless steel, support-
coated, open-tubular column (SCOT OV-101;
non-polar silicone oil*) operated with nitrogen
carrier gas with a flow rate at the column out-
let of 240 ml/s. Samples were temperature
programmed from 75° to 300 °C at 6°C/min
with isothermal operation at 300°C for 12
min.
Waste crankcase oil GC profiles exhibited a
large envelope area above the baseline at-
tributable to polynuclear and polycyclic aro-
matic ring compounds; a "light-end" of hydro-
carbons with carbon numbers under C12; C17-
pristane/C18-phytane peak-pairs; various peaks
atop the envelope portion indicative of
branched paraffinic, olefinic, and hetero-com-
pounds over a wide range of boiling points.
The chromatograms generated by refined petro-
leum products differed from those of crude or
fuel oils (Figure 2). GC-retention times and
relative peak heights were used to identify
petroleum hydrocarbons in environmental sam-
ples. A Cfl through C36 n-paraffin hydrocarbon
"spike" (Figure 2F) was added to samples
after noting the original samples' profiles and
retention times for resolved peaks. Increases
in peak heights of previously noted sample
components indicated its presence in the sam-
ple extract. Organism sample sub-fractions
were analyzed by retention times generated in
the GC-MS system.
uv-fluorescence spectroscopic method. Re-
cent investigators 19 have exhibited the ability
of fluorescence spectroscopy to detect trace
quantities of petroleum-derived hydrocarbons
in oceanic waters. Goldberg and Devonald 20
have been able to differentiate between a
lubricating oil and a crude or fuel oil using
fluorescence spectroscopic techniques. All
petroleum products fluoresce when excited
by uv light because of the presence of aromatic
hydrocarbons with multi-ring configurations
such as fused ring polynuclear aromatics.21
A uv-fluorescence spectrophotometer with two
independent monochromators (150 watt xenon
arc light source), and a constant temperature
cell bath that maintained a 10 mm path length,
quartz cell at 20° ± 0.5°C were used for all
fluorescence analyses. Each standard oil was
excited at 290 m/j, while scanning the emission
spectrum from 240 to 540 m^. Thurston and
Knight22 had used 340 m^ as the excitation
wave length for the characterization of petro-
"Perkin-Elmer Corp.
218 Journal WPCF
-------
Petroleum Hydrocarbons
leum entities; however, it has been recently
demonstrated 23 that Raman scattering at that
frequency obscures the fluorescence profiles,
making sample differentiation difficult.
A relatively new fluorescence technique 24
was also used to excite each sample at suc-
cessive excitation wavelengths from 240 m^u, to
440 uifj. (at 20-mp, intervals) while scanning
for the maximum fluorescence emission (Figure
3). Each maximum peak was used as a point
to be plotted graphically; this process gener-
ated a "fluorescence maxima profile" (FMP)
for each sample. Correlation was determined
by visually comparing the maxima profile plots
of known oil standards to the maxima profile
plots of environmental samples. When these
maxima profiles "fit" (Figure 4) in addition to
exhibiting the other qualitative characteristics
established by standards, detection was estab-
lished for the particular sample under in-
vestigation.
When the profile for a sample met the
qualitative criteria established by standard oils,
it was recorded in a "correlates" column. When
profiles generated were not totally consistent
with the qualitative criteria for a correlation, it
was recorded in a "slight-correlation" column.
Slight correlations were felt to be the result of
mixtures of petrochemical entities. It should
be emphasized that the correlation criteria are
essentially qualitative in that source identifica-
tion, (such as gas stations or individuals dump-
ing crankcase oil into a sewer), of the detected
waste petrochemical cannot be directly estab-
lished by this technique. This method was
100-
90-
80-
70-
60 —
KEY
> = WCCO Standard
= 26th. Ward
A (mn)
FIGURE 4. Fluorescence Maxima profile
(FMP) "fit" for waste crank-
case oil with pollution control
facility final effluent extract
samples collected on September
7, 1973.
260 280 300 320 340
380 400 420
EXCITATION FREQUENCY
FIGURE 3.
Fluorescence emission maxima
peaks for the standard waste
automobile crankcase oil.
originally intended to match spilled weathered
petroleum products with already-known stock
oils. When a positive correlation exists in all
fluorescence categories, it is an extremely
strong indication that a waste automotive
petroleum product is present; however, the
correlation arrays are based solely upon gener-
ated profiles and fluorescence analysis of stan-
dard oils.
cc-mass spectroscopic method. The final
analytical phase of this project used a com-
puterized cc-mass spectrometer combination
with 1.52 m X 2 mm ID glass, packed with
3 percent OV-1 on a Chromsorb W column.
Samples were temperature programmed from
100° to 280°C at 6°C/min with isothermal
operations at 280°C for approximately 10 min.
Helium carrier gas at 90 ml/s was measured
'at the column outlet. Only organism sub-
February 1977 219
-------
Tanacredi
fractions were analyzed for specific petroleum-
derived aromatic compounds by this method.
RESULTS
When a waste petroleum product is dis-
carded and enters the environment, it is sub-
jected to a variety of weathering phenomena.
Volatiles are lost as a result of evaporation and
bacteria sequentially degrade different hydro-
carbons.25 To see if detection parameters
established for standard oils would be appre-
ciably affected by weathering, standard oils
were weathered in filtered sea water for a
period of 32 days. Profiles generated indicated
no significant changes in fluorescence detection
criteria even though some profiles exhibited
decreases in intensity because of concentration
factors.26 Waste crankcase oil chromatograms
did lose "light-ends"; however, weathering had
little effect on other detection parameters such
as the unresolved envelope portion.
Treated wastewater samples. Thirty-nine
treated effluent samples were analyzed for total
CC14 extractable hydrocarbons (Table I).
Regular and spiked (C6 through C38 n-paraffm
mix) GC runs were conducted and qualitative
characteristics and retention time data of re-
solved peaks were noted. In all cases, chro-
matograms indicated an unresolved envelope
portion above 200°C. Spiked samples ex-
hibited increases in peak heights for a major
portion of previously resolved peaks. Those
peaks that occurred between standard peaks
were tentatively labeled as being a series of
isomeric or branched chain compounds.27
Clrpristane, C18-phytane hydrocarbon peak
pairs, indicative of petroleum contamination,
were observed in some effluent chromatograms,
but not in all cases.
uv-fluorescence analysis and correlation re-
sults for treated effluent extracts are shown in
Table II. Figure 5 exhibits fluorescence maxi-
mum profiles for wastewater effluents and their
relation to standard waste crankcase oil and
No. 2 fuel oil fluorescence maxima profiles.
Surface water samples. By using a similar
solvent extraction method, average background
levels of petroleum hydrocarbons in oceanic
waters have been established at approximately
2 fj.g/1; greater than average, or excessively
high levels of petroleum hydrocarbons for
oceanic waters, ranged between 10 and 20
iug/l. Table III reveals that the values ob-
tained for the' Jamaica Bay surface waters are
significantly above normal background oceanic
levels of hydrocarbon concentrations. Non-
petroleum, biologically derived compounds are
extracted with CC14 and are not all lost during
the jet-evaporation step. Extensive care and
consideration was taken to prevent contamina-
tion from sample containers, extraction opera-
tions, solvents or from the research vessel
during collection. The January 8 Jamaica Bay
surface water extracts (Figure 6) were an-
alyzed by gas chromatography using the same
sample injection quantities and attenuation
conditions in order to observe a gradient re-
sponse of increased petroleum hydrocarbons as
one moves in and through the Bay.
Table IV exhibits fluorescence results on sur-
face water samples and reveals the presence
of petroleum fractions in those samples taken
from the Bay proper. Again, the degree of
weathering to which the oil had been sub-
jected probably dictated the intensity of the
fluorescence response, yet in no instance did
profiles correlate with other types of petroleum
entities such as fuel or crude oils.
Organism samples. Mya II (HPP) and Mya
III (LPP) extracts were analyzed by GC, uv-
fluorescence and GC-MS methods outlined
previously. Results of analyses clearly demon-
strate the presence of petroleum-derived hydro-
carbons from Mya arenaria tissue extracts con-
sidered to be of high pollution or contamination
potential. The aromatic portion of the Mya II
TABLE I. IR Quantification of Total Extractable Hydrocarbons from Treated Effluents.
Water Pollution
Facility
Coney Island plant
26th Ward plant
Jamaica plant
Rockaway plant
9/10
1.50
29.7
—
—
9/15
16.4
20.0
10.7
—
9/17
7.1
34.9
12.0f
4.9
9/24
2.0
28.9f
7.2
1.3
9/29
3.5
22.9
5.3
4.7
Date
10/1
15.6
12.3
4.7
0.5
10/8
3.0
19.2
9.6
lO.Oj
10/15
39.8f
19.1
4.6
13.8
10/22
10.5
9.3
9.4
8.5
10/29
416
—
14.2
7.7
11/5
8.6
—
18.8
3.2
* All values in mg/1.
t = Gas chromatographic analysis.
220 Journal WPCF
-------
Petroleum Hydrocarbons
TABLE II. Effluent Sample Correlation Data.
Fluorescence Maxima
Profile Fit.
Maxima Region Fit
332 HIM Peak Fit
Date Correlates Slight Correlates Slight Correlates Slight
1973 Sample With WCCO Correlation With WCCO Correlation With WCCO Correlation
7 Sept.
10 Sept.
15 Sept.
17 Sept.
24 Sept.
29 Sept.
1 Oct.
8 Oct.
15 Oct.
22 Oct.
29 Oct.
5 Nov.
26th Ward
CIP
Jamaica
Rockaway
CIP
26th Ward
26th Ward
CIP
Jamaica
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
CIP
Jamaica
Rockaway
CIP
Jamaica
Rockaway
extract (Figure 7A) was shown to contain a
wide range of aromatic compounds with boiling
ranges above 250°C. Mass spectral analysis
of this aromatic portion indicated the presence
of substituted akyl-benzenes, a group of
aromatic hydrocarbons found in extremely
small amounts or not at all in marine organ-
isms.28' 29 Chromatograms of the Mya II-
saturated portion revealed the presence of iso-
meric compounds or other members of the
homologous series. The Mya III aromatic
portion (Figure 7B) was considerably less
complex than the Mya II aromatic fraction;
this suggested that there was less exposure of
the organisms to petroleum hydrocarbons out-
side the Bay than within. Brown and Huff-
man 80 showed this when they observed lower
concentrations of aromatic hydrocarbons in
February 1977 221
-------
Tanacredi
tu
I-
o
- No. 2 Fuel Oil
x = 9/7/73 26th Ward
o= 9/7/73 Jamaica
* = 9/7/73 Rockaway
o-9/7/73 Conev Is.
a = 9/24/73 Rockaway
» - 9/24/73 Jamaica
240 260 300 320 340 . 360 380 400
X (mju)
420 440 460
FIGURE 5.
UV-Fluorescence Maxima profiles for pollution control facility extracts
of September 1973 in correlation with standard crankcase oil and No. 2
fuel oil.
ocean waters than would be found during
petroleum pollution incidents.
The results of the fluorescence analysis on
Mya II extracts, (Table V) indicate that sam-
ples collected from the Bay proper correlated
excellently with standard crankcase oil waste;
however, the Mya III extract did not correlate
successfully with these standards. When the
Mya III extract was excited at 290 m/n, an
additional peak of greater intensity occurred at
370 niju,. This peak could be attributable to
contamination from other petroleum pollutants
such as residual fuel oil, which peaks between
350 and 400 m/x.31 Further investigation is
needed to determine whether or not similar
findings are obtained from non-petroleum ma-
terials in such samples.
DISCUSSION
Chromatograms generated by waste auto-
motive lubricating oil and refined petroleum
have been shown to be characteristic and dif-
ferentiable from chromatograms of other petro-
leum entities. Once a waste oil enters the
environment, it seems that weathering phe-
nomena such as evaporation and bacterial de-
gradation will have little effect upon the less
soluable aromatic and higher molecular weight
components; thus, detection parameters are
TABLE III. Jamaica Bay Surface Water Total
Extractable Hydrocarbons.*
Sample
Site
NYN16
NYN09A
NYJ01
NYJ02
NYJ03
NYJ05
NYJ07
7 November
1973
0.94
1.20
2.10
1.17
3.10
0.50
1.08
8 January
1974
1.13
0.88
2.16
2.20
5.10
1.50
1.40
' All values in mg/1.
222 Journal WPCF
-------
Petroleum Hydrocarbons
preserved. The wide-boiling range, variety of
substituents separated, and the unresolved en-
velope portions of chromatograms indicate the
presence of crankcase oil, although they are
not conclusive. These chromatographic data
may be considered as preliminary findings for
crankcase oil in wastewater and will require a
more quantitative investigation.
Though the specific sources of the detected
waste petroleum could not be established, the
accumulated evidence from all analyses
strongly indicates a crankcase oil origin. Gas
chromatograms of pollution control plant ex-
tracts did show a wide range of hydrocarbon
compounds above C2o, a characteristic of lube
oils.29 They did exhibit unresolved envelope
portions with some samples revealing the C17-
pristane/C18-phytane peak-pairs. Gas chro-
matograms generated by organism extracts
strongly exhibited the presence of aromatic
compounds in body tissue. Tentative GC-MS
identification of organism subfractions indicated
the presence of alkyl-substituted benzene struc-
tures, which are highly toxic substances indica-
tive of petroleum contamination. uv-fluo-
rescence analysis furnished dramatic evidence
for the presence of crankcase oil in environ-
mental samples and greatly strengthened the
other analytical results obtained. Emission
spectra of environmental samples consistently
demonstrated the presence of polynuclear
aromatics (PNA), compounds which could only
FIGURE 6. Gas chromatographic profiles
generated by Jamiaca Bay sur-
face water samples collected on
January 8, 1974.
TABLE IV. Fluorescence Correlation Data for Jamaica Bay Surface Water Samples.
Fluorescence Maxima
Profile Fit Maxima Region Fit 332 mM Peak Fit
Date
7 November
1973
9 January
1974
Slight
Correlates Corre- Correlates
Source With WCCO lation With WCCO
NYN16 + +
NYN09A + +
NYJ01 + +
NYJ02 + +•
NYJ03 + +
NYJ05 + +
NYJ07 + +
NYN16 + +
NYN09A + +
NYJ01 + +
NYJ02 + +
NYJ03 + +
NYJ05 + +
NYJ07 + +
Slight Slight
Corre- Correlates Corre-
lation With WCCO lation
+
+
-f
+
+
+
+
+
-).
+
-f
+
+
+
February 1977 223
-------
Tanacredi
FIGURE 7. Gas chromatograms of (A)
Mya II aromatic sub-fraction,
and (B) Mya III aromatic sub-
fraction.
be attributable to petroleum pollution.32 Due
to crankcase oil's unique fluorescence and
characteristic profiles, its presence in environ-
mental samples was tentatively established,
even though the fluorescence techniques used
for this project were originally to be used for
the comparative identification of unknown oils
to standard oils.
The results presented give a strong indica-
tion that hydrocarbons that are discharged
with treated wastewater into Jamaica Bay and
that ultimately accumulate in biological tissue
are of a waste automotive petroleum origin.
Whether this is from intentional or accidental
dumping into sewers, cannot be determined
here. Because of the unique characteristics of
the study area and the fact that the only fresh
water input comes from pollution control
plants, there is a good indication that a major
portion of detected hydrocarbons originate
from combined wastewater effluents. Further
investigations are necessary to determine the
contributions of hydrocarbons from atmospheric
washings, recreational boating, and biotic
sources.
CONCLUSION
The results of this project indicate the
existence of an unusually large hydrocarbon
burden in Jamaica Bay. Continued addition of
these hydrocarbons can only lead to a further
deterioration of this ecosystem. Tidal-wetland
areas provide food and shelter for a variety of
indigenous and migratory wildlife, and thus
provide critical support to marine food chains
reaching all the way to man. It seems that
problems of aquatic pollution in Jamaica Bay
will be magnified in the future even though
portions of the Bay may be included in the
Gateway National Park. Added pollution from
new housing, continued land-fill operations, and
off-shore oil drilling will continue to pollute,
perhaps irreversibly, this coastal area. In-
creased efforts should be aimed at restoring
these waters so that they may support a much
greater variety of marine life. The idea of
fostering a shell-fishery in Jamaica Bay is a
good one.
The analytical results of this study suggest
that appreciable quantities of hydrocarbons at-
tributable to waste automotive petroleum prod-
ucts are present in treated wastewater effluents
entering Jamaica Bay. The discharge of petro-
leum hydrocarbons in the effluent is chronic.
Significant quantities of detectable hydrocar-
bons remain in solution in the surface waters
of the Bay, while aromatic hydrocarbons from
waste petroleum are found in tissue extracts of
marine benthic organisms collected in the Bay.
The establishment of a national waste oil re-
cycling program would not only reduce the
pollution already developing in the Bay, but
would be an exemplary step towards the most
efficient use of U. S. energy sources.
TABLE V. Fluorescence Data for MYA Extracts.
Fluorescence Max
Profile
Date
Source
Corre-
lation
Slight
Correlation
Max Region
Fit
Slight
Corre- Corre-
lation lation
Excitation
290 HIM
Slight
Corre- Corre-
lation lation
12/3/73
12/3/73
Mya Ilf
Mya IIIJ
f Collected at Diamond Point, Jamaica Bay.
t Collected at Rockaway point, Atlantic Ocean.
224 Journal WPCF
-------
Petroleum Hydrocarbons
ACKNOWLEDGMENTS
Credits. George Kupchik, Jack Foehren-
bach, Michael Gruenfeld, Uwe Frank and
Michael Alavanja reviewed and commented on
this project. This project was made possible
through the cooperation of the Environmental
Protection Agency, Region II, Industrial Waste
Treatment Research Laboratory, Edison, New
Jersey; the Department of Environmental
Health Sciences, Hunter College, of the City
University of New York; and the New York
City Department of Water Resources. B.
Dudenbostel conducted the GC-MS analyses.
Author. John T. Tanacredi was a lecturer
with the Department of Environmental Health
Sciences at Hunter College, New York, N. Y.
He is currently Environmental Protection Ad-
ministrator with the U. S. Coast Guard, N. Y.
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15, 4 (Oct. 1972).
24. Frank, U., "Identification of Petroleum Oils
by Fluorescence Spectroscopy." Proc. of
Joint Conf. on the Prevention and Control
of Oil Pollution, EPA/API/USCG, San
Francisco, Calif., 87 (1975).
25. Kator, H., "Utilization of Crude Oil Hydro-
carbons by Mixed Cultures of Marine Bac-
teria." Ahearn, D. G., and Meyers, S. P.
(Eds.), The Microbial Degradation of Oil
Pollutants. Workshop at Georgia State
University, Atlanta, 60 (1972).
26. Frank, U., "uv-fluorescence Spectroscopy.''
AQCS Newsletter (EPA, Cincinnati, Ohio)
18, 9 (1973).
27. Seminar and personal communications with
Dr. B. Dudenbostel, Region II, Surveillance
and Analysis Lab., EPA, Edison, N. J., on
"Computerized Gas Chromatography/Mass
Spectroscopy" presented October 9, 1973.
February 1977 225
-------
Tanacredi
28. Meinschein. W. G.. "Origin of Petroleum."
Bull Am. Assoc. Petrol Gcol. 43. 925
(1959).
29. Meinschein, W. G.. "Hydrocarbons: Saturate.
Unsaturated and Aromatic." In Elington.
G,, and Murphy, M, I. J. (.Eds,1! Organic
Geochemistry ^Nevr York: S^•ineer-Yerla^V
346 (1969)."
30. Brown, R. A., and Huffman, H. L.. "Hvdro-
carbons In Open Ocean "\Yaters." Science.
191. S47 (1976).
31. Zitko. V., "Determination of Residual Fuel Oil
Contamination of Aquatic Animals." Bull.
Env. Contain, and Tox.. 5, 560 (1971).
32. Wedge\vod. P., and Cooper, R. L., "Detec-
tion and Determination of Traces of Poly-
nuclear Hydrocarbons in Industrial Effluents
and Sewage." Analyst. SO. 651 (19551
•226 Journal \YPCF
-------
BIBLIOGRAPHY OF RECENT METHODS FOR THE FLUORESCENCE
ANALYSIS OF PETROLEUM OILS
Uwe Frank
Industrial Waste Treatment Research Laboratory
U.S. Environmental Protection Agency
Edison, New Jersey
I. Qualitative Methods
1. Thruston, A.D., Jr., and Knight, R.W. 1971. Characterization of Crude
and Residual-Type Oils by Fluorescence Spectroscopy. Env. Sci. and
Tech. 5:64-69.
2. Coakley, W.A. 1973. Comparative Identification of Oil Spills by Fluor-
escence Spectroscopy Fingerprinting. Proceedings of Joint Conference
on Prevention and Control of Oil Spills, pp. 215-222. Washington,
D.C.: American Petroleum Institute.
3. Jadamec, J.R., and Porro, T.J. 1974. Identification and Fingerorinting
of Oils by Fluorescence Spectroscopy. Abstracts of the Pittsburgh
Conference on Analytical Chemistry and Applied Spectroscopy.
Cleveland, Ohio.
4. Lloyd, J.B.F. 1971. The Nature and Evidental Value of the Luminescence
of Automobile Engine Oil and Related Materials - I. Snychronous Ex-
citation of Fluorescence Emissions. J. Fores. Sci. Soc. 11:83-94
5. Lloyd, J.B.F. 1971. The Nature and Evidental Value of the Luminescence
of Automobile Engine Oil and Related Materials - II. Aggregate Lu-
minescence. J. Fores. Sci. Soc. 11:153-170.
6. Lloyd, J.B.F. 1971. The Nature and Evidental Value of the Luminescence
of Automobile Engine Oil and Related Materials - III. Separated Lu-
minescence J. Fores. Sci, Soc. 11:235-253.
7. Drushel, H.V., and Sommers , A.L. 1966. Combination of Gas Chromato-
graphy with Fluorescence and Phosphorescence in Analysis of Petroleum
Fractions. Anal. Chem. 38(1):10-19.
8. McKay, J.F., and Latham, D.R. 1972. Fluorescence Spectrometry in the
Characterization of High-Boiling Petroleum Distillates. Anal. Chem.
44(13):2132-2137.
9. McKay, J.F., and Latham, D.R. 1973. Polyaromatic Hydrocarbons in High-
Boiling Petroleum Distillates. Anal. Chem. 45 (7) :1050-1055.
10. Frank, U. 1972. Analysis for Crankcase Oil in Water by Fluorescence
Spectrophotometry. Analyt. Quality Contr. Newsl., U.S. Environmental
Protection Agency, Cincinnati, Ohio' 13:3.
-------
11. Frank, U. 1972. Passive Tagging of Oils by Fluorescence Spectrophoto-
metry. Analyt. Quality Contr. Newsl., U.S. Environmental Protection
Agency, Cincinnati, Ohio 15:4-5.
12. Frank, U. 1974. Passive Tagging of Oils by Fluorescence Spectrophoto-
metry. Analyt. Quality Contr. Newsl., U.S. Environmental Pro-
tection Agency, Cincinnati, Ohio 20:8.
13. Frank, U., and Jeleniewski, H. 1973. Solvent Impurities and Fluores-
cence Spectrophotometry. Analyt. Quality Contr. Newsl., U.S. Environ-
mental Protection Agency, Cincinnati, Ohio 18:10-11.
14. Frank, U. 1974. An Improved Solvent for Fluorescence Analyses of Oil.
Analyt. Quality Contr. Newsl., U.S. Environmental Protection Agency,
Cincinnati, Ohio 21:10.
15. Frank, U. 1974. Effect of Fluorescence Quenching on Oil Identification.
Analyt. Quality, Contr. Newsl., U.S. Environmental Protection Agency,
Cincinnati, Ohio 22:5.
16. Sawicki, E. 1969. Fluorescence Analysis in Air Pollution Research.
Talanta 16:1231-1266.
17. Gruenfeld, M. 1973. Identification of Oil Pollutants: A Review of
Some Recent Methods. Proceedings of Joint Conference on Prevention
and Control of Oil Spills, pp. 179-193. Washington, B.C.: American
Petroleum Institute.
18. Freegarde, M., Hatchard, C.G., and Parker, C.A. 1971. Oil Spilt at
Sea: Its Identification, Determination and Ultimate Fate. Labora-
tory Practice 20(1):35-40.
19. Frank, U. 1975. Identification of Petroleum Oils by Fluorescence Spec-
troscopy. Proceedings of Joint Conference on Prevention and Control
of Oil Pollution, pp. 87-91. San Francisco, California: American
Petroleum Institute.
20. Hornig, A.W. and Brownrigg, J.T. 1975. Total Luminescence Spectroscopy
as a Tool for Oil Identification. Abstracts of the Pittsburgh Con-
ference on Analytical Chemistry and Applied Spectroscopy, No. 400.
Cleveland, Ohio.
21. Jadamec, J.R., Saner, W.A., and Porro, T.J. 1975. Identification of
Spilled Petroleum Oils by Combined Liquid Chromatographic and Fluo-
resence Spectroscopic Techniques. Abstracts of the Pittsburgh Con-
ference on Analytical Chemistry and Applied Spectroscopy, No. 456.
Cleveland, Ohio.
22. Hornig, A.W., and Eastwood, D. 1971. Development of a Low Temperature
Molecular Emission Method for Oils. Progress Report, Program No.
16020 GBW, U.S. Environmental Protection Agency, Water Quality
Office. Washington, D.C.
-------
II. Quantitative Methods
1. Goldberg, M.C., and Devonald, D.H. 1973. Fluorescent Spectroscopy,
A Technique for Characterizing Surface Films. J. Res. U.S. Geol.
Survey 1(6):709-717 .
2. Gordon, B.C., Jr., and Keizer, P.D. 1974. Estimation of Petroleum
Hydrocarbons in Seawater by Fluorescence Spectroscopy: Improved
Sampling and Analytical Methods. Fisheries and Marine Services
Technical Report No. 481. Environment Canada.
3. Frank, U. 1973. A Method for Quantitating Oil Directly in Water by
Fluorescence Spectrophotometry. Analyt. Quality Contr . Newsl.,
U.S. Environmental Protection Agency, Cincinnati, Ohio 18:9-10.
4. Keizer, P.D., and Gordon, D.C., Jr. 1973. Detection of Trace Amounts
of Oil in Sea Water by Fluorescence Spectroscopy. Journal of the
Fisheries Research Board of Canada 30(8) :1039-1046.
5. Zitko, V., and Tibbo, S.N. 1971. Fish Kill by an Intermediate Oil
From Coke Ovens. Bulletin of Environmental Contamination and
Toxicology 6(l):24-25.
6. Zitko, V., and Carson, W.V. 1970. The Characterization of Petroleum
Oils and Their Determination in the Aquatic Environment. Fisheries
Research Board of Canada Technical Report No. 217.
7. Zitko, V. 1971. Determination of Residual Fuel Oil Contamination of
Aquatic Animals. Bulletin of Environmental Contamination and Toxi-
cology 5(6) :559-564.
8. Hornig, A.W. 1974. Identification, Estimation and Monitoring of Petrole-
um in Marine Waters by Luminescence Methods. NBS Special Publication
409.. Proceedings of Symposium and Workshop on Marine Pollution Mon-
itoring (Petroleum) , pp, 135-144. Gaithersburg, Maryland.
9. Cretney, W.J., and Wong, C.S. 1974. Fluorescence Monitoring Study at
Ocean Weather Station P. NBS Special Publication 409. Proceedings
of Symposium and Workshop on Marine Pollution Monitoring (Petroleum) ,
pp. 175-177. Gaithersburg, Maryland.
-------
SECTION II
OTHER ANALYTICAL PUBLICATIONS
AQC Newsletter Articles
EXTRACTION OF OIL FROM WATER FOR QUANTITATIVE ANALYSIS BY IR
M. Gruenfeld
COMPARISON OF HYDROCARBONS IN MARINE ORGANISMS
FROM UNPOLLUTED WATER WITH PETROLEUM OILS
B. F. Dudenbostel
PREPARATION OF HEAVY OILS FOR INFRARED ANALYSIS
M. Gruenfeld
QUANTITATIVE ANALYSIS OF OIL BY IR
M. Gruenfeld
STORAGE AND TRANSPORT OF OIL CONTAINING SAMPLES IN PLASTIC BOTTLES
B. F. Dudenbostel, M. Gruenfeld
A TLC METHOD TO FACILITATE THE QUANTITATION OF OIL BY IR
U. Frank
ULTRASONIFICATION FOR PREPARING STABLE OIL IN WATER DISPERSIONS
M. Gruenfeld, F. Behm
USE OF GAS CHROMATOGRAPHIC PEAK HEIGHT RATIOS
FOR PASSIVE TAGGING OF PETROLEUM OILS
B. F. Dudenbostel
STORAGE AND TRANSPORT OF OILS IN SOLVENTS
FOR QUANTITATIVE ANALYSIS BY IR
M. Gruenfeld, J. Puglis
CALCULATION OF ABSORBANCE FROM IR ORDINATE EXPANSION MEASUREMENTS
M. Gruenfeld
GLASSWARE CLEANING FOR THE QUANTITATION OF OIL IN WATER
J. Puglis, M. Gruenfeld
SOLVENT EXTRACTION OF OIL FROM WATER
U. Frank
SULFUR INTERFERENCE IN U.V. ANALYSIS
M. Gruenfeld, J. Lafornara
REMOVAL OF CHARRED OIL DEPOSITS FROM GLASSWARE
H. Jeleniewski, U. Frank
SOLVENT FOR OIL ANALYSIS
M. Gruenfeld
EVALUATION OF A PORTABLE IR SPECTROPHOTOMETER
M. Gruenfeld, U. Frank
IDENTIFICATION OF MILLIGRAM QUANTITIES OF PETROLEUM OILS
M. Gruenfeld, R. Frederick
SEPARATION OF PETROLEUM AND NON-PETROLEUM OILS
M. Gruenfeld
REPLICATE OIL CHROMATOGRAMS
M. Gruenfeld, M. Urban
DETERMINATION OF OIL IN SEDIMENT BY NMR
U. Frank, M. Gruenfeld
-------
Research Papers
IDENTIFICATION OF OIL POLLUTANTS: A REVIEW OF SOME RECENT METHODS
M. Gruenfeld
EXTRACTION OF DISPERSED OILS FROM WATER FOR QUANTITATIVE
ANALYSIS BY INFRARED SPECTROPHOTOMETRY
M. Gruenfeld
QUANTITATIVE ANALYSIS OF PETROLEUM OIL POLLUTANTS
BY INFRARED SPECTROPHOTOMETRY
M. Gruenfeld
PRELIMINARY OBSERVATIONS ON THE MODE OF ACCUMULATION OF NO. 2 FUEL OIL
BY THE SOFT SHELL CLAM, MYA ARENARIA
D. Stainken
THE ULTRASONIC DISPERSION, SOURCE IDENTIFICATION, AND
QUANTITATIVE ANALYSIS OF PETROLEUM OILS IN WATER
M. Gruenfeld, R. Frederick
THE EFFECT OF A NO. 2 FUEL OIL AND A SOUTH LOUISIANA CRUDE OIL
ON THE BEHAVIOR OF THE SOFT SHELL CLAM, MYA ARENARIA L
D. Stainken
A DESCRIPTIVE EVALUATION OF THE EFFECTS OF NO. 2 FUEL OIL
ON THE TISSUES OF THE SOFT SHELL CLAM, MYA ARENARIA L
D. Stainken
Bibliography
BIBLIOGRAPHY OF PETROLEUM OIL ANALYSIS METHODS
M. Gruenfeld
-------
NEWSLETTER *
t 0 I I 0
Analytical
U S ENVIRONMENTAL PROTECTION AGENCY
LNVIRONMENTAL MONITORING AND SUPPORT LABORATORY
CINCINNATI , OHIO 48200
PHONf 5IJ-6J4- 7301
SELECTED REPRINTS
OTHER ANALYTICAL PUBLICATIONS
These selected reprints describe work that was performed at the
Industrial Environmental Research Laboratory - Ci., Oil and Hazardous
Materials Spills Branch, Edison, New Jersey 08817.
-------
Analytical Quality Control Newsletter #12 January 1972
Extraction of Oil from Water for Quantitative Analysis by IR A
preliminary study was made to compare the extraction efficiency of
two solvents that are commonly used to separate oil from water. This
study also examined the changes in extraction efficiency that result
from additions of acid and salt. Trichlorotrifluoroethane (Freon 113)
and carbon tetrachloride (CC14) were the solvents of interest, and
both are usable for the quantitative analysis of oils by infrared
spectrophotometry. Carbon tetrachloride exposure to laboratory
personnel can be extremely hazardous. This material is highly toxic
when inhaled (10 ppm TLV), or when absorbed through the skin, and
should be handled with gloves in well ventilated laboratory hoods.
Freon 113 is much safer (1,000 ppm TLV) and is better, particularly
in situations where adequate ventilation may be lacking, such as in
mobile laboratory and field use. The solvents were found to be
about equally efficient for extracting a broad range of oils from
water; i.e., light and heavy; processed and unprocessed oils. Freon
113 is therefore generally recommended as the solvent of choice.
Tests were performed with 1 liter emulsions prepared from tap water
and No. 2 and No. 6 fuel oils, which are light and heavy processed
oils, and South Louisiana and Bachaquero crudes, which are light
and moderately heavy crude oils. Addition of acid and salt dramat-
ically improved extraction efficiency. When acid and salt were not
present, complete separation of the oils was not achieved even after
fifteen 25 ml solvent extractions; however, complete separation was
achieved with only four extractions when 5 gms sodium chloride and
5 ml 50% sulfuric acid were added to the 1 liter synthetic samples.
Additional salt yielded no further improvement. During the latter
determinations, more than 90% of each emulsified oil was separated
in the first extract. Quantitative analyses were performed by
measuring the oil absorbance band intensity at 2,930 cm"-'- in the
infrared spectral region, using 10 nun and 100 mm path length po-
tassium bromide cells and near infrared silica cells. (M. Gruenfeld)
-------
Analytical Quality Control Newsletter #13 April 1972
Comparison of Hydrocarbons in Marine Organisms from Unpolluted Water
with Petroleum Oils - Passive tagging and quantitative analysis of
petroleum oil pollutants in fish and other pelagic marine organisms are
under investigation. Rapid and accurate methods are needed for analyzing
these pollutants of low concentrations in pelagic organisms. Previous
investigations examined mainly benthic organisms such as shellfish. As
part of the investigation a summary table has been prepared comparing the
hydrocarbons found in crude petroleum oils with those oils normally
present in organisms taken from "unpolluted" waters. A table showing
comparisons and the references from which the information was obtained are
available from the Edison Laboratory. (B. F. Dudenbostel)
-------
Analytical Quality Control Newsletter #15 October 1972
PREPARATION OF HEAVY OILS FOR INFRARED ANALYSIS
Although Freon is adequate for extracting dispersed oil from
water, it is not usable for preparing IR standard solutions of
heavy oils. Bachaquero Crude and No. 6 Fuel oils do not readily
dissolve in Freon. Satisfactory "simulated" standard solutions
were prepared by mixing 2.0 ml carbon tetrachloride containing
known amounts of oil with 98 ml of Freon in a 100 ml volumetric
flask. The preferred amounts of oil for measurements in 10 mm
and 100 mm cells are 1 - 40 mg and 0.2 - 2.5 mg, respectively.
The El%cm values (absorbances in 10 mm cells normalized to 1%
w/v dissolved oil) of No. 2 Fuel and South Louisiana Crude oils
in 98% Freon - 2% carbon tetrachloride are virtually identical
to these values in Freon. All the Ei%cm values of the oils in the
solvents are listed below. This parameter may merit inclusion in
passive tagging profiles; e.g. Bachaquero Crude oil is clearly
distinguishable from the other oils by virtue of its diverging
Ei%cm value.
Carbon Freon 2% - 98%
Tetrachloride 113 Solvent Mix
No. 2 Fuel Oil 23.3 21.5 21.5
South Louisiana 25.3 22.5 23.1
Crude Oil
Bachaquero Crude Oil 17.8 — 16.5
No. 6 Fuel Oil 26.2 — 23.7
(M. Gruenfeld,, 201-548-3347)
-------
QUANTITATIVE ANALYSIS OF OIL BY IR
A-preliminary evaluation of infrared spectrophotometry for
quantitating petroleum oils nas been completed. No. 2 and
No. 6 fuel oils and South Louisiana and Bachaquero Crude oils
were examined. Solutions naving known concentrations of these
light and heavy, processed and unprocessed oils in carbon
tetrachloride, Freon 113, and mixtures of these solvents were
prepared. Trichlorotrifluoroethane and Freon-TF are synony-
mous with Freon-113 which is obtainable from E. I. DuPoat de
Nemours, Co. Oil absorbances were measured at 2930 cm"1 in
10 mm path lengths silica cells at ordinate expansions 1 and 5.
Absorbance versus concentration plots derived from the measure-
ments were linear and passed through the origin (Lambert-Beer
Law). Concentrations of 0.2 - 2.5 mg/100 ml and 1-40 mg/100
ml were preferred for measurements at ordinate expansion 1 in
100 mm and 10 mm cells, respectively. The detection limit of
these oils by IR at ordinate expansion 5 using 100 mm cells was
estimated to be 0.05 mg/100 ml. This represents 0.05 mg/liter
oil in water when using the extraction method described in the
Analytical Quality Control Laboratory Newsletter #12, January
1972. A final volume of 100 ml was selected to facilitate
direct correlation with the previous method.
(M. GruenfeZd, 201-548-3247)
STORAGE AND TRANSPORT OF GIL CONTAINING SAMPLES IN PLASTIC BOTTLES
HALAR plastic bottles manufactureed by Vanguard Plastics, Inc.
(104-118 Wagaraw Road, Hawthorne, New Jersey 07506) were tested
for storing and transporting oil containing samples for labora-
tory analysis. Preliminary findings indicate that these bottles
are generally usable for this purpose. Hexane, chloroform,
carbon tetrachloride, and Freon 113, used as solvents for quan-
titative analysis of oil by gravimetric and infrared spectro-
scopiq methods, did not extract any materials from the plastic
that interfere with: (a) infrared analysis of oil at concen-
trations exceeding 2 mg/liter water (slight interference was
found at lower levels), or (b) gravimetric analysis of oil.
Exposing the plastic bottles to methanol and acetone yielded
similar results. The HALAR bottles prevented volatile oil com-
ponents from escaping and thereby assured sample integrity as
effectively as glass stoppered Pyrex bottles. They also cleansed
free of oil, for reuse, as easily as Pyrex containers. A slight
"memory" effect for polar solvents was noted, however. Traces
of methanol and acetone were not as readily removed as from Pyrex.
Following exposure to polar solvents the plastic bottles should
be carefully cleansed before reuse. These findings are based on
the very limited number of plastic bottles that were available
for testing and that were repeatedly exposed to the solvents.
The reported findings and conclusions should therefore be con-
sidered preliminary. (B. F. Dudenbostel/M. Gruenfeld. 201 -548-XZ47)
-------
Analytical Quality Control Newsletter #16 January 1973
A TLC Method to Facilitate
the Quantitation of Oil by IR
Approximately 200 water samples containing dispersed JP-4 jet fuel
were recently analyzed by the IR method outlined in AQC Newsletter
Number 15. The initial sample extracts differed widely in petro-
leum content, and often required repeated dilutions for measurement.
Estimation of these dilutions by trial and error IR measurements,
proved to be very time consuming and cumbersome. Therefore, a thin
layer chromatographic method was developed to reduce analysis time
and simplify sample extract handling. TLC plates coated with
cellulose (Chromagram Sheet 6064, Eastman Kodak Company) were simply
spotted (optimum spot diameters ca. 5 mm) with equal volumes of the
sample extracts. Similarly, a set of standards with known concen-
trations of JP-4 was spotted onto the same plate. Since no
chromatographic development was needed the entire plate surface
was utilized, thereby accommodating up to 80 samples. By comparing
the fluorescence intensity of the sample spots with the standard
spots, it was possible to estimate needed dilutions for IR measure-
ments. A UV box containing long and short wavelength light sources
was used_ (Chromato-Vue, Ultra Violet Products, Inc., San Gabriel,
California). Spot fading due to evaporation did not occur for
several hours, even though JP-4 jet fuel is quite volatile. The
same procedure was also attempted with a number 2 fuel oil and
proved successful. It is therefore thought that this TLC method
can also be readily extended to yield semi-quantitative analyses
of water dispersed oils which would be simpler and less costly
than instrumental methods such as IR, but less accurate.
(U. Frank, 201-548-3347)
-------
Ultrasonification for Preparing Stable Oil
in Water Dispersions
The use of ultrasonification to prepare stable concentrated oil in
water dispersions is currently being investigated. Resulting stock
solutions can be readily diluted further to yield any desired con-
centration of oil in water, and these dispersions are useful for
evaluating analytical methods, environmental cleanup procedures,
and for performing bioassay evaluations of oil toxicity. Most
previous methods for preparing oil in water dispersions involved
addition of known quantities of oil to water, followed by vigorous
agitation. Such procedures are considered unsatisfactory because
rapid oil separation can occur, yielding a system that no longer
represents a dispersion. Ultrasonification can yield stable
dispersions and is therefore more satisfactory. In an effort to
determine whether ultrasonification causes changes in oil that
interfere with its analysis, a low viscosity South Louisiana crude
oil (4.8 centistokes at 100°F) was examined by gas chromatography,
and by infrared, ultraviolet, and fluorescence spectrophotometry.
These measurements were made after ultrasonification at 23°C -
36°C in tap water, solvent extraction, and recovery after solvent
stripping. Comparison was made to the chromatogram and spectra
of a portion of this oil not exposed to ultrasonification, but
otherwise subjected to the same treatment. No significant
spectral or chromatographic changes were evident. Stability of
oil in water dispersions, appears to diminsh with increasing oil
viscosity. The South Louisiana crude oil yielded 1% dispersions
that remained stable for more than four days. But a more viscous
Bachaquero crude oil (1,070 centistokes at 100°F) yielded less
stable dispersions. A model W185 Sonifier Cell Disrupter, sold
by Heat Systems-Ultrasonics, Inc., Plainview, L.I., N.Y., was
used.
(M. Gruenfeld/F. Behm, 201-548-3247)
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Analytical Quality Control Newsletter #18 July 1973
USE OF GAS CHROMATOGRAPHIC PEAK HEIGHT RATIOS
FOR PASSIVE TAGGING OF PETROLEUM OILS
A novel GC data handling system is under investigation as a simple
method to utilize both the quantitative and qualitative aspects of
gas chromatograms for passive tagging of petroleum oils, both
weathered and unweathered. Samples are compared by using only the
"heart fraction" of the gas chromatograms, eliminating volatile
components and heavy-end components from consideration to reduce
the possible effect of environmental change, such as weathering.
A well-resolved GC peak, present in all samples under consideration,
is chosen as the reference and the other peak heights in the
"heart fraction" are ratioed to this reference peak height. By
comparison of the data obtained in this fashion on all samples
under consideration, it appears possible to "match" an environ-
mental sample with possible source mathematically to those obtained
from the environmental sample and the closest match determined.
This approach has been shown to give almost identical results on
an authentic No. 2 fuel oil and a sample of the same oil that had
been naturally weathered in the Sandy Hook area for one month. The
method was also successful in identifying the source gasoline
sample (from among several possibilities) in two contamination cases
handled for the New Jersey State Department of Environmental
Protection. (B.F. Dudenbostel, FTS 201-548-3419, Coml. 201-548-3347)
STORAGE AND TRANSPORT OF OILS IN SOLVENTS
FOR QUANTITATIVE ANALYSIS BY IR
Dispersed oil in water samples is susceptible to evaporative and
microbial degradation losses. Prolonged transport and storage is
therefore not recommended. Prompt solvent extraction, and sub-
sequent transport and storage in the extraction solvent is a
promising alternate approach. A preliminary evaluation of the sta-
bility of oils in some common solvents was therefore performed
A No. 2 fuel oil and a South Louisiana Crude Oil, which are non-
viscous, moderately volatile oils, were used. Ten mg/100 ml solu-
tions were prepared in carbon tetrachloride (CC14) and Freon 113,
while 2 mg/100 ml solutions were prepared in a 2% CC14 - 98%
Freon 113 solvent mixture. The solvents were selected to correlate
this work with oil quantitation procedures in the Analytical
Quality Control Laboratory Newsletters, Nos. 12 and 15. All solu-
tions were stored in stoppered 100 ml volumetric flasks for eight
days, at normal room temperature and lighting conditions. Solution
stabilities were monitored by measuring the 2930 cm'1 absorbance
band of oil. All the solutions proved to be stable; less than 1%
change in absorbance occurred during the test period. This pre-
liminary study suggests that dispersed oils should be promptly
extracted from water, prior to prolonged transport or storage.
(M. Gruenfeld/J. PugUs, FTS 201-548-3543, Coml. 201-548-3347)
-------
CALCULATION OF ABSORBANCE FROM IR ORDINATE EXPANSION MEASUREMENTS
An equation was developed in order to transpose into absorbance
units the 5X ordinate expansion readings obtained with a Perkin-
Elmer Model 457A (now sold as Model 467) infrared spectrophoto-
meter. This instrument expands any 20% portion of the transmit-
tance range to cover the entire chart ordinate. The equation can
also be applied to similar data output of other IR instruments
with ordinate expansion features that operate in the same manner.
Transposition of expansion factors other than 5XP into absorbance
units, can be accomplished by substituting this new expansion
factor for 5 in the numerator and denominator of the equation.
Absorbance = log 5B.
10 5B+D-C
B = % T of the unexpanded baseline
C = % T of the expanded baseline
D = % T of the expanded peak maximum.
(M. Gruenfeld, FfS 201-548-3542 - Coml. 201-548-2347)
GLASSWARE CLEANING FOR THE QUANTITATION OF OIL IN WATER
Carbon tetrachloride is frequently used for cleaning glassware to
remove trace organic impurities. This solvent is expensive and
its excessive handling is hazardous. A comparison of the ability
to remove oil from glassware was therefore made between carbon
tetrachloride and a powdered detergent (Tide Laundry Detergent).
A small quantity of detergent and warm water was added to a
separatory funnel coated with oil. Light and heavy crude and
processed oils were used. The funnel was thoroughly shaken, and
then rinsed repeatedly with water. After the elimination of suds
no interference was encountered at 2930 cm"-'-, which is the in-
frared region used for quantitating oil. The detergent appears
essentially equal to carbon tetrachloride for removing the oils.
It is believed that other detergents will yield similar results.
(J. Puglish/M. Gruenfeld, FTS 201-548-3542, Coml. 201-548-3347)
SOLVENT EXTRACTION OF OIL FROM WATER
Difficult emulsion problems were encountered while analyzing 200
water samples for oil, by the IR method described in the AQCL
Newsletter #15, October 1972. These samples originated from the
overboard discharges of a refined cargo tanker during its ballast
run. It was determined by visual inspection that many of the
samples contained more than 2 ml of oil as a surface layer. This
exceeded the upper concentration limit of the method and often
yielded difficult-to-break emulsions. Use of the IR method was
possible only after additions of 100 ml portions of concentrated
hydrochloric acid to the sample - carbon tetrachloride mixtures
in the separatory funnels. This caused the emulsions to separate
readily into two distinct layers.
(U. Frank, FTS 201-548-3510, Coml. 201-548-334?)
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Analytical Quality Control Newsletter #20 January 1974
SULFUR INTERFERENCE IN U.V. ANALYSIS
The U.V. absorption spectrum of elemental sulfur has been found to
resemble the absorption spectra of some petroleum oils. As a
consequence, leaching of elemental sulfur from the environment
(sediments, et al), by spilled oils, can result in erroneous
analyses. This possibility should be considered whenever quan-
titating oil in water by a U.V. absorption technique (Harva, 0.,
Somersalo, A., Suomen Kern, 3_1 (B) : 384-7 (1958)), and whenever
using a U.V. method for "passive tagging" oils (Levy, E. M.,
Water Research, 6_, 57-69 (1972); also - Shields, D. C., "Forensic
Aspects of Oil Pollution", presented at the 1973 CIC-CCIW
Symposium on Water Quality Parameters, Author's address - R.C.M.P.
Chemistry Section, Crime Detection Laboratory, Vancouver, B.C.,
Canada). These conclusions derive from our limited measurements
of South Louisiana and Bachaquero Crude, and No. 2 and No. 6 Fuel
Oils. These are viscous and non-viscous crude and processed oils
that are considered to be representative of many petroleum oils.
Solutions of the oils and sulfur, in cyclohexane, were prepared
for U-V. measurement. The spectrum of sulfur was found to exhibit
absorption maxima at approximately 225 mp and 260 my. The oil
spectra also exhibit absorption maxima or shoulders at these
approximate wavelengths. The spectral profiles of South Louisiana
Crude and No. 6 Fuel Oils most closely resemble that of elemental
sulfur, but the spectral profile of No. 2 Fuel Oil differs from
it significantly. (J. Lafornara/M, Gruenfeld, FTS 202-548, 3543,
Coml. 201-548-3347)
REMOVAL OF CHARRED OIL DEPOSITS FROM GLASSWARE
During our laboratory's recent participation in an ASTM collabora-
tive study of methods for "passive tagging" oils, difficulties
arose in cleaning charred oil deposits from distillation equipment
and from GC injector sleeves. These items we.re constantly reused
and, therefore, required frequent cleaning. Scrubbing with steel
wool and detergent, and soaking in cold Chromerge solution
(sulfuric acid and sodium dichromate) proved ineffective. Hot
Chromerge solution was effective, but is considered too dangerous
for routine use. Complete removal of all visible traces of oil
deposits was achieved by heating the glassware for 10 minutes at
500°C, in a muffle furnace. It is thought that this technique
can be used to remove other carbonaceous deposits from glassware
that can withstand this temperature.
(H. JeZeniewski/U. Frank, FTS 201-548-3510.. Coml. 201-548-3347)
-------
Analytical Quality Control Newsletter #21 April 1974
SOLVENT FOR OIL ANALYSIS
Recurring questions have been raised about the Identity and
commercial source of tr1chlorotrlf1uorethane, a solvent that
Is often recommended for the quantitative analysis of water
dispersed oils. While this term describes two isomers, the
Isomer that Is actually the recommended solvent Is 1,1,2-
trIchloro-1,2,2-trlfluoroethane. It is available from E. I.
DuPont De Nemours and Company as a refrigerant (Freon-113)
and as a cleaning agent (Freon TF). It is also available
from a number of chemical supply companies, in several
grades of purity. (M. Gruenfeld., FTS 201-548-3543, Coml. 201-548
3347)
EVALUATION OF A PORTABLE IR SPECTROPHOTOMETER
A preliminary evaluation of a compact portable infrared
spectrophotometer that Is suitable for the quantitative
analysis of oMs In the field, Is described. This unit, a
MIran I Fixed Filter Infrared Analyzer (Wllks Scientific
Corporation, South Norwalk, Connecticut) is a single beam
device with a fixed 3.4 micron filter. It accommodates a 10
mm path length sample cell, Incorporates ordlnate expansion
capability 5x and 20x, and can operate off a 12-volt auto-
mobile battery. Battery operation was used during most of
this study, but AC operation was found to yield identical
readings. Solutions of four oils (South Louisiana and
Bachaquero Crude and No. 2 and No. 6 Fuel Oils) with known
concentrations In carbon tetrachloride were measured in 10
mm path length cells without ordlnate scale expansion, and
by using expansion settings 5x and 20x. Absorbance versus
concentration curves were generated, and these prove to be
nonllnean. Mlran "absorbances" do not match absorbance
readings of a double beam IR Instrument. In accordance with
a recommendation by Wllks, appropriate correction factors
were determined as the difference between linearity and the
curve of one oil. These correction factors, when applied to
the curves of the oils, translated them into, linear plots
that passed through the origin. This demonstrates the
utility of the Instrument for single point analysis of oils.
Similar curves were also derived by using ordinate scale
expansions, but no attempt was marie to correct these to
linear plots. At ordlnate expansion 20x, points were quite
scattered and did not yield a smooth curve. The concen-
tration range 0.1 - 150 mg/100 ml oil in carbon tetra-
chlorlde was examined in this study, and the lower concen-
tration figures Is considered to be the reasonable detection
limit of the Instrument at 20x expansion. This represents
an oil In water concentration of 0.1 ppn when 1 liter water
samples are extracted with 100 ml carbon tetroch1 oridp.
(M. Gruenfeld/U. Frank, FTS 201-548-3543, Coml. 201-548-334?)
-------
Analytical Quality Control Newsletter #24 January 1975
IDENTIFICATION OF MILLIGRAM QUANTITIES OF PETROLEUM OILS
Several new methods for fingerprinting petroleum oils have
been published in the 19714 Annual Book of ASTM Standards, Part
31, Water. They include a procedure for sample preparation
and laboratory weathering (D332G-71+T), and for gas
chromatographic correlation (D3328-74T). Fairly large volumes
of oil (50 ml) are required by D332G, and this amount may be
difficult to collect at the scene of a spill. This is also an
.excessive amount of oil to extract from the water column for
those analyses requiring fingerprinting of water dispersed
oils. A procedure has therefore been developed for laboratory
weathering and gas chromatographic correlation of small
amounts of oil, i.e., 0.5-30 mg. Our procedure yields
chromatograms that closely resemble those of D3328, Method A,
(packed column GO; work with SCOT columns (Method B) will
soon be undertaken. Carbon tetrachloride or 1,1,2 - trichloro
- 1,2,2, - trif1uorethane (Freon 113, et al.) solutions of the
spilled oils are quantitatively analyzed by IR. Solution
volumes of 100 ml are then prepared, which contain either 0.5
or 30 mg oil. A steam table and a filtered air stream are
used to strip the solutions to a final 1-2 ml volume in 150 ml
beakers, and these concentrates are transferred to 10 x 30 nm
vials. The vials that contain the 30 mg oil portions are
suspended in a kQ°C water bath, and a filtered air stream is
used to remove the final trace of solvent. This condition is
then maintained for an additional 10 minutes. The vials that
contain 0.5 mg portions of oil are held at room temperature,
the airflow is stopped just before total solvent removal, and
final solvent evaporation is achieved spontaneously. This
condition is then maintained for an additional 10 minutes.
Distilled solvent is used for the lower concent ratior
determinations. Small amounts of CC1 4 (10-20 VI ) are then added
to each vial prior to GC injection. Laboratory weathering of
the reference oils Is achieved by suspending vials containing
70 mg of each reference oil in a kQ° C water bath, for 15
minutes, in the presence of a filtered air stream. Portions
of these reference oils are then injected onto the GC.
(Michael Gruenfeld/R. Frederick, FTS 201-548-35U3 Coml. 201-
5U8-33i*7)
-------
Analytical Quality Control Newsletter #26 July 1975
Separation of Petroleum and Non-Petroleum Oils
The direct addition of silica gel to freon has been recommended
(Newsletter #22, p 7) for separating petroleum oils from animal
oils and vegetable oils. Silica gel removes the non-hydrocarbons
from the solution while leaving the hydrocarbons unaffected.
This technique avoids column chromatographic and solvent
stripping steps and is therefore very straightforward. We
examined this method as part of a project to develop a procedure
for quantitatlng 1 ppb levels of petroleum oils in water. The
Interaction of silica gel with petroleum and non-petroleum oils
In carbon tetrachloride was monitored. The consequence of silica
gel deactivatlon was also examined. This information was then
plotted versus the degree of oil removal, and the Influence of
different stirring times. The resulting graph suggests optimum
operating conditions, and Is available on request. (M.
Gruenfeld, FTS 201-5^8-351*3, Coml. 201-51+8-3347).
-------
Analytical Quality Control Newsletter #29 April 1976
Replicate Oil Chromatograms
Replicate high resolution gas chromatograms of four weathered
and unweathered crude oils were obtained in order to evaluate
a pattern recognition computer program for fingerprinting oils.
This program was previously described in AQC Newsletter No. 25
April 1975, Page 8. Ten replicate runs of each oil (total of
80 chromatograms) were prepared, using a 50 ft OV-101 SCOT
column. The injection and quality assurance techniques used
were in accordance with Technical Report 72-55 of the Woods
Hole Oceanographic Institution (1972) by Zafiriou, 0., Blumer,
M., and Myers, J. Photocopies of the chromatograms are avail-
able on loan, for duplication, together with information re-
garding oil identity and weathering history. (M. Gruenfeld,
FTS 342-7543, Coml. 201-548-3347, M. Urban, FTS 342-7517)
-------
Analytical Quality Control Newsletter #30 July 1976
Determination of Oil in Sediment by NMR
Nuclear magnetic resonance (NMR) spectrometry was found to be a
rapid and useful technique for determining the presence of
petroleum contamination in aquatic sediment samples. Deter-
minations were performed in support of a study conducted by
Region II to establish whether a major oil spill, that occurred
in 1973, caused long term irreversible damage and plant mor-
talities in a mangrove swamp in Puerto Rico. Column chroma-
tographic "clean-up11 procedures and gas chromatography (GLC-FID)
were initially used to analyze carbon tetrachloride extracts of
the sediments, but failed to provide conclusive results because
of interferences from possible biogenic organics. Extracts of
sediments from both the spill site and a similar uncontaminated
location (control) were therefore examined with our 60MHz NMR
spectrometer. The extracts from the spill site were found to
exhibit absorptions in the range 6.5 - 8.0 ppm (ring aromatic
protons) and 0.5 - 2.0 ppm (paraffinic protons) relative to
tetramethylsilane (TMS); the control extracts exhibited no
aromatic absorptions. The low-field aromatic absorptions found
only in sediments from the spill site were ascribed to the pre-
sence of petroleum oil suspected of originating from the past
spill incident. (U. Frank^ FTS 342-7510, Coml. 201-548-3347,
M. Gruenfeld, FTS 342-7543, Coml. 201-548-3347)
-------
Reprinted from: Proceedings 1973 Conference on Prevention and Control of Oil
Spills, March 13-15, 1973, Washington, B.C., API, Wash., DC
IDENTIFICATION OF OIL POLLUTANTS:
A REVIEW OF SOME RECENT METHODS
Michael Gruenfeld
U.S. Environmental Protection Agency
Edison Water Quality Research Laboratory
National Environmental Research Center (Cincinnati)
Edison, New Jersey
ABSTRACT
Some recent studies of methods for identifying weathered
oils through chemical fingerprints (passive tagging) are re-
ported. These studies were performed by Esso Research and
Engineering Company, Phillips Scientific Corporation, Woods
Hole Oceanographic Institution, and Baird Atomic Corpora-
tion under U.S. Environmental Protection Agency (EPA)
sponsored grants and contracts. A broad range of analytical
instruments and techniques were used, e.g., adsorption
chromalography, molecular emission and absorption spec-
trophotometry, atomic absorption spectrophotometry, gas
chromalography, computerized mass spectrometry, el. al.
Some of the oil parameters evaluated as potential finger-
print indices are vanadium, nickel, sulfur and nitrogen con-
tent, gas chromatographic profile appearance, carbon and
sulfur isotope ratios, API gravity, and pour point. Several
promising methods for passive tagging oils are suggested
by these studies.
INTRODUCTION
Discharges and spills of crude, residual, and distillate
oils are occurring with ever increasing frequency in coastal
waters. They cause extensive damage to marine life, coastal
life, recreational beaches, and to their dependent industries,
and are therefore of special concern to the U.S. Environ-
mental Protection Agency (EPA). Establishing the true
source of the pollutant, where several suspect sources exist,
and proving identity between the weathered residue and an
unweathered authentic often requires extensive analytical
instrumental procedures. Many methods are now used, but
there is a lack of agreement among laboratories about the
advantage or necessity of any single technique or combina-
tion of techniques. This variety of methods is illustrated in a
recent publication1 that reviews some current procedures for
correlating weathered and unweathered oils (passive tagging).
Some techniques such as infrared spectrophotometry require
only simple inexpensive instrumentation that is readily avail-
able and usable by most laboratory personnel. But other tech-
niques, such as neutron activation analysis, require such
expensive instruments and highly specialized personnel that
they are available in very few laboratories.
The Edison Water Quality Research Laboratory, a
Laboratory of EPA's National Environmental Research
Center in Cincinnati (NERC), investigated numerous
methods for passive tagging of oils that occur as slicks and
shoreline residues. Several analytical systems that integrate
simple techniques such as gas chromatography with more
sophisticated ones such as mass spectrometry were developed
through grants and contracts. These techniques were eval-
uated singly and in combination as a basis for recom-
mending reasonable analytical approaches to oil identifica-
tion problems. Funding was provided to the Esso Research
and Engineering Company2, the Phillips Scientific Corpora-
tion3, the Woods Hole Oceanographic Institution4, and the
Baird Atomic Corporation^. Resulting analytical methods
and oil parameters measured are summarized (Table 1).
The Esso System
Esso examines a variety of crude and processed oils
(Table 2) by mass spectrometry and gas chromatography to
determine high molecular weight paraffins, naphthenes
(cyclic paraffins), and polynuclear aromatics. Emission
spectroscopy is used to determine bulk vanadium-nickel
content; X-ray spectroscopy and Kjeldahl analysis are used
to determine bulk sulfur-nitrogen content. Adsorption
chromatography with several different columns is used to
isolate paraffins, naphthenes, and aromatics from the oil
matrix and to separate the aromatics from the aliphatics.
A schematic presentation of these analysis steps is pro-
vided (Figure 1).
Portions of the authentic and weathered, samples are
first separated for determination of bulk nickel-vanadium
and sulfur-nitrogen content. Separate portions are distilled
'The Baird Atomic study was funded through the EPA Analytical
Quality Control Laboratory, NERC, Cincinnati.
'Weathering of oils is simulated by using a continuously recirculat-
ing salt water system with controlled temperature, agitation, light
and wind conditions.
179
-------
180
IDENTIFICATION OF OIL
Table 1: Summary of Techniques, Fingerprint Indices, and Investigators
Oil Parameter
Vanadium & Nickel Content
Nitrogen Content
Sulfur Content
Total Profile
N-Paraffins
Isotopic Composition
Carbon & Sulfur in:
Total High Boiling Residue
Carbon in:
Saturate Fraction
Aromatic Fraction
Asphaltic Fraction
Polynuclear Aromatics
Naphthenes
Saturates
Polynuclear Aromatics
Asphaltics
API Gravity
Pour Point
Method of Determination
Emission Spectrometry
Atomic Absorption Spectrophotometry
Kjeldahl Method
Dumas Method & Gas Chromatography
X-Ray Spectroscopy
ASTM Method3
Gas Chromatography (Flame lonization):
Packed Column
Open Tubular Column
Gas Chromatography/Adsorption Chromatography
Gas Chromatography/Urea Adduction
Isotope Mass Spectrometry
Isotope Mass Spectrometry
Isotope Mass Spectrometry
Isotope Mass Spectrometry
Gas Chromatography
Mass Spectrometry (Low Voltage)
Ultraviolet Absorption Spectrophotometry
Fluorescence and Phophorescence Spectrophotometry (77°K)
Mass Spectrometry (High Voltage)
Gravimetric/Adsorption Chromatography
Gravimetric/Adsorption Chromatography
Gravimetric/Adsorption Chromatography
ASTM Method3
ASTM Method3
Investigator
1
2
1
2
1
2
2
3
1
2
1-Esso Research and Engineering Company
2-Phillips Scientific Corporation
3-Woods Hole Oceanographic Institute
4-Baird Atomic Corporation
3ASTM Method cited in text.
to remove components boiling below 400°F; these volatiles
are discarded. Accurately weighed portions of the higher
boiling residues are dispersed in 50 cc n-pentane, insolubles
are separated by centrifugation and discarded. Each solu-
tion is then fed to a clay column and successively eluted with
n-pentane, benzene-acetone, and acetone. The n-pentane
eluate preferentially contains the paraffins, naphthenes, and
aromatics, and the acetone-benzene and acetone eluates
contain the more polar sample fractions. These polar ma-
terials are thought to be more water soluble and therefore
unreliable indices; they are not used in the analysis scheme.
The n-pentane eluate is stripped of solvent, and yields a con-
centrate rich in paraffins, naphthenes and aromatics which is
fed to a silica gel column. The aliphatic components are
selectively eluted with n-pentane, and the aromatic com-
ponents with acetone. The two solvents are then stripped and
the residues weighed. These material balance data may be
useful for oil characterization (Table 3). The saturate frac-
tion is subsequently analyzed by gas Chromatography for n-
paraffins and by computerized high voltage mass spec-
trometry for various naphthene types. The aromatic fraction
is analyzed by low voltage computerized mass Spectrometry
Table 2: Oil Samples Examined by Esso
Sample No. Sample Type Oil Field Location
1 Crude oil Tia Juana Venezuela
2 Crude oil Lago Venezuela
3 Crude oil Grande Isle Indonesia
4 Crude oil Nigeria Nigeria
5 Crude oil Zuitina Libya
6 No. 2 Heating oil
7 No. 4 Fuel oil Refined and
8 No. 5 Fuel oil formulated from
Venezuelan stock
-------
POLLUTANTS
181
Sulfur Analysis
X-Ray
Spectroscopy
Pentane 1
Benzene + Acetone 2
Acetone 3
Pentane 1
to
400°F
ition
F
\
Nickel/ Vanadium
Analysis
Emission
Spectroscopy
\
!
Nitrogen
Analysis
Kjeldahl
Residue
Centrifugation
Clay Separation
Insolubles .
Acetone + Polars
Benzene
Pentane
Paraffins + Naphthenes
Aromatics
Aromatics,
Acetone
n-Paraffin Aromatic Analysis - Low Voltage
Analysis Naphthene Analysis (P + N fraction) - High Voltage
Figure 1: Analysis Schematic of the Esso System.
-------
182
IDENTIFICATION OF OIL
Table 3: Typical Material Balance Data from Esso Sample Separation Processing3
Oil Type
Tia Juana Medium
Crude Oil
Lago Crude Oil
Grande Isle Mix
Crude Oil
Nigerian Crude Oil
Zuitina Crude Oil
No. 2 Heating Oil
No. 4 Heating Oil
No. 5 Heating Oil
Replicate Tia Juana
Medium Crude Oils
Paraffins
+
Naphthenes
46.0
39.8
73.0
61.2
66.1
49.7
63.2
64.2
48.2
47.7
47.4
Aromatics
21.6
21.8
16.1
20.7
12.1
46.6
26.2
20.4
19.5
20.8
18.6
Weight
Polars
19.3
24.7
12.3
10.6
15.2
0.0
8.6
12.1
18.4
20.4
21.5
% of Sample
Pentane
Insolubles
9.0
15.5
1.2
1.0
3.1
0.2
1.0
5.8
10.9
9.4
10.9
2 Fractions
95.9
101.8
102.6
93.5
96.5
96.5
99.0
102.5
97.0
97.9
98.4
aData based on unweathered samples.
for various polynuclear aromatic types. Twenty-six promising
fingerprint indices derived by these techniques (Table 4) are
subjected to Discriminant Function Analysis to identify the
best discriminators for the test oils (Table 5). This statistical
treatment is needed to isolate five indices having the lowest
probability of mismatching two oils (Table 5). Estimation
of confidence levels for oil classification could also have
been performed with the Bonferroni "t" Statistics.
In this study, Esso uses only four fingerprint indices
and, with high statistical confidence, distinguishes among
any possible pairs of the oils, even after the oils have ex-
perienced extensive laboratory weathering. These indices
are generally unaffected by simulated weathering, but their
applicability to other oils has not been established. Other
indices may be required for individual situations.
Another Esso Approach
In a supplemental study Esso evaluates a combined gas
chromatographic-ultraviolet spectrophotometric procedure
for passive tagging oils (Figure 2). Individual polynuclear
aromatics (PNA's) separated by gas chromatography are col-
lected and measured by ultraviolet absorption spectro-
photometry. After 1-gram portions of the weathered and un-
weathered oils are extracted with caustic and dissolved in
cyclohexane, triphenyl benzene is added as internal
standard. Each solution is fed to a deactivated alumina
column (2%H O) that is successively eluted with cyclohexane,
cyclohexane/benzene, benzene, and benzene/methanol. The
cyclohexane/benzene fraction, which is enriched in three-
ring and heavier PNA's, is stripped of solvent and the residue
dispersed in toluene. A gas chromatogram of the PNA's is
then obtained (Figure 3). Selected components appearing as
gas chromatographic peaks are trapped and their ultraviolet
absorption spectra obtained. These spectra together with
appropriate calibration coefficients are used to quantitate the
individual polynuclear aromatics in the oils (Table 6). Esso
uses these quantitative results and visual examination of the
chromatograms for passive tagging oils. Preliminary data in-
dicate that this approach is promising.
The Phillips Study
Phillips surveys a multitude of parameters that may be
useful for passive tagging oils. Main emphasis is on iden-
tifying promising "fingerprint" indices; extensive controlled
weathering experiments are not performed. Seventy-seven
crude oils representing world-wide production (Appendix A)
are stripped of components having boiling points below 600°F
and analyzed by mass spectrometry for carbon and sulfur iso-
tope compositions, by atomic absorption spectrophotometry
for bulk vanadium-nickel content and by gas chromatography
for general profile appearance and odd-even n-paraffin pre-
dominance. Total sulfur, nitrogen, saturates, aromatics,
and asphaltics, as well as API gravity and pour point are also
determined. A schematic presentation of these analysis steps
is provided (Figure 4).
The oils are first freed of insolubles by centrifugation at
20,000 RPM for 90 minutes. The volatiles are then stripped by
heating to 214°F at 0.15 mm Hg. This is equivalent to ap-
proximately 600°F atmospheric. The 600° F + residuals are
analyzed for total nitrogen (Dumas method followed by gas
chromatography); sulfur content (ASTM method D 1552-
64); API gravity and pour point (ASTM methods D287-67
and D97-66, respectively); total nickel and vanadium (in-
cineration in the presence of sulfur and subsequent analysis
by atomic absorption spectrophotometry); carbon and sulfur
isotope ratios (mass spectrometry); and gas chromatographic
profile appearance. A novel classification scheme is used for
coding gas chromatographic information in a form amenable
to computer input: a straight line is drawn between the €20
and Cso peak tips, and the locations of the intermediate nine
n-paraffin peaks are noted as "above" or "under" the line
(Figure 5). Designating the gas chromatographic profile as
Type A (above) or Type U (under) is based on the location of
the majority of peaks. The number of peaks in the minority
is designated by a digit following the type letter. For example,
Type U-2 indicates that seven peaks are under the line and
two above (Figure 5). A Type B profile has a broad envelope
with n-paraffin peaks poorly defined, which prevents the con-
struction of the characterization line between peaks (Figure
6). A summary of all these "fingerprint" indices is presented
(Table 7).
Phillips examines still more potential "fingerprint"
indices. The 600° F + residuals are separated by adsorption
chromatography over silica into three fractions: saturates,
aromatics, and asphaltics (Figure 4). Pentane, dichloro-
methane, and methanol-dichloromethane, respectively, are
-------
POLLUTANTS
183
Table 4: Some Promising Esso Finger
V
Ni
2—3 Ring Naphthenes
2 (P + N)
N
2—4 Ring Naphthenes
2 (P + N)
-20
UnParaffin
c = 20
c = 40
£—5 Ring Naphthenes
2(P + N)
C
21
EnParaffin
c = 20
c = 40
CnH2n-6
CnH2n-i8
C24
2nParaffin
c = 20
c = 40
2 CnH2n -14
2nParaffin
c = 20
c = 40
2-1 Ring + 2 Ring Naphthenes
2—5 Ring + 6 Ring Naphthenes
-26
2nParaffLn
c=20
c = 40
2CnH2n_6 (Benzenes)
2 Aromatics
C
27
2nParaffin
c = 20
c = 40
2 CnH2n_6 c- 20
2 Aromatics
2nParaffin
C
31
2nParaffin
2C
20
2 C30
c = 20
c = 40
c = 20
c = 40
-1\
-22
C32
2 CnH2n_i0 (Indenes)
2 Aromatics
2CnH2n_10 c = 20
2 Aromatics
2CnH2n_14 (Acenopthenes)
2 Aromatics
2 C20 + C21 + C22 + C30 + C31 + C32
2 C
24
25
4- C27
28
2CnH2n_16 (Acenopthalenes)
2 Aromatics
2nParaffins
2 (P+N)
2 Aromatics
(Phenanthrenes)
used for elution. Percent composition of each fraction in the
600° F + residuals, and the carbon isotope ratios in these
fractions are measured as additional indices. The n-paraffins
in the saturates fraction are further separated by urea adduc-
tion and measured by gas chromatography for relative abun-
dance according to carbon number distribution. Odd-
numbered to even-numbered n-paraffins are calculated as a
function of carbon number. A summary of these additional
"fingerprint" indices is presented (Table 8). A limited
assessment is also made of measurement repeatability and
weathering effects.
The Woods Hole Oceanographic
Institution System
Woods Hole Oceanographic Institution (WHOI) uses gas
chromatography with support-coated open tubular (SCOT)
columns for passive tagging oils. The oils are merely dis-
-------
184 IDENTIFICATION OF OIL
Table 5: Most Promising Esso Fingerprint Indices
V_
Ni
SnParaffins
S-5 Ring Naphthenes
2 (P + N)
_ _c=
HnParaffinc=4o
S-l Ring+2 Ring Naphthenes
2-5 Ring+6 Ring Naphthenes
solved in carbon disulfide (ca 5-10% W/V) and injected;
exact concentrations need not be known. The gas chroma-
tograph injector is equipped with a glass liner that is re-
movable for cleaning. Column specifications are 50 feet
x 0.02 inch packed with nonpolar liquid silicone OV-101
and rated at 25,000 effective plates. Oil chromatograms
are compared visually, and certain features are abstracted
and tabulated when analyzing many samples and for sta-
tistical purposes. These features are measured as vertical
distances between superimposed baselines and appropriate
peak heights (Figure 7, Table 9).
The WHOI method requires frequent evaluation of sys-
tem performance by injecting a standard oi!3 that yields a
repeatable known chromatogram. The extent of weathering
undergone by oils is estimated from the onset of the gas
chromatographic signal: if below n-C]4 then the tabulated
indices are considered unaffected by weathering, but if at
or above n-C]5 then weathering alterations are implied.
Tabulated indices of oils that exhibit considerable weath-
ering should not be compared directly with the indices of
unweathered oils. Instead, each unweathered suspect is
artificially weathered in a rotary evaporator by dropping
pressure slowly to a high vacuum while heating to 60° C or
warmer. Small portions are withdrawn periodically for GC
analysis and the procedure continued until the oil resembles
the environmental sample. Similarity is judged by the shape
of low-boiling — range signals, or by comparing background
ratios at Q5 to any signal parameter at CI7 or Qg. The re-
maining indices (Table 9) are then tabulated and compared
to those of the weathered sample. SCOT column lifetimes
generally exceed 200 oil analyses (injections), and tabulated
3A Number 2 Fuel Oil "stabilized" by passage through an alumina
and silica gel column.
Add Cyclohexane and
Internal Standard
to Sample
Separate on A1203 ( + 27. H20)
Elute by Cyclohexane,
Cyclohexane/Benzene, Benzene
Benzene/Mechanol
Cut 1A
125 ml
eye lohexane
Colorless
Fractions
Front Cue Point
a c Appearance
o£ Color
Evaporate, Add
Toluene
157.
Figure 2: Analysis Schematic of Another Esso Approach.
-------
POLLUTANTS 185
Figure 3: Gas Chromatogram by Esso of Polynuclear Aromatics in a Crude Oil. Marked Peaks Are Identified in Table 6. Triphenyl Benzene
(TPB) Is Used as Internal Standard.
Table 6: GC Components Trapped by Esso for UV Analysis
Peak Aa fluorene
Peak B methyl fluorene
Peak C phenanthrene
Peak D 2-methyl phenanthrene
Peak E 1-methyl phenanthrene
Peak F dimethyl phenanthrene
aGC peaks are identified in Figure 3
-------
186
IDENTIFICATION OF OIL
CRUDE OIL
Isothermal
Distillation
0,15 nm
RESIDUE, WT. %
(600 F+ Fraction)
Liquid-Solid
Chromatography
Silica
Measxrra
API Gravity
Pour Point
Sulfur Content
Nitrogen Content
Nickel + Vanadium Content
Carbon Isotopic Composition
Sulfur Isotopic Composition
GLC Profile
DISTILLATE
SATURATES
AROMATICS
ASPHALTICS
Measure
%(w/w) In 600°F+ Fraction
Carbon Isotopic Composition
Urea Adduction
Measure
Measure
600 F+
%(w/w) In
Fraction
Carbon Isotopic
Composition
600°F+
Fraction
Carbon Isotopic
Composition
n-Paraffins
Measure
GLC Carbon Nimber Distribution-
Calculate OEP Ratio
Figure 4: Analysis Schematic of the Phillips Study.
-------
POLLUTANTS
187
Typo U-2 Profile
Figure 5: Coding of Gas Chromatogram Profile by Phillips — Type U-2 System.
indices of individual oils remain quite stable from column
to column.
WHOI reports on a preliminary evaluation of their system
with oils exposed by the Edison EPA Laboratory to various
weathering conditions for different time intervals. Common
spill-control chemicals were also added to some of the oils.
Unweathered portions of 16 out of 17 oils used for this test
accompanied 35 simulated spill samples. All the samples
were number coded and identified only as potential sources
or simulated spills. Test results are reported as "definite
correlation", "probable correlation", or "no definite or
probable correlation" Correct "definite correlation" is
achieved in 74% of the cases, and only one "probable correla-
tion" is incorrect (Table 10). An evaluation of the system's
ability to distinguish among similar unweathered oils and oil
products is also reported; in the majority of cases the oils
were uniquely identifiable (Table 11). It is thought that since
short term weathering does not significantly affect the cor-
relation ability of the method, these results obtained with
unweathered oils also apply to many weathered oils.
The Baird Atomic Study
Baird-Atomic provides a preliminary assessment of
molecular emission (fluorescence and phosphorescence) at
low temperatures as a method for identifying oils. Their ef-
-------
188 IDENTIFICATION OF OIL
C29
18
Figure 6: Coding of Gas Chromatogram Profile by Phillips — Type B System.
fort is a probe to evaluate the utility of the technique for
discriminating among oils and to determine whether an ad-
vantage derives from using cryogenic temperatures. This
study deals mostly with unweathered heavy crude and
processed oils (Table 12); only two weathered oils were
tested and no substantial effort was made to correlate them
with the appropriate unweathered oils. Emphasis is on asses-
sing the utility of low temperature molecular emission for
distinguishing among different oils and not to demonstrate
the utility of this technique for correlating appropriate
weathered and unweathered oils; i.e., passive tagging.
The oils are dissolved in methylcyclohexane and cooled to
77° K. Methylcyclohexane is a good solvent that forms a clear
glass at the cryogenic temperature. The use of other solvents
and different oil concentrations is described; 10 ppm oil in
methylcyclohexane yields the most satisfactory results.
290 mju and 340 mji are the diagnostic excitation wave-
lengths used to compare the oils. In every case oil emission
spectra resulting from excitation at 340 m^t are more intense
than those resulting from excitation at 290 m/j., but excitation
at this latter wavelength yields broader emission spectra; all
the oils yield characteristic emissions in the 380-400 m^t
region when excited at 340 mju. Dramatically improved spec-
tral resolution is achieved by cooling to 77°K (Figures 8a
and 8b). These emission spectra at cryogenic temperature
are generally a mixture of fluorescence and phosphorescence,
the latter occurring at the longer wavelengths. Phos-
phorescence and fluorescence spectra of several oils are com-
pared; they differ sufficiently to distinguish the oils. This
study also attempts to identify major luminescing compound
types in the oils. Porphyrins, compounds similar to 4-methyl-
pyrene, et. al., are identified. Several solvents in addition
to methylcyclohexane are also evaluated; they include pen-
tane, hexane, heptane, and octane. Initial difficulties with
spectral resolution were traced to oxygen-quenching that was
more evident in these normal paraffin solvents. The difficulty
was resolved by degassing the solutions with dry nitrogen.
The Baird Atomic study demonstrates the potential
utility of low temperature molecular emission for passive
tagging oils. Cooling to cryogenic temperature enhances spec-
tral resolution and the technique can differentiate among
very similar oils; e.g., several number 6 f"°l oils (Figure 9).
Table 7: Initial Phillips Fingerprint Indices
API Gravity
Pour Point
Carbon Isotopic Composition
Sulfur Isotopic Composition
Nickel/Vanadium Content (Ratio)
Sulfur/Nitrogen Content (Ratio)
GC Profile
Table 8: Additional Phillips Fingerprint Indices
% (w/w) Saturates
% (w/w) Aromatics
% (w/w) Asphaltics
Odd-Even N-Paraffin Predominance
Carbon Isotopic Composition (Saturates)
Carbon Isotopic Composition (Aromatics)
Carbon Isotopic Composition (Asphaltics)
-------
POLLUTANTS
189
DEFINITION OF TERMS
CHROM'ATOGRAM SERIES NO:
216
-^ ^ ^~r
COLUMN
NO.
INJECTION
NO.
CHART SPEED
I/2"=IMIN= 6°C !
i • '
-PROGRAM START!
CS2 SOLVENT PEAK
BASE LINE
-INCREASING TEMPERATURE
INJECTIONJ
Figure 7: Gas Chromatogram by Woods Hole Oceanographic Institution with Terms Defined.
Table 9: Woods Hole Oceanographic Institute
Fingerprint Indices3
Pristane/Phytane
C17/ Pristane
C18/Phytane
C17/Background
C17/C17-Pristane Valley
a - Ratios measured as in Figure 7.
This study does not evaluate the susceptibility of low
temperature fluorescence spectra to changes that result
from weathering.
Discussion
These EPA sponsored studies are an effort to develop
passive tagging methods having broad scope of application,
and to appraise the usefulness of many analytical techniques
for this purpose. Methods are sought that permit identifying
weathered oils by comparing them with libraries of unn-
weathered oil indices, but that can also be readily simplified
for comparing a weathered oil with only a few unweathered
oils. Although comparison with a few unweathered oils is now
the common procedure, libraries of standard oil indices may
come into use around harbors and other high density oil
storage and handling locations.
Of these studies, only those by Esso and WHOI yield
ready-to-use passive tagging methods. The others mostly
evaluate promising oil parameters and analytical techniques,
and determine their utility for pairing and discriminating
among unweathered oils. Phillips and Baird Atomic do not
perform extensive weathering tests and, therefore, do not con-
firm the reliability of their parameters and techniques for
passive tagging oils. A considerable difference also exists
in the usability of the instrumental techniques in these
studies by "average" analytical laboratories.
The Esso System yields a ready-to-use passive tagging
method that can be applied, in whole or in part, according to
analysis needs and available instrumentation. For example, if
Table 10: Woods Hole Oceanographic Institute Test Results of EPA Weathered Samples
Prepared by EPA
17 Source Oils: 35 Simulated Spills:
8 Crudes Exposures:
5 Fuels
4 Re-refined
1,3, 30 Days
Freshwater, Saltwater,
Beach Sand
With or Without 1 of 4
Spill Control Chemicals
Added.
Correlated by Woods Hole
26 Unique Correlations
6 "Probable" Correlations
(A Second Source Possible)
3 No Definite or Probable
Correlations Found
Result
All Correct
1 Error2
No Correlation
Existsb
aError attributable to presence of oil-based spill control chemical.
bThe source of these samples was not supplied to Woods Hole by EPA.
-------
190
IDENTIFICATION OF OIL
Table 11: Ability by Woods Hole Oceanographic Institute
to Distinguish Among 30 Oils and Oil Products
from Greater New York Harbora
Product Type
Gasoline
#2 Fuel Oil
#4 Fuel Oil
#6 Fuel Oil
Miscellaneous:
Kerosene
Marine Diesel
Nigerian Crude
Asphalt
Overall:
No. of Samples
12
14
Assignment
Not Analyzed
7 Unique
3 Pairs of Indistinguishable Oils
1 Trio of Indistinguishable Oils
All Unique
All Unique
Unique
Unique
Unique
Unique
77% Unique
20% Indistinguishable Pairs
3% Indistinguishable Triplets
aEach oil was analyzed and compared with the 29 others to determine distinguishability.
pairing a weathered oil to one of only a few unweathered oils
is required, then nickel-vanadium, sulfur-nitrogen, and GC
analyses may suffice; but, where comparison with many simi-
lar unweathered oils is to be made, the entire Esso Method
should be used. This assumes, of course, that a computerized
mass spectrometer is available. Therefore, the extent of
method utilization depends on problem complexity and avail-
able instrumentation.
The Esso Method is somewhat inconvenient because
Adsorption chromatography is used repeatedly and because
new selections among the 26 fingerprint indices (Table 4)
may have to be made for different analyses. Additionally,
computerized mass spectrometry is not available in most
laboratories, thereby limiting the usability of the Esso
Method. The method should permit unique correlations with
extensive libraries of oil indices, however. It also benefits
from the extensive weathering studies that were performed in
its development by Esso.
Another Esso approach evaluates a combined GC-UV
procedure that may provide additional fingerprint indices
for passive tagging oils. Weathered and unweathered portions
of the same oils are used in order to establish method integrity
in the presence of weathering. If successful, this technique
should find wide acceptance because gas chromatographs and
ultraviolet spectrophotometers are generally available in
analytical laboratories. It could also be combined to ad-
vantage with those portions of the Esso Method that require
more commonly available instruments. The chromatograms of
polynuclear aromatics differ considerably from the aliphatic
Table 12: Oil Types3 Tested by Baird-Atomic
Five Crude Oils
Two Weathered Crudes
Seven Number 6 Fuel Oils
Two Asphalts
a - Individual oils not identified.
GC profiles of the Esso Method. These aromatic profiles are
easily obtained and may facilitate passive tagging oils, even
without further UV analysis.
Use of adsorption chromatography and GC fraction col-
lection make this procedure somewhat inconvenient. Losses
of oil components can also occur during these steps, thereby
yielding erroneous results. But, Esso's work at the time of
this writing is still too preliminary for meaningful conclusions.
Usable chromatograms of polynuclear aromatics could per-
haps also have been obtained in the previous Esso study if
the aromatic fraction, following silica gel separation (Figure
1), had been analyzed by GC.
The Phillips Study evaluates the suitability of many
parameters and techniques for distinguishing and correctly
pairing unweathered crude oils that represent worldwide
production. Emphasis is directed toward identifying those
parameters that distinguish oils and that may retain their
integrity in the presence of weathering. No in-depth evalua-
tion of weathering effects is done, however, and therefore,
no ready-to-use passive tagging method results.
The potential fingerprint indices examined in this study
include those that require only minimal equipment (API
gravity and pour point) and those that require unusually
expensive instruments (carbon and sulfur isotope ratios).
The suitability of some commonly used passive tagging
indices (sulfur, nitrogen, nickel, and vanadium content)
for distinguishing among a large number of crude oils is
also determined. Phillips aims to evaluate a sufficiently
broad range of parameters to yield a comprehensive method,
such as the Esso Method.
Phillips' general scheme (Figure 4) suggests a compre-
hensive passive tagging method. Identification of parameters
that are unaffected by weathering and extension of the study
to processed oils (distillates and residuals) must necessarily
precede acceptance of this outline as a method. Use of ad-
sorption chromatography and urea adduction are thought to
be somewhat inconvenient.
The WHOl System is a ready-to-use passive tagging
method requiring minimum sample pretreatment and using
only one instrumental technique. WHOI provides explicit
-------
POLLUTANTS 191
CONCENTRATION Hi l'i"i
SLITS 22
TIME CONSTANT ".3
GAIN 30/0 u 0.01/MAX
TEMPERATURE
EXCITATION WAVELEr-GTH 2''UMU
EXCITATION WAVELENGTH 310HU
GAIN 30/0
FLUORESCENCE WAVELENGTH 3UOMU
SLITS
GAIN 3'./" K 0.01/HAX
TEMPERATURE ROUM
EX SOMU FI. E*
FL. 3<40MU
200
CONCENTRATION IOPPM
SLITS 22/11
TIME CONSTANT 0.3
GAIN 30/6 R 0.1 MAX
TEMPERATURE 77°K
EXCITATION WAVELENGTH 290
EXCITATION WAVELENGTH 3M
GAIN 30/0
FLUORESCENCE WAVELENGTH
SLITS 11/22
GAIN 30/6 R 0.01 MAX
TEMPERATURE 77°K
300
507T
WAVELENGTH (nanometers)
600
Figure 8: Fluorescence Excitation and Emission Spectra by Baird Atomic of a Crude Oil (a) at Room Temperature,
and (b) at 77°K. Concentrations Are 10 ppm in Methylcyclohexane.
-------
192
IDENTIFICATION OF OIL
CONCENTRATION 10 PPM
SLITS 22/12 R 0.01 MAX
TIME CONSTANT 0.3
GAIN 30/10
TEMPERATURE " K
EXCITATION WAVELENGTH 290MU
PHOSPHOROSCOPE 16.5 VOLTS
OIL (A)
200
300
400 500
WAVELENGTH (ncmomuluis)
600
700
Figure 9: Phosphorescence Spectra of Three Different Number 6 Fuel Oils at 77"K by Baird Atomic. Concentrations Are 1U ppm in
Methylcyclohexane.
instructions for separating oils from water, sand, and animal
tissue matrices. The oils are then injected directly into the
GC instrument in carbon disulfide solution. Unweathered
suspect oils are also merely dissolved in carbon disulfide
and injected. No preweathering, high speed centrifugation,
adsorption chromatographic, or multi-instrumental tech-
niques, such as those used by Esso and Phillips, are used by
WHOI. The method requires some modification of most GC
instruments, however. These are now generally available
instruments in most analytical laboratories.
The WHOI Method was evaluated by using weathered
and unweathered portions of oils supplied by EPA's Edison
Laboratory. Most, but not all of the weathered oils were
uniquely correlated; several correlations were only "prob-
able" The WHOI Method, therefore, seems usable for com-
parisons to small numbers of standard oils and possibly for
correlations with small libraries of oil indices. But, it may not
achieve the "definite" correct correlations with extensive
libraries of oil indices that should be achievable by the Esso
Method, and by a method that may derive from the Phillips
study. Use of additional fingerprint indices, such as nickel,
vanadium, sulfur, and nitrogen content, in conjunction with
the WHOI Method, should yield further improvement.
The requirement for support-coated open tubular (SCOT)
columns, capillary injectors, glass liners, etc., may diminish
the acceptability of this method. SCOT columns are unusually
expensive GC columns (ca. $160) and have useful lifetimes
that may not greatly exceed 200 injections. Excellent resolu-
tion and reproducibility of fingerprint indices are achieved.
however; the usual packed GC columns yield inferior results.
The use of capillary injectors and glass liners will require
modification of many GC instruments.
The Baird Atomic Study is an evaluation of low tempera-
ture molecular emission as a technique for distinguishing
among oils. Efforts are also made to identify individual
fluorescing and phosphorescing components in oil and to
maximize spectral differences among oils. An enhanced
ability to distinguish between oils and to yield more detailed
spectra, by cooling to 77°K, is demonstrated. But, Baird
Atomic does not demonstrate the usability of their technique
for correctly pairing weathered and unweathered oils; i.e.,
passive tagging.
Although this study does not yield a passive tagging
method, it does demonstrate that low-temperature molecular
emission is promising for this purpose. Equipment cost and
operating complexity is commensurate with other commonly
used analytical instruments. The technique should, therefore,
find ready acceptance. From this point of view, the Baird
Atomic technique, like the WHOI Method, appears more
attractive for normal use than the Esso Method or Phillips
-------
POLLUTANTS
193
approach in their entirety. But, the effect of weathering on
the low temperature molecular emission of oils has not yet
been determined.
CONCLUSION
These EPA-sponsored studies were performed to evaluate
many existing and suggested techniques for passive tagging
oils. The gas chromatographic Method of WHOI and the
fluorescence spectroscopic approach of Baird Atomic are
readily usable in many "normal" laboratory facilities; the
Esso Method and Phillips study encompass techniques that
range beyond the capabilities of most laboratories. Considera-
tion is given in these studies to "simple" methods that permit
correct pairing of weathered and unweathered oils when
dealing with a small selection of oils, and to more complex
methods that may permit identification of totally unknown
weathered oils by using extensive libraries of appropriate
"fingerprint" indices. Evaluation of so many analytical tech-
niques by these studies should facilitate the selection by other
personnel of the most appropriate passive tagging procedures
that suit individual situations and available facilities. These
descriptions of the Esso and Phillips projects are preliminary
and subject to change; final reports are not yet available to
EPA at this time. Most tables and figures presented were
extracted in whole or in part from investigators' reports.
REFERENCES
1. E. R. Adlard, J. Inst. Petrol. 58, 63 (1972).
2. Esso Research and Engineering Company (EPA Project
Number 68-01-0058).
3. Phillips Scientific Corporation (EPA Project Number
68-01-0059).
4. Woods Hole Oceanographic Institution (EPA Project
Number 15080 HEC).
5. Baird Atomic Corporation (EPA Project Number 16020
GBW).
APPENDIX A
Crude Oils Tested By Phillips
Crude Oil
Sample
Crude Oil Number County j Region
Abu Dhabi
Abu Dhabi
Abu Dhabi
Alaska
Alaska
Alaska
Alaska
Alaska
Alaska
Algeria
Algeria
Algeria
Algeria
Argentina
California
California
California
20
59
60
1
2
3
4
5
6
21
22
23
24
70
7
Offshore
Cook Inlet
Cook Inlet
Cook Inlet
Gulf of Alaska Shore
North Slope
North Slope
Off Shore St. Barbara
Los Angeles Co.
St. Barbara Co.
Crude Oil
California
California
Canada
Colombia
Cuba
Cuba
Cuba
Florida
Florida
Florida
Florida
Florida
Gabon
Gabon
Indonesia
Indonesia
Iran
Iran
Iran
Iran
Israel
Israel
Kuwait
Kuwait Neu
Kuwait Neu
Libya
Libya
Libya
Louisiana
Louisiana
Louisiana
Louisiana
Louisiana
Louisiana
Louisiana
Mississippi
Mississippi
Mississippi
Mississippi
Mississippi
Nigeria
Nigeria
Norway
Norway
Norway
Qatar
Qatar
Saudi Arabia
Saudi Arabia
Saudi Arabia
Texas
Texas
Texas
United Arab Republic
United Arab Republic
United Arab Republic
Venezuela
Venezuela
Venezuela
Venezuela
Crude Oil
Sample
Number
75
76
47
71
72
73
74
51
52
53
54
55
57
58
25
61
26
28
29
30
62
63
64
31
27
32
33
34
13
14
77
78
79
80
81
15
16
17
18
19
35
36
37
56
82
65
66
67
68
69
10
11
12
41
42
43
44
45
46
50
County 1 Region
St. Barbara
St. Barbara
Alberta
Collier
Collier
Hendry
Lee
Hendry-Lee
Brunei
Offshore
Offshore
Offshore
Khasji
Cyrenaica
Timbalier Off Shore
Jackson Parish
Claiborne Parish
Offshore
Offshore
Offshore
Offshore
Wayne
Wayne
Clark
Jones
Jasper
North Sea
North Sea
Yoakum & Gaines Co.
Nueces Co.
Brazoria Co.
Lake Maracaibo
Lake Maracaibo
Anzoatequi
-------
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Work supported by the Connecticut Research Commission, Grant
RSA-71-20. Supplementary Material Available. Tablet, 2, 3, and
4, in complete form, will appear following these pages in the mi-
crofilm edition of this volume of the journal. Photocopies of the
supplementary material from this paper only or microfiche (105 x
148 mm, 20X reduction, negatives) containing all of the supple-
mentary material for the papers in this issue mav be obtained
from the Journals Department, American Chemical Society, 1155
16th St., N.W., Washington, B.C. 20036. Remit checks or money
order for $3.00 for photocopy or $2.00 for microfiche, referring to
code number ES&T-73-631.
NOTES
Extraction of Dispersed Oils from Water for Quantitative
Analysis by Infrared Spectrophotometry
MICHAEL GRUENFELD
Environmental Protection Agency, Edison Water Quality Research Laboratory, National Environmental
Research Center (Cincinnati), Edison, N.J. 08817
• Some parameters that optimize the extraction of dis-
persed oils from water for quantitative analysis by in-
frared Spectrophotometry (ir) are examined, and an im-
proved extraction procedure is recommended. Trichloro-
trifluoroethane, an essentially nonpoisonous solvent (1000
ppm TLV), is compared with carbon tetrachloride, which
is highly poisonous to operating laboratory personnel (10
ppm TLV). Both solvents are usable for extracting dis-
persed oils from water, and for their quantitative analysis
by ir, but trichlorotrifluoroethane is preferred. Changes in
extraction efficiency following small additions of sulfuric
acid and sodium chloride are examined. Great improve-
ment results in extraction efficiency, but no further im-
provement derives from addition of more salt. Absorbance
measurements are at 2930 cm"1 in 10-mm path length
cells.
Many biological processes necessary for the survival of
aquatic organisms may be adversely affected by the pres-
ence of extremely low levels—0.1 mg/1.—of petroleum oils
in water. Jacobson (1972), for example, has shown that
kerosine extracts in water, in the part-per-billion range,
upset the chemotactic response of Nansarius obsoletus to
oyster and scallop tissue. Identification of the particular
oil and its quantitative determination in the water col-
umn are essential properly to monitor and assess potential
biological damage resulting from oil spill incidents.
The development of a method that can be used to rap-
idly and completely extract dispersed oils from water for
quantitative analysis by infrared Spectrophotometry, and
one that can be safely used in a mobile laboratory operat-
ing at the scene of a spill incident is described in this
paper. Methods currently available for the quantitative
analysis of petroleums dispersed in water are broadly
identified as gravimetric and spectroscopic procedures.
636 Environmental Science & Technology
-------
Table I. Fraction of Oil Removed by Individual Extracts from Synthetic Dispersions Containing 5 Ml of 50% H2S04
and 5 Grams of NaCI
Percent recovered0
a
No. of
25-ml
extrac-
tions
1
2
3
4
South Louisiana
Crude Oil
Freon 113
92.6
99.3
99.8
100
Determined as A/ A, X 100 + 8: XI.
tracts (2/1); B,
CCU
94 4
99.7
100
No 2
Fuel Oil
Freon 113
97.2
99.5
100
infrared absorbance at 2930 cm
total percent oil recovered by the
previous extracts.
Bachaquero
ecu
97.8
100
-' due to
Crude Oil
Freon 1 13
90.0
98.7
99.7
100
the extract of
ecu
95.4
99.5
99.9
Freon
91.
98.
100
No. 6
Fuel Oi
113
1
7
I
CCU
92.2
98.6
100
100
nterest, XI;
, sum of absoi
bances at 2930
cm ' of all the ex
The gravimetric methods produce losses of the more vola-
tile petroleum fractions making their use questionable for
measuring light oils and distillates. Spectroscopic meth-
ods are inherently more sensitive and accurate, as indicat-
ed by Harva and Somersalo (1958). Infrared and ultravio-
let procedures therefore seem to hold greater promise for
yielding sensitive and accurate techniques.
The extraction of petroleum pollutants from water is a
necessary part of quantitative analysis by either gravimet-
ric or spectroscopic methods. Parameters that influence
these extractions are evaluated in this study, and an opti-
mum extraction scheme is presented. These parameters
include the degree to which addition's of acid and salt in-
fluence extraction efficiency and the utility of trichlorotri-
fluoroethane (Freon 113) and carbon tetrachloride (CC1J
for such extractions; the latter solvent is highly toxic
when inhaled (10 ppm TLV) or when absorbed through
the skin (Sax, 1968).
The literature contains reports on the extraction of oils
from water with carbon tetrachloride and trichlorotrifluo-
roethane, and acid and salt have previously been used to
increase extraction efficiency. The American Petroleum
Institute's spectroscopic procedure (1958) utilizes carbon
tetrachloride as the extracting solvent, after addition of
sulfuric acid and salt. Carbon tetrachloride is also used in
a spectroscopic procedure developed by the Beckman In-
strument Co. (1968) without, however, the addition of
acid or salt. Freon 113 is used as the extracting solvent in
a gravimetric procedure reported by the American Public
Health Association (1971), following addition of sulfuric
acid, but not salt. These methods suggest that there is a
lack of uniformity and general agreement about the ad-
vantages of using acid and salt. They also do not examine
the extent to which additions of these materials influence
extraction efficiency, or the possibility of using Freon 113
for spectroscopic analyses. Freon is safer than carbon
tetrachloride from the analyst's viewpoint.
In the present study four oils were used to compare the
efficiencies of the two solvents and the influence of acid
and salt: No. 2 Fuel Oil, which is a low-viscosity distillate
oil (2.4 cSt at 100°F); No. 6 Fuel Oil, which is a high-vis-
cosity residual oil (2300 cSt at 100°F); South Louisiana
Crude Oil which has a low viscosity (4.8 cSt at 100°F);
and Bachaquero Crude Oil which has a moderately high
viscosity (1070 cSt at 100°F). Consecutive extractions of
each oil from synthetic dispersions in water were carried
out with each solvent; the quantity of oil in the individual
extracts was monitored by measuring the oil absorbance
band intensity at 2930 cm-1 in the ir spectral region (Fig-
ure 1). This band is not unique to oils, but derives from
the CH2 group that is common to many organics. Freon
113 and carbon tetrachloride yield minimal absorbance in
the 2930 cm-1 region and are amenable for such analyses.
0.70 -
0.80
090-
1.0
3200 3000 2BOO
WAVENUM»E« CM'1
Figure 1. Infrared absorbance band of No. 2 Fuel Oil dissolved in
Freon 113 (0.034% W/V), using 10-mm path length silica cells;
Freon is in the reference beam
Absorbance at 2930 cm"1 is determined as the difference between
points A and 8
The changes in extraction efficiency that accompanied
additions of sulfuric acid and sodium chloride were estab-
lished by monitoring resulting changes in the quantity of
oil separated by the individual extracts. The degree to
which the dispersed oils were separated by each extract,
following addition of acid and salt, is also estimated
(Table 1). Since the tabulated results are derived solely
from our synthetic oil-water dispersions however, they
should not be extrapolated to other types of dispersions
without further study.
Experimental
Apparatus. Perkin Elmer Model 457A and Beckman
IR-33 infrared grating spectrophotometers were used for
the determinations. Absorbance of the solutions was mea-
sured in 10-mm path length glass-stoppered rectangular
silica cells (Beckman Instruments, Inc., Catalog No.
580015).
Reagents. Extractions were performed with Freon TF
(Freon 113) solvent (E. I. Du Pont De Nemours and Coin-
Volume 7, Number 7, July 1973 637
-------
A FREON 113
• C Cl,
10 11 12
Figure 2. Number 2 Fuel Oil extracted from 1-liter duplicate
synthetic oil-water samples containing no added acid or salt
LEGEND
NO ACID OR SALT
5
-------
ing the ratio of its absorbance to the sum of the absorban-
ces of all the extracts (Table I).
Results and Discussion
Freon 113 and carbon tetrachloride were found to be
about equally effective for extracting the dispersed oils
from water. Virtually the same number of extractions
with each solvent effected removal of the oils (Figures 2
and 4). Additions of sulfuric acid and sodium chloride
dramatically improved extraction efficiency. In the ab-
sence of these materials, the complete separation of No. 2
Fuel Oil was not possible even after 15 separate 25-ml ex-
tractions with carbon tetrachloride and Freon 113. How-
ever, complete separation of oil was achieved with only
four extractions when 5 grams of sodium chloride and 5
ml of 50% sulfuric acid were added to the 1-liter synthetic
samples. The addition of more than 5 grams of salt yield-
ed no further improvement (Figures 2 and 3). Four 25-ml
extractions with either solvent achieved complete separa-
tion of all the test oils when these quantities of acid and
salt were added to the synthetic 1-liter samples (Figure
4). In these latter determinations, more than 90% of each
emulsified oil was removed by the first extract (Table I).
Freon 113 is recommended as the solvent of choice for
extracting dispersed oils from water, because it is virtual-
ly as efficient for these extractions and as usable for the
infrared determination of oil as carbon tetrachloride, but
is much less poisonous to laboratory personnel. It is espe-
cially preferable to carbon tetrachloride in situations
where adequate ventilation may be lacking, such as in
some mobile laboratory and field use.
The recommended procedure for extracting dispersed
oils from water is the addition of 5 ml of 50% sulfuric acid
and 5 grams of sodium chloride to 1-liter samples. Extrac-
tion should be carried out with four 25-ml portions of
Freon 113 in 2-liter separatory funnels. Checks for acidity
(below pH 3) and completeness of extraction should be
performed. Initial dilution is to 100 ml. Seawater samples
are an exception because they already contain adequate
salt and can therefore probably be analyzed without addi-
tion of sodium chloride. Such samples were not examined
in the present study, however. A "blank" determination of
the reagents and water should be performed to prevent in-
terference with the oil measurement at 2930 cm"1 by ex-
traneous solvent extractable organics.
Acknowledgment
Special thanks are given to Joseph Lafornara for provid-
ing valuable background information, and to Henry Jelen-
iewski, Midhael Killeen, Susan Rattner, and Peter Furth
for their assistance.
Literature Cited
American Petroleum Institute, "Manual on Disposal of Refinery
Wastes," Vol. IV, Method 733-58, 1958.
American Public Health Association, "Standard Methods for the
Examination of Water and Wastewater," 13th ed., APhA,
AWWA, and WPCF, New York, N.Y., 254-6, 1971.
Beckman Instruments, Inc., Mountainside, N.J., Infrared Appli-
cation Note 68-2, 1968.
Harva, O., Somersalo, A., Suomen Kern., 3l(b), 384-7 (1958).
Jacobson, S., Woods Hole Oceanographic Institution, Woods
Hole, Mass., personal communication, June 7, 1972.
Sax, I. N., "Dangerous Properties of Industrial Materials," 3rd
ed., pp 535, 1192, Reinhold, New York, N.Y., 1968.
Received for review August 10, 1972. Accepted March 26, 1973.
Volume 7, Number 7, July 1973 639
-------
Reprinted from: ASTM Special Technical Publication 573
Nov. 1973, ASTM, Phil., PA
Michael Gruenfeld*
Quantitative Analysis of Petroleum
Oil Pollutants by Infrared
Spectrophotometry
REFERENCE: Gruenfeld, Michael, "Quantitative Analysis of Petroleum Oil Pollutants
by Infrared Spectrophotometry," Water Quality Parameters, ASTM STP 573, American
Society for Testing and Materials, 1975, pp. 290-308.
ABSTRACT: The accuracy and sensitivity of infrared Spectrophotometry are evaluated
for the quantitative analysis of water dispersed oils, by single point analysis. Absorbance
versus concentration (Beer-Bouguer Law) plots are prepared for viscous and nonviscous
crude and processed oils in Freon 113, carbon tetrachloride, and in a mixture of these
solvents. Absorbances at 2930/cm are measured in 10 and 100-mm path length cells,
with and without ordinate scale expansion. Solution concentrations in the range 0.5 to
40 mg/100 ml oil in solvent yield linear plots that pass through the origin. The concen-
tration 0.05 mg/100 ml oil in solvent yields a recognizable absorption band at approx-
imately 2930/cm when measured in 100-mm path length cells with ordinate scale
expansion X5. This is considered the practical detection limit of these oils by the
infrared (1R) technique. Stability of oil absorptivities following solution storage, and use
of IR absorptivities for oil identification are also examined briefly.
KEY WORDS: water quality, oils, infrared spectrophotometers, Spectrophotometry,
environmental tests, water pollution
Improved methods for the quantitative analysis of water dispersed
petroleum oils are under investigation by the Industrial Waste Treatment
Research Laboratory of the National Environmental Research Center
(NERC), Cincinnati. Such methods are needed to evaluate the per-
formance of oil-water separator devices, to measure the dispersed oil con-
centration in water below surface floating oil slicks, to monitor oil
concentrations in effluent waters of various industrial plants and water-
craft, and to support studies of the effect of water dispersed petroleum
oils on the biosphere. That low concentrations of water dispersed oils
adversely affect living organisms is well known: for example, Jacobson [I]2
has shown that kerosene extracts in water, in the parts-per-billion range,
'Supervisory chemist, U.S. Environmental Protection Agency, Industrial Waste Treatment
Research Laboratory, National Environmental Research Center, Edison, NJ. 08817.
'The italic numbers in brackets refer to the list of references appended to this paper.
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS 291
upset the chemotactic response of Nassarius Obsoletus to oyster and
scallop tissue.
Most methods that are currently used for the quantitative analysis of
water dispersed oils can be classified as gravimetric or spectroscopic pro-
cedures. The determinative step of gravimetric methods is simple weigh-
ing, while the determinative step of spectroscopic methods is the measure-
ment of radiation absorption by dissolved oils. .Solvent extraction is
commonly used by gravimetric and spectroscopic methods to isolate and
concentrate water dispersed oils for quantitative measurement. Solvent
evaporation (stripping), which precedes weighing in gravimetric methods,
has a drawback because the more volatile petroleum fractions are lost. A
gravimetric method by the American Public Health Association [2]
illustrates yet another drawback: questionable sensitivity and accuracy are
achieved by weighing minute oil residues in "large" (approximately 125
ml) distilling flasks. Harva and Somersalo [3] conclude that the spectro-
scopic methods are more accurate and sensitive—a justifiable conclusion.
Infrared spectrophotometry (IR) is the most commonly used spectro-
scopic technique for the quantitative analysis of water dispersed oils. In an
American Petroleum Institute procedure [4] dispersed oils are extracted
from water with carbon tetrachloride; a mechanical shaking apparatus
and a 5-liter extraction flask are used. The absorption band maxima of oil
at 2850 and 2930/cm are measured, using 10-mm path length quartz
cells. Oil concentrations are determined by comparing the sum of these
absorbances to the sum of absorbances derived from a prepared carbon
tetrachloride solution containing an accurately known concentration of the
oil. The latter solution is the standard solution, and the determination is
performed by single point analysis. These terms are discussed later. In
this method a blend of hydrocarbons (37.5 percent isooctane, 37.5
percent cetane, and 25 percent benzene) that is thought to approximate
the IR absorptivity of an average petroleum oil is used to prepare the
standard solution when a portion of the dispersed oil is not available.
In an IR method by Beckman Instruments, Inc. [5], 100-ml of
dispersed oil in water samples are extracted with 2-ml carbon tetra-
chloride, using hand-held separatory funnels. The absorption band max-
imum of oil at 2930/cm is measured as in the previous method, using
10-mm path length near IR silica cells. The 2850/cm band is not used.
Reference solutions are prepared as oil in water dispersions having known
oil content, and each of these dispersions is extracted with 2-ml carbon
tetrachloride. The extracts are then measured at 2930/cm, and a concen-
tration versus absorbance plot is derived. This plot is used to quantitate
dispersed oil in water samples.
The two preceding methods utilize the same extraction solvent, but they
differ in most other respects. The method by the American Petroleum
Institute uses a large oil extraction flask and a mechanical shaker; salt
-------
292 WATER QUALITY PARAMETERS
and acid are added; extraction is performed with successive 100-ml
portions of carbon tetrachloride; two IR absorption band maxima are
measured; and comparison is made to only a single standard solution. The
Beckman Instrument method uses a small hand-held separatory funnel;
one 2-ml portion of carbon tetrachloride is used to extract a 100-ml
sample volume; acid or salt are not added; one IR absorption band
maximum is measured; and a concentration versus absorbance plot
derived from several oil-in-water reference solutions is used for sample
determination. These methods thus differ in the type of apparatus used,
sample and solvent volumes, addition of salt and acid, and the method of
IR measurement and data handling. Gruenfeld [6] recently investigated
some of these parameters in detail. He examined the influence of salt and
acid, and compared the extraction efficiencies of carbon tetrachloride and
Freon 113 (l,l,2-trichloro-l,2,2-trifluoroethane). Freon 113, like carbon
tetrachloride, can be used for IR measurement of oil at 2930/cm.
Gruenfeld [6] extracts 1-liter dispersed oil in water samples with four
consecutive 25-ml portions of solvent, using 2-liter hand-held separatory
funnels. Use of Freon 113 is of special interest because it is far less
poisonous to exposed personnel than carbon tetrachloride. According to
Sax [7], carbon tetrachloride is highly toxic (10 ppm TLV) when inhaled
or absorbed through the skin, while Freon 113 is much safer (1000 ppm
TLV).
The present paper describes a study of the accuracy and sensitivity of
the IR technique, when used for the quantitative determination of
petroleum oils by single point analysis. Carbon tetrachloride, Freon 113,
and a mixture of these solvents are used. The detection limit of oils by IR,
the stability of oil absorptivities during prolonged solution storage, and
the utility of these absorptivities for oil identification are also examined.
Four oils were used: No. 2 Fuel Oil, which is a low viscosity distillate oil
(2.4 cSt at 100°F); Number 6 Fuel Oil, which is a high viscosity residual
oil (2300 cSt at 100°F); South Louisiana Crude Oil, which has a low
viscosity (4.8 cSt at 100°F); and Bachaquero Crude Oil, which has a
moderately high viscosity (1070 cSt at 100°F). IR measurements were
made in 10 and 100-mm path length silica cells, at approximately
2930/cm. The oils, solvents, final solution volumes, and cells were selected
in order to correlate this work with the previously reported investigation
by Gruenfeld [6].
Atwood et al [8] reported a somewhat similar, though more limited
study of IR for the quantitative analysis of oils. A blend of hydrocarbons
(isooctane, hexadecane, and benzene) was used to represent "typical" oils,
and carbon tetrachloride solutions of this blend were measured in 10 and
100-mm path length quartz cells. No actual oils, or solvents other than
carbon tetrachloride were used. An effort was not made to establish
whether concentration versus absorbance (Beer-Bouguer Law) plots of oils
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS 293
are linear and pass through the origin; that is, whether oils can be quanti-
tatively determined by single point analysis.
Experimental
Apparatus
A Perkin Elmer Model 457A infrared grating spectrophotometer3 was
used for the determinations. Solution absorbances were measured in
10-mm (Beckman Instruments, Inc., Catalog No. 580015) and 100-mm
(Fisher Scientific Co., Catalog No. 14-385-930F) path length cells. These
are rectangular silica and cylindrical Supracil cells, respectively. Cell
holders obtained from the Perkin Elmer Corporation (Catalog No. 186-
0091) were used for the 10-mm path length cells, while holders obtained
from International Crystal Laboratories (Catalog No. R 100-22 with Teflon
gaskets as spacers) were used for the 100-mm path length cells.
Reagents
The oil solutions were prepared in spectroanalyzed carbon tetrachloride
(Fisher Scientific Co., Catalog No. C-199), Freon 113 solvent (E. I.
DuPont De Nemours and Company, Inc.), and in a 98 percent Freon
113/2 percent carbon tetrachloride (by volume) solvent mixture. Freon 113
is a DuPont refrigerant. It is l,l,2-trichloro-l,2,2-trifluoroethane, of
specified purity. This isomer of trichlorotrifluoroethane is available from
several manufacturers, under a variety of trade names.
Procedure
Oil solution concentrations were adjusted to yield absorbances that were
within the ordinate scale range of the IR chart paper. Measurements were
made with matched 10 and 100-mm path length cells, without ordinate
scale expansion, and with ordinate scale expansion x5. Measurements
without scale expansion required oil concentrations of 2 to 40 mg/100 ml
and 0.5 to 4.0 mg/100 ml for measurements in the 10 and 100-mm path
length cells, respectively. Measurements with ordinate scale expansion x5
required concentrations of 0.5 to 4.0 mg/100 ml and 0.05 to 0.4 mg/100
ml for measurements in the 10 and 100-mm path length cells, respectively.
All the solutions contained accurately known oil concentrations. The
reference cell was filled with solvent from the same reagent bottle that was
used to prepare the oil solution. Absorbances derived from measurements
without scale expansion were read directly from a nonlinear absorbance
type chart paper. These absorbances were measured as vertical distances
between the 2930/cm absorption band maximum of oil and a baseline
'Mention of trade names or commercial products does nut constitute endorsement hy the
U.S. Government.
-------
294 WATER QUALITY PARAMETERS
drawn tangent to absorption minima adjoining the band maximum (Fig.
1). Absorbances that were determined by using ordinate scale expansion
x5 required use of a linear transmittance type chart paper, and calculation
by special equation (Fig. 2). The solvents used were Freon 113, carbon
tetrachloride, and 98 percent Freon 113/2 percent carbon tetrachloride
(by volume). Absorbance versus concentration (Beer-Bouguer Law) plots
of the oils in the solvents were derived.
0.70
O.SO
0.90
1.0
3200 3000 2800
WAVENUMBER CM'1
FIG. I—In/rared absorption band of No. 2 Fuel Oil dissolved in Freon 113 (0.034% w/v),
using I0-mni path length silica cells: Freon 113 is in the reference beam. Absorbance at
2^30/cm is determined as the difference between Points A and B.
The utility of IR absorptivities as a parameter for identifying weathered
oils was also briefly investigated. Diverging slopes of Beer-Bouguer Law
plots illustrate differences in oil absorptivities. Eiomm1''0 values (absorb-
ances measured in 10-mm path length cells, normalized to 1 percent
weight/ volume dissolved oil) of the oils were calculated and evaluated for
stability, following loss of volatile oil components as a consequence of
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS 295
100-
90-
Z
<
60-
50-
2930cm'1
Absorbance = Iog10
2930cm"1
5B
5B 4- D — C
B = % T of the unexpanded baseline
C = % T of the expanded baseline
D = % T of the expanded peak maximum
FIG. 2—Infrared absorption band of No. 2 Fuel Oil dissolved in Freon 113: (/) without
nrdinate scale expansion, and (2) with ordinate scale expansion x5.
solvent distillation (stripping). An oil solution was stripped free of solvent,
following IR measurement in 10-mm path length cells. The solvent was
discarded and the oil residue weighed. The residue was then redissolved,
diluted to the previous volume (100-ml), and the solution measured again
by IR. The new Eiomm1"7' absorptivity was compared to the original value.
The solvent stripping procedure of the American Public Health Associa-
tion [2] was used.
A brief investigation was also made of the stability of oil absorptivities
following prolonged solution storage under normal room light and tem-
perature conditions. Oil solutions were prepared in carbon tetrachloride,
Freon 113, and in the 98 percent/2 percent solvent mixture. South
Louisiana Crude and No. 2 Fuel Oils were used; solution concentrations
were 2 and 10 mg/100 ml. Following the initial absorbance measure-
ments, the solutions were stored for eight days on an exposed laboratory
shelf, in clear-glass tightly sealed 100-ml volumetric flasks. The absor-
bance measurements were then repeated and compared with the original
values.
Results and Discussions
The major purpose of this study was to assess the utility of IR for the
-------
296 WATER QUALITY PARAMETERS
quantitative determination of petroleum oils, by single point analysis.
Solutions of four representative oils in two solvents, and in a mixture of
these solvents, were used to prepare the appropriate absorbance versus
concentration (Beer-Bouguer Law) plots. Quantitative determination by
single point analysis requires linear plots that pass through the origin. Use
of single point analysis usually offers a considerable time saving because
only one standard solution is used for each analysis, rather than a
Beer-Bouguer Law plot derived from several solutions. The term standard
solution designates a solution containing an accurately known concen-
tration of oil with the same identity as the dispersed oil in the water
sample. The following equation is used for single point analysis
where
Cx = the unknown oil concentration of the sample extract used
for IR measurement;
Ax and As = the absorbances of the sample extract and standard
solution, respectively; and
Cy = the standard solution concentration used for IR measure-
ment.
The measurements were made in cells with identical path lengths, and
using identical ordinate scale expansion settings. The four oils selected for
study were thought to be fairly representative of viscous and nonviscous
crude and processed petroleum oils. The choice of solvents was determined
by the previously cited publications, and by consideration of oil solubility,
IR absorptivity, and solvent toxicity.
Carbon tetrachloride is used in the previously described IR methods by
the American Petroleum Institute [4], Beckman Instruments, Inc. [5], and
Atwood et al [8]. As previously indicated, this solvent is highly toxic.
Freon 113 is used in a gravimetric procedure by the American Public
Health Association [2], and is much safer than carbon tetrachloride.
Freon 113 is evaluated in the present study for IR measurement of oils.
Gruenfeld [6] compared the ability of these solvents to extract dispersed
oils from water. He found them equally effective for extracting small
quantities (less than 200 ppm) of water dispersed oils. Although Freon 113
is adequate for extracting low concentrations of water dispersed oils, it
does not readily dissolve (cut) undispersed viscous oils such as No. 6 Fuel
and Bachaquero Crude oils. Freon 113 is therefore not recommended for
preparing IR standard solutions of viscous oils. This problem is solved by
first dissolving a sufficient amount of viscous oil in carbon tetrachloride to
yield an accurately known concentration approximating 20 mg/ml. This
solution (2.0-ml) is then diluted to 100-ml with Freon 113, to yield a final
98 percent Freon 113/2 percent carbon tetrachloride mixture that contains
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS 297
approximately 40-mg oil. A fresh portion of the 98 percent/2percent
solvent mixture is used for further dilutions. These standard solutions will
be discussed further.
The oils used in the present study were dissolved directly in the solvents,
to yield solutions having accurately known concentrations. These solutions
simulate solvent extracts of actual dispersed oil in water samples. Beer-
Bouguer Law plots were prepared and evaluated for compliance with the
criteria for single point analysis; that is, plots that are linear and pass
through the origin. Solutions of the four oils in the two solvents and in the
solvent mixture were measured by IR. A range of concentrations was
examined in 10 and 100-mm path length cells, with and without ordinate
scale expansion. Linear plots that pass through the origin were obtained
for the four oils in carbon tetrachloride (Fig. 3) and in 98 percent Freon
NO 1 FUEL OIL
NO 6 FUEL OIL
BACHAQUERO CRUDE OIL
SOUTH LOUISIANA CRUDE OIL
10 20 30 40 50
CONCENTRATION |mg/100 ml) OIL IN SOLVENT
FIG. .1—Oil solutions in carbon tetrachloriJe measured in 10-mtn path length cr//i with-
out ordinale scale expansion.
-------
298 WATER QUALITY PARAMETERS
113/2 percent carbon tetrachloride (Fig. 4), and for the two less viscous
oils in Freon 113 (Fig. 5). Number 6 Fuel and Bachaquero Crude oils
were not readily soluble,, in Freon 113. Therefore, they were dissolved
initially in carbon tetrachloride, and then diluted further with Freon 113,
by the previously described-procedure, to yield a final 98 percent Freon
113/2 percent carbon tetrachloride solvent mixture. The potential absorp-
tivities of viscous oils in Freon 113 are thought to match their absorptivi-
ties in the solvent mixture, because Beer-Bouguer Law plots of the
nonviscous oils in Freon 113 yield slopes that match the slopes of these
oils in 98 percent Freon 113/2 percent carbon tetrachloride (Figs. 4, 5,
and 6). Preparation of standard solutions in 98 percent Freon 113/2
KEY
NO 2 FUEL OIL
NO 6 FUEL OIL
BACHAQUERO CRUDE OIL
SOUTH LOUISIANA CRUDE OIL
10 20 30 40 50
CONCENTRATION (mg/100 ml) OIL IN SOLVENT
FIG. 4—Oil solutions in 98 percent Freon 113/2 percent carbon tetrachloride measured in
10-mm path length cells without ordinate scale expansion.
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS 299
KEY
NO 1 FUEL OIL
SOUTH LOUISANA CRUDE OIL
10 20 30 40 50
CONCENTRATION (mg/100 ml) OIL IN SOLVENT
FIG. 5—Oil solutions in Frcon 113 measured in W-mm path length cells without ordinate
scale expansion.
percent carbon tetrachloride is recommended whenever Freon 113 is used
as the extraction solvent for quantitating dispersed oil in water samples.
Use of IR for the quantitation of water dispersed oils at concentrations
below 1 ppm, by single point analysis, was also examined. Appropriate
Beer-Bouguer Law plots were derived from measurements in 100-mm path
length cells without ordinate scale expansion, using solutions of the four
oils in carbon tetrachloride (Fig. 7), in 98 percent Freon 113/2 percent
carbon tetrachloride (Fig. 8), and solutions of the two less viscous oils in
Freon 113 (Fig. 9). While some of the plots exhibit deviations from
linearity above 0.7 absorbance, all the plots are linear below this value
and pass through the origin. In some cases, portions of an oil used for
-------
300 WATER QUALITY PARAMETERS
9B% FREON-113 -
2% CARBON IETRACHLORIDE
1.0 2.0 3.0 4.0 5.0
CONCENTRATION (mg/100 ml] OIL IN SOLVENT
FIG. 6—Solutions oj No. 2 Fuel Oil measured in 100-mm path length cells without
animate scale expansion.
measurements in 10 and 100-mm path length cells were inadvertently
taken from different drums. This caused the slopes of some plots deriving
from measurements in 10-mm path length cells (Figs. 3, 4, and 5) to
differ from slopes of plots deriving from measurement of the same oils in
100-mm path length cells (Figs. 7, 8, and 9). Use of Freon 113 or 98
percent Freon 113/2 percent carbon tetrachloride in 100-mm path length
cells results in a sluggish response of the IR instrument recorder pen;
Freon 113 has a higher absorptivity at 2930/cm than carbon tetrachloride.
The IR instrument gain setting should be increased sufficiently for these
measurements to yield a properly shaped absorption band at approxi-
mately 2930/cm (Fig. 1). Linear Beer-Bouguer Law plots that pass
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS 301
z
2 05-
oc
O
KEY
NO 2 FUEL OIL
NO. 6 FUEL OIL
BACHAQUERO CRUDE OIL
SOUTH LOUISANA CRUDE OIL
1.0 20 30 4.0 5.0
CONCENTRATION (mg/100 ml) OIL IN SOLVENT
FIG. 1—Oil Mtliitinns in carbon Ictrachlnride measured in 100-mm path length cc//.v with-
nl nntiiitili' .sra/r expansion.
through the origin were also obtained for measurements of the four oils in
carbon tetrachloride, using 10-mm path length cells with ordinate scale
expansion x5 (Fig. 10). But, considerable deviation from linearity resulted
from the measurement of Bachaquero Crude oil in 100-mm path length
cells with ordinate scale expansion *5 (Fig. 11).
Accurate quantitative determination of water dispersed oils by single
point analysis can be accomplished in the concentration range 2 to 40
mg/liter (ppm) oil in water, by using 10-mm path length cells without
ordinate scale expansion (Figs. 3 to 5). This concentration range assumes
use of the extraction procedure by Gruenfeld [6|, whereby 1-litcr oil in
water samples are extracted with four 25-ml portions of solvent. Improved
-------
302 WATER QUALITY PARAMETERS
KEY
NO 1 FUEL OIL
NO 6 FUEL OIL
BACHAOUERO CRUDE OIL
SOUTH LOUISIANA CRUDE OIL
10 20 30 4.0 5.0
CONCENTRATION (mg/100 ml) OIL IN SOLVENT
FIG. 8—Oil solutions in 98 percent Freon 113/2 percent carbon tetrachloride measured in
100-mm path length cells without ordinate scale expansion. A higher than normal IR instru-
ment gain setting was used.
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS 303
KEY
NO 2 FUEL OIL
SOUTH LOUISIANA CRUDE OIL
1.0 2.0
CONCENTRATION I
3.0 40 50
ng/100 ml) OIL IN SOLVENT
FIG. 9—Oil solutions in Freon 113 measured in 100-mm path length cells without or-
dinal? scale expansion. A higher than normal 1R instrument gain setting was used.
sensitivity can be achieved by using less solvent. Accurate quantitation of
oils in the concentration range 0.5 to 3 ppm oil in water can be achieved
by single point analysis when using 100-mm path length cells without
ordinate scale expansion (Figs. 7 to 9), of 10-mm path length cells with
ordinate scale expansion x5 (Fig. 10). The measured absorbances in
100-mm path length cells should not exceed" 0.7. Use of 100-mm path
length cells with ordinate scale expansion x5 yields nonlinear Beer-
Bouguer Law plots (Fig. 11). While accurate quantitation of oils by single
point analysis is not possible under these conditions, useful information is
gained about the detection limit of the 1R technique. These measurements
-------
304 WATER QUALITY PARAMETERS
0 10 T
KEY
NO 1 FUEL Oil
NO. 6 FUEL OIL
BACHAQUERO CRUDE Oil
SOUTH LOUISIANA CRUDE OIL
10 20
CONCENTRATION (
3.0
g/100
4.0 5.0
I OIL IN SOLVENT
FIG. 10—Oil solutions in carbon tetruchloride measured in I0-mni path length cells with
tmliiititt' scale expansion*5.
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS 305
0.) 02 03 04 05 06
CONCENTRATION (m9/IOO ml) OIL IN SOLVENT
FIG. 1 \—Carbon tetrachloride solutions of Bachaquero Crude Oil measured in 100-mm
path length cells with ordinate scale expansion X5.
yield a recognizable absorption band at approximately 2930/cm (Fig. 1)
for an oil solution containing 0.05 mg/100 ml oil in solvent. This is
equivalent to 0.05 ppm dispersed oil in water when the extraction
procedure by Gruenfeld [6] is used. This detection limit of the IR
technique can be improved further by reducing the volume of extraction
solvent.
Consideration of the Beer-Bouguer Law plots demonstrates that the
lines diverge and, therefore, that the oils have different absorptivities.
These absorptivities appear to remain reasonably stable, despite loss of the
more volatile oil components (Table 1). They are solvent dependent,
however, as demonstrated by the diverging Beer-Bouguer Law plots that
-------
306 WATER QUALITY PARAMETERS
• IN CARBON TETRACHLORIDE
• IN FREON-II3
10 20 30 40 50
CONCENTRATION (mg/100 ml) OIL IN SOLVENT
FIG. 12—Solutions oj No. 2 Fuel Oil measured in 10-mm path length cells without
ontinule scale expansion.
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS 307
• IN CARBON TETRACHLORIDE
• IN FREON-113
10 20 30 40 50
CONCENTRATION (ma/100 ml) OIL IN SOLVENT
FIG. 1.1—Solutions of South Louisiana Crude Oil measured in 10-mm path length cells
without nnlintilf scale expansion.
are obtained from the same oil in different solvents (Figs. 12 and 13): No.
2 Fuel and South Louisiana Crude oils yield absorptivities in Freon 113
that differ from their absorptivities in carbon tetrachloride. These
differences are also apparent when comparing Figs. 3 and 4, and others.
IR absorptivities are thus a promising parameter for "passive tagging" oils
because they differ from oil to oil, and yet remain reasonably stable. Their
solvent dependence may also be useful for distinguishing among similar
oils. The term "passive tagging" describes a procedure whereby a
weathered oil residue, that is, an environmental pollutant, is correlated
(matched) with an unweathered portion of the same oil.
The stability of solution absorbances, and therefore oil absorptivities,
-------
308 WATER QUALITY PARAMETERS
TABLE 1—Effect uj so/vent distillation on ad absorptivity. (No. 2 Fuel and South Louisiana
Crude Oils are in Freon 1/3 solution: No. t> Fuel and Bachauiiero Crude Oils are in 98%
Freon 113/2% carbon tetrachloride solutum).
Before Distillation After Distillation"
Oil in Solvent Absorptivity" % Loss Absorptivity %
Oil (nig/100 ml) (EiOmm1%> (by weight) (H1omml%) Change
No. 2 Fuel
South Louisiana
Crude
No. 6 Fuel
Bachaquero
Crude
104
14.5
101
10.5
10. fa
103
21.4
22.4
23.8
16.1
11.5
23.0
3.0
14.3
0.0
4.4
21.1
19.6
21.6
21.7
23.8
15.3
1.4
8.4
3.6
3.1
0.0
5.0
"Solvent distilled (slripped) from 100-ml solutions having known oil content, using the
procedure of the APHA |2|. The weighed residues are redilmed to 100-ml and the solution
absorbances measured at 2930/cm.
^E|0 mm " values are absorbances at 2930/cm, measured in 10-mm path length cells,
normalized to 1% (weight/volume) dissolved oil.
following prolonged solution storage was also examined. Oil solutions in
carbon tetrachloride, Freon 113, and 98 percent Freon 113/2 percent
carbon tetrachloride were stored under normal room light and tempera-
ture conditions, in clear glass flasks for eight days. The absorbances
following storage matched those prior to storage, within 1 percent. Prompt
solvent extraction of dispersed oil in water samples is therefore recom-
mended. Oil transport and storage in carbon tetrachloride or Freon 113
solution should prevent biological degradation and evaporative losses that
can occur in the water matrix.
References
|/] Jacobson, S., personal communication, Woods Hole Oceanographic Institution, Woods
Hole, Mass., 7 June 1972.
[2| Standard Methods for the Examination of Water and Wastewater, 13th ed., American
Public Health Association, New York, 1971. pp. 254-256.
|.?| Harva, O. and Somersalo, A., Suomen Kemistilehti, Vol. 31 (b), 1958, pp. 384-387.
[4\ Manual on Disposal of Refinery Wastes. Vol. IV, Method 733-58, American Petroleum
Institute, 1958.
151 Infrared Application Note 68-2, Beckman Instruments, Inc., Mountainside, N.J., 1968.
|6| Gruenfeld, M., Environmental Science and Technology. Vol. 7, 1973, pp. 636-639.
|7| Sax. I. N., Dangerous Properties of Industrial Materials. 3rd ed., Reinhold, New York,
1968, pp. 535. 1192.
|,V| Atwood, M. R., Hannah, R. W., and Zeller, M. V., Infrared Bulletin No. 24, The
Perkin-Klmer Corp.. Norwalk, Conn. 1972.
-------
Reprinted from: Proceedings 1975 Conference on Prevention and Control
of Oil Pollution, March 25 - 27, 1975, San Francisco, API, Wash., DC
PRELIMINARY OBSERVATIONS ON THE MODE OF
ACCUMULATION OF #2 FUEL OIL BY
THE SOFT SHELL CLAM, MYA ARENARIA
Dennis M. Stainken
Rutgers University
Department of Zoology and Physiology
Newark, New Jersey
ABSTRACT
Chemical analysis has shown that various components of oils can
accumulate within marine invertebrates. Several mechanisms by
which this may occur have been conjectured. This paper offers
experimental verification of a mechanism by which a commercially
important bivalve, Mya arenaria, can accumulate oil within its tis-
sues. The paper also documents the behavioral response of Mya
arenenaria and deleterious ecological side effects resulting from oil
accumulation.
Young Mya (25-35 mm) were exposed to #2 fuel oil and an oil
soluble dye (Oil Red 0) which were ultrasonically emulsified in
water. The concentrations tested were 50 ppm, 100 ppm and 150
ppm. Exposures were done in both natural and artificial seawater at
4°C and 22° C. Exposure periods ranged from 3 hours to 4 days.
Macroscopic observations were performed to determine the
effects of the dyed oil contacting the gill surfaces and the means by
which the oil was either ingested or ejected. Definite patterns of
response to the dyed oil were established. Essentially, the clams
treat oil micelles and globules as food or detritus particles. The
smallest oil micelles are passed by ciliary currents directly to the
stomach. Larger globules are bound by mucus secreted by the gill
ctenidia. Gas chromatography and mass spectrometry confirmed the
binding of oil-mucus. The oil-mucus is ingested or rejected by means
of the clam ciliary pathways. Implications of the oil-mucus mecha-
nism and the ejection of this mucus into the environment are dis-
cussed.
INTRODUCTION
Oil spillage continues to be a serious problem in coastal and
estuarine waters. Many spills reported from barges, tankers, and
industrial installations involved fuel oils [8,2,32,22,20] . Spilled oil
may be altered by many factors including evaporation and photo-
oxidation. The water soluble portions of the oil, mostly aromatics
[5,21], will dissolve and be dispersed. Many of the petroleum
hydrocarbons may be dispersed throughout the water column by
formation of emulsions, either by turbulent wave action [13,12] or
chemical dispersion. Morris [23] documented the occurrence of oil
emulsions in the eastern Mediterranean. Some of the oil may be
adsorbed to particulate matter within the water column or mixed in
the sediment. Boehm and Quinn [4], experimenting with #2 Fuel
Oil, have demonstrated the existence of accomodated (particulate or
droplet), solubilized (colloidal micelles), and soluble hydrocarbon
components in fuel oil-seawater mixtures.
The solubilized petroleum hydrocarbons dispersed throughout
the water column and sediments may be ingested by benthic marine
filter feeders. These animals filter the water to obtajn planktonic
food, maintain respiration, and excrete wastes. Chemical analyses
have shown that various components of oils can accumulate within
marine bivalves [11,6,7,33,10].
Figure 1. Gas Chromatograms: (a) 8 ppm #2 fuel oil in hexane,
attenuation 1 X 128Mb) Carbon tetrachloride extract of clam mucus,
attenuation 10 X 1024
Number two fuel oil was chosen for study because it is com-
monly shipped in coastal waters, used in coastal industrial installa-
tions, and has already been involved in a well-documented spill [2].
The #2 fuel oil was supplied by the United States Environmental
Protection Agency, Industrial Waste Treatment Research Labora-
tory in Edison, New Jersey. Two temperatures, 4°C and 22°C, were
used to represent the winter and summer temperature regime found
in the New York region. The bulk of the study was performed at
4°C assuming that the winter with its accompanying wind and
463
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464
CONFERENCE ON PREVENTION AND CONTROL OF OIL POLLUTION
storms would be the most likely time for spills to occur. Oil was
added as an emulsion assuming that initially much of the spilled oil
is naturally emulsified through turbulent wave action. Subsequent
treatment by oil dispersants would increase emulsion formation.
Mya arenaria were chosen to study because they are a. commer-
cially valuable bivalve commonly found in the littoral and sublitto-
ral zones on the American eastern Atlantic coast from the coast of
Labrador to the region of Cape Hatteras, North Carolina, and on the
Pacific coast from Monterey, California, to Alaska. Mya occur on
the European coast from Norway to the Bay of Biscay, France, and
along the western Pacific coast from the Kamchatka Peninsula to
the southern regions of the Japanese Islands [16]. Young Mya with
a mean length of 25 mm [25,27] were utilized because young
bivalves tend to have higher respiratory and filtration rates than
older bivalves [28,29,19,9,35]. Young clams would therefore prob-
ably accumulate oil present in the water column faster than older
clams. They would also be prone to recurrent exposure to oil during
their maturation process with the concurrent possibility of continu-
ous low-level accumulation of petroleum hydrocarbons within their
tissues. A continuing re-exposure to oil can occur in cases where the
oil is mixed into the sediments and slowly released again. Farrington
and Quinn [ 11 ], Zafiriou [36], Blumer et al. [2], and Scarratt and
Zitko [31] have reported the occurrence of petroleum hydrocar-
bons from marine sediments. Blumer et al. [2] demonstrated that
oil-contaminated sediments can continue to release relatively unde-
graded oil for extended periods of time.
Numerous studies have reported the occurrence of petroleum-
derived hydrocarbons in bivalved molluscs, and several mechanisms
by which this may occur have been conjectured. This paper offers
experimental verification of a mechanism by which a commercially
important bivalve, Mya arenaria, can accumulate oil within its tis-
sues. The paper also shows the behavioral response of Mya arenaria
and the possible deleterious ecological side effects that result from
oil accumulation.
Methods and Materials
The mode of accumulation of #2 fuel oil and behavioral
response of Mya was followed by using an oil soluble dye, Oil Red
0. Adapting a procedure developed by Gruenfeld and Behm [14],
#2 fuel oil and Oil Red 0 (5 mg) were emulsified in 100 ml of
chilled tap water by ultrasonification to produce a stable 20,000
ppm dyed oil in water emulsion. This was diluted to the desired
concentrations for each test. After exposure to the dyed oil, each
clam was removed from the water and its left valve was removed.
Dyed oil accumulation was then followed by observations using a
binocular dissecting scope. A color photographic record was main-
tained of selected specimens.
A preliminary series of four experiments was run at different
exposure periods (3-24 h) and concentrations at 22°C to determine
procedures and patterns. In the first experiments, lab acclimated
clams were placed in specimen dishes with natural filtered seawater
(26%) or artificial Rila seawater mix (26%). The dyed oil emulsion
was added either by syringe when the clam was siphoning or by
adding the desired concentration to the water. In subsequent experi-
ments, clams were placed in 1,600 ml of Rila artificial seawater mix
(26%), and enough dyed oil emulsion was added to achieve the test
concentration. The salinity chosen for the tests was the salinity of
the water at the time the clams were collected. In those experiments
in which clams were kept in 1,600 ml seawater, aeration was pro-
vided through a 1 ml pipet with a moderate airflow.
In the experiments performed at 4°C, clams were kept in 1,600
ml of artificial seawater (26%) and exposed to concentrations of 50
ppm, 100 ppm, and 150 ppm dyed oil emulsion for periods of 1, 2,
3, and 4 days.
The dye was determined to be totally insoluble in mucus by
taking human mucus and mixing the raw dye in. The dye and mucus
remained insoluble. A drop of #2 fuel oil instantly solubilized both.
After mixing and standing for several minutes in seawater, the
mucus-oil-dye combination had the same appearance as seen in the
clams in the experiments.
Results
The dyed oil emulsions were very stable in the water column and
did not crack readily. Whenever the dyed oil emulsion cracked,
small red oil globules would appear on the water surface. Even when
some of the dyed oil emulsion accumulated on the surface, there
was still a visible red tint in the water column. This may have been
due to the solubilized (colloidal micelles) and soluble hydrocarbon
components in the dyed fuel oil emulsion-seawater mixture [4].
Because some of the dyed emulsion did crack with time, actual
exposure concentrations are relative only to time 0 when the emul-
sion was added. The Oil Red 0 (C.I. 26125) is a neutral dis-azo dye
which does not contain water solubilizing groups and hence is insol-
uble in aqueous media but is soluble in oils, fats, waxes, etc. [15].
The Oil Red 0 dye was totally insoluble in aerated and nonaerated
control mixtures of dye and artificial seawater and dye and
natural seawater. Earlier toxicity and ancillary experiments had
confirmed that the patterns of mucus secretion and behavior
response were elicited by the emulsified fuel oil. The dye had no
effect on the clam.
In preliminary experiment I at 22°C, seven clams (25-35 mm)
were used. Tests were run for 3 hours. All clams behaved similarly.
The dyed oil emulsion present in the water while the clam was
filtering resulted in periodic "coughing" and periodic rejection of
dye-oil in a mucus binding. This periodic coughing is a rapid con-
traction of the adductor muscles which forces water out the inhal-
ant siphon. It is a mechanism commonly used by bivalves to rid
themselves of accumulated detritus and pseudo feces.
Within 2 to 3 hours, two clams had dyed oil-mucus visible in the
stomach. One of these plus two others now had dyed oil-mucus in
the digestive diverticula and around the style. Most commonly, the
dyed oil was found bound in mucus near the palps. The behavior of
the dyed oil on the gills and palps was closely followed. It was noted
that the oil droplets and globules were passed to the edge of the
gills, bound in mucus as a food or detritus particle, and passed
towards the palps. Smaller oil droplets passed directly to the palps.
The oil usually followed the ciliary pathways outlined by Kellog
[18].
The results from preliminary experiments II and III are listed in
tables 1 and 2 respectively. In preliminary experiment IV at 22°C,
clams were exposed to 50 ppm and 100 ppm of dyed #2 fuel oil
emulsion for 24 hours. Large clams were used (40-50 mm), four per
concentration. At 50 ppm exposure, one clam accumulated dyed oil
and oil-dye-mucus in the stomach and diverticula. All had much
oil-dye-mucus on the mantle, gills, and some on the palps. At 100
ppm exposure, three died. One accumulated oil in the stomach and
diverticula, with oil-dye-mucus on gills and mantle.
The first accumulation experiment at 4°C was a 24-hour expo-
sure of clams (20-25mm) to the dyed-oil emulsion at concentrations
of 50 ppm and 100 ppm. Fourteen clams were used in the test at 50
ppm exposure. Four clams accumulated dyed oil in the stomach and
diverticula. Two clams had no oil visible and ten had dyed oil in
mucus on the gills, palps, mantle, and foot. Some dyed oil-mucus
exited through the pedal aperture. Seven clams were exposed to the
100 ppm concentration. Five clams accumulated dyed oil in the
stomach and digestive diverticula. All clams had oil-dye-mucus on
gills, mantle, and palps.
The results from accumulation experiment II at 4°C are listed in
table 3. In these experiments, clams were exposed to the dyed oil
emulsion for 1, 2, 3, and 4 days. Figure 2 illustrates the results of
accumulation experiment II at 4°C. Experimental problems pre-
cluded the measurement of the incorporation of the 150 ppm con-
centration of the dyed oil emulsion on day one.
Chemical confirmation of the oil being bound or adsorbed to
mucus is shown in figure 1. The data was obtained from a different
series of experiments in which Mya were exposed to sublethal con-
centrations of #2 fuel oil. Complete results from these experiments
will be published at a later date. The mucus extracted was secreted
by clams exposed to an initial concentration of 100 ppm emulsified
#2 fuel oil. Four weeks later some of the mucus was collected. It
had a gray-green flocculent appearance and formed flocculent
clumps on the surface and in the water column. A sample of water
and mucus was taken from the water column (there was no surface
-------
EFFECTS
465
Table 1. Preliminary experiment //-22°C, natural filtered
seawater, clams (20-25 mm) were placed in specimen dishes
and dyed oil added
Group 1 —66 ppm, exposure 5 hours (3 clams)
Much of the dyed oil was visibly bundled in mucus.
appearing as semisolid viscous globs on the surface. No oil
was readily visible in the gut. Most oil was bundled in
mucus near pedal and palp area or following general cili-
ary rejection pathways.
Group 2 — 83 ppm, exposure 5 hours (5 clams)
All clams had packaged th( ill-dye within mucus. This
oil-dye-mucus was always found on and around the palps.
A small amount was sometimes found on the gills.
Group 3 — 83 ppm, exposure 5 hours (kept anaerobic l'/i hours
before test by placing on wet towel to induce filtering
when placed in oil and water) (7 clams)
Oil-dye-mucus strings were near, on, and around the palps
in 5 clams. None had oil visible in the visceral mass; some
oil-dye-mucus was being shunted out the pedal aperture
and siphon in several clams.
Group 4 — 83 ppm, exposure 8 hours (kept anaerobic 6 hours before
test to induce filtering when placed in oil and water) (8
clams)
Three clams showed no trace of oil. One clam's digestive
diverticula were tinged red. Five clams had oil-dye-mucus
on, near, and around palps and siphon and very small
amounts in mantle rejection tracts.
Group 5-83 ppm, exposed 9 hours (kept anaerobic 6 hours before
test) (8 clams)
Two clams showed no trace of oil. The other 6 clams had
oil-dye bound in mucus near the palps and/or siphon. A
few had some in the mantle rejection tracts.
Group 6—Kept anaerobic l'/2 hours prior to test, placed in seawater,
and fed 2,000 ppm oil-dye emulsion from syringe when-
ever pumping for 8 hours (5 clams)
Four clams had accumulated oil in stomach. No oil was
visible in the digestive diverticula. Several clams had some
oil-dye bound in mucus near the palps or siphon.
Groups 7 & 8-Kept anaerobic 6 hours prior to test, then placed in
seawater and fed dye-oil emulsion by syringe for 9 hours.
Four clams were fed a 100 ppm emulsion. All accumu-
lated oil in the stomach and in mucus around palps and on
mantle. Four clams were fed a 2,500 ppm emulsion. Three
accumulated oil in stomach; all accumulated oil in mucus
on palps, near siphon, and some on gills and mantle.
slick) and filtered through no. 1 Whatman paper. Infrared analysis
of the water at the time of sampling indicated a concentration of
approximately 0.466 ppm oil in the water column. Solvents were
glass distilled and checked for impurities. All glassware was pre-
washed with solvents. Approximately 40 ml of mucus-water sample
was filtered and then washed with artificial seawater. The filter was
then extracted in an erlenmeyer flask with 50 ml of carbon tetra-
chloride with constant shaking for ten minutes. The sample was
then filtered through prewashed sodium sulfate and concentrated to
a volume of 0.15 ml under nitrogen. During this procedure, all
carbon tetrachloride evaporated. Gas chromatographic analysis was
done using a Perkin Elmer Model 900 gas chromatograph equipped
with a flame ionization detector and a six-foot stainless steel column
packed with 8% Dexsil on 80/100 mesh of Chromosorb W. Standard
operating conditions were as follows: carrier gas N2, 2 cc/min;
detector H2, 20 cc/min, air 40 cc/min; injector temperature 200°C,
detector temperature 300°C, manifold temperature 300°C; column
temperature 70°C with two-minute hold, programmed at an increase
of 8°C per minute to 300°C; injection sample 0.1 micro liter. The
mucus sample gas chromatographic tracing was quantitated gravi-
metrically by compaiison to the peak aiea of 16 micrograms of
octacosane dissolved in hexane. The calculated quantity of oil
derived from the mucus was 833 micrograms. The hydrocarbon con-
tent of the water was 4.66 ppm or 0.466 micrograms per milliliter.
Therefore, the most the hydrocarbon background of the water
Table 2. Preliminary experiment I1J-22°C, clams (25-34 mm)
placed in 1,600 ml Rila seawater mix (26%), 12-houi exposure
cone.: 25 ppm (6 clams), 50 ppm (6 clams), 100 ppm
(5 clams)-all in #2 fuel + dye
50 ppm (5 clams) in So. Lo. crude oil
25 ppm (#2 Fuel):
50 ppm (#2 Fuel):
100 ppm (#2 Fuel):
50 ppm (So. Lo. Crude):
Five clams had oil-dye and or dye-mucus
accumulated in the stomach and divertic-
ula. All had oil-dye wrapped in mucus
near and on palps, and some on mantle
and gills.
All clams had oil-dye and or oil-dye-
mucus accumulated in the stomach and
diverticula. In some the style tip was
slightly tinted. All had oil-dye-mucus on,
near, and around palps, some near foot
and on mantle, gills, and pedal aperature.
Two clams accumulated oil-dye in stom-
ach and diverticula, but no oil was visible
in three. All had large amounts of oil-dye-
mucus on gills, palps, mantle, siphon, and
pedal aperture.
All 5 clams accumulated dyed oil and
dyed oil-mucus in the stomach and diges-
tive diverticula. Only 2 clams had small
amounts of oil-dye-mucus visible on gills,
mantle, and pedal aperture.
could have contributed by adhering to the filter was i 8.64 micro-
grams, and the remainder was from the oil bound in mucus. Mass
spectrometric analysis of the extracted sample was provided by the
U.S. Environmental Protection Agency, Industrial Waste Treatment
Research Laboratory. Edison. New lersey. Analysis demonstrated
that the sample was predominantly dimethylnaphthalenes and tri-
methylnaphthalenes with paraffins, mainly in the C-14 and C-15
regions.
Discussion
When dyed oil initially contacts the gill surfaces, the beating of
the gill frontal cilia often disintegrates the film or globules. The
resultant small oil globules and micelles then pass along ciliary tracts
to the palps directly, or enwrap in mucus secreted by the gills. The
clams treat the oil globules and micelles as food or detritus particles.
The mucus covered particles will then either fall onto rejection
paths of the mantle or enter the esophagus and stomach. When the
dyed oil is bound in mucus it follows the ciliary pathways as out-
lined by Kellog [18]. The movement is similar to the general pat-
terns described for bivalves by Morton [24] and J^rgensen [17], If
the mucus strings accumulate on the gills and palps and fall onto the
mantle, they may be ejected out the siphon or pedal aperture. As oil
concentration and exposure time increases, production of mucus
binding the oil increases. This tends to accumulate in the anterior
portion of the clam around the palps causing the clams to increas-
ingly shunt the bulk of the rejecta out the pedal aperture.
Bernard [ 11 described particle sorting and labial palp function in
the Pacific oyster. Suspended particles, both organic and inorganic.
which impinge on the gill ctenidium are entrapped m mucus and
transported by the action of the frontal cilia. Stimulation of two or
more adjacent ctenidial filaments results in a copius secretion. The
ctenidial surfaces are normally covered with a thin watery fluid not
subject to ciliary movements. A definite band of mucus overlays the
frontal cilia. Tactile stimulation of the ctenidia results in the abun-
dant production of "rejection" mucus forming a sheet over the
ctenidium. The mucus masses are then carried to the free margins of
the ctenidia. Overstimulation of the filaments by particles wili cause
the release of rejectory mucus which is removed from the ctenidium
by muscular action or its inability to enter the food grooves. Much
of the mucus is carried to the palps. In the normal state the inner
surfaces of the palpi are always pressed together. The complicated
ciliation of the palps brings any large mucus masses to the free edges
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466
CONFERENCE ON PREVENTION AND CONTROL OF OIL POLLUTION
Table 3. Accumulation experiment 11-4°C, clams (20-25 mm)
exposed 1, 2, 3, and 4 days to #2 fuel oil-dye emulsion.
Cone.: 50 ppm, 100 ppm, and 150 ppm in 1600 ml Rila s.w. (26%)
Day 1
100 ppm: (6 c!ams)-Four clams accumulated dyed oil in stomach
and digestive diver ticula. One had no oil visible. All
others had varying degrees of oil-dye-mucus on palps,
gills, mantle, and in mantle rejection tracts near siphon
and pedal aperture.
Day 2
50 ppm: (6 clams)-Three clams accumulated dyed oil in the
stomach and digestive diverticula. All clams had varying
amounts of oil-dye-mucus on gills, palps, mantle, and
foot, and near siphon and pedal aperture, and in mantle
rejection tracts. There were many visual observations of
oil following ciliary pathways.
100 ppm: (6 clams)-Two clams had dyed oil and oil-dye-mucus in
stomach and diverticula. All had varying amounts of oil-
dye-mucus on gills, palps, mantle, and foot. Oil globules
in all clams followed standard ciliary pathways, [18].
Day 3
50 ppm: (6 clams)—Four clams accumulated dyed oil in the stom-
ach and diverticula. All had varying amounts of oil-dye-
mucus on gills, palps, mantle, and foot, and out siphon
and pedal aperture. There were many visual observations
of oil being bound in mucus and passing along ciliary
paths.
100 ppm: (6 clams)-Four clams accumulated dyed oil in stomach
and diverticula. Oil-dye-mucus observations were the
same as for 50 ppm, except more was present.
150 ppm: (6 clams)-Two clams accumulated dyed oil and oil-dye-
mucus in stomach and diverticula. All had varying
amounts of oil-dye-mucus on gills, palps, etc., as did 100
ppm and 50 ppm, except much more was present for 150
ppm. Many observations were made of oil-dye being
passed along ciliary tracts, wrapped in mucus, and pass-
ing to palps and into stomach or along rejection tracts.
Day 4
50 ppm: (6 clams)-All accumulated dyed oil and dye-oil-mucus in
either the stomach, digestive diverticula, or both. All had
varying amounts of oil-dye-mucus on gills, palps, mantle,
etc., as outlined. There were many visual observations of
oil following ciliary tracts and being enwrapped in
mucus.
100 ppm: (6 clams)-Four clams accumulated dyed oil and dye-oil-
mucus in stomach. All had much oil-dye-mucus passing
out the pedal aperture, near palps and siphon, and some
was on gills, mantle, and foot. There were many observa-
tions of oil being ensnared in mucus and passing along
ciliary paths.
150 ppm: (6 clams)-Four clams accumulated some dyed oil and
oil-dye-mucus in stomach, digestive diverticula, or both.
All were relatively unresponsive and slow. Much oil-dye-
mucus was in each clam on gills, mantle, and near siphon.
It was also passing out the pedal aperture, and it was near
the siphon and on visceral mass and foot. There were
many observations of oil following ciliary paths and
being ensnared in mucus.
of the palps for rejection. The mechanism is presumably the cause of
,oi]-dye-mucus commonly occurring in large masses on, near, and
around the palps of My a. When sufficient rejecta accumulate in the
palp region, the clam contracts the tip of the siphons and then
rapidly contracts the anterior adductor muscle forcing water and
rejecta out the pedal aperture. There also is a slow ciliary beat on
(he mantle expelling rejecta out the pedal aperture. This pattern of
rejection may not be a primary pathway in the field. When the clam
is burrowed in the sand mud substrate, it is extremely difficult to
Figure 2. Accumulation experiment
#2 fuel oil emulsion at4°C
Clams exposed to dyed
expel rejecta out the pedal aperture. The bulk of the rejecta accu-
mulates within the mantle cavity. If much rejecta accumulates, the
clam has to do more work to expel it out the inhalant siphon. In the
interim, the oil-dye-mucus may possibly release some of the oil
again. In essence, this would constitute an internal re-exposure to
oil. Other detrimental effects are the increased energy demand on
the clam for mucus secretion, ciliary beating, and muscular contrac-
tions and the synergistic effects of narcotization by the oil and
clogging of gills, palps and digestive tract. Concentrations of #2 fuel
oil above 100 ppm over a 24-hour or longer exposure cause an
increasingly greater degree of general narcotization with increasing
oil concentration.
Figure 3 illustrates the general ciliary pathways which dyed #2
fuel oil globules and micelles follow in Mya, as they are bound in
mucus. The oil enters through the inhalant siphon into the pallial
cavity. Here the oil follows pathways depicted where the mi-
celles pass to the edge of gill ctenidia to the marginal groove.
Ciliary currents then sweep the material along the marginal groove
towards the palps. Some of the rejecta passes slowly along mantle
rejection tracts towards the inhalant siphon and accumulates at the
base of the inhalant siphon. Occasionally, the clam contracts expel-
ling the dye-oil-mucus out the inhalant siphon.
Figure 3. General ciliary pathways which dyed #2 fuel oil droplets
follow as they are bound in mucus
Whether a clam will accumulate the dyed oil as dyed oil-mucus
in the stomach and diverticula seems to be related to prior feeding,
oil concentrations, length of exposure, and temperature. Figure 2
illustrates the results of accumulation experiment II at 4°C. At all
oil concentrations, 50 ppm, 100 ppm, and 150 ppm, there was a
general trend for an increasing number of clams to visibly accumu-
late and concentrate low concentrations of oil from the water col-
umn, since test concentrations in the water column were relative
-------
EFFECTS
467
only to time zero at the beginning of the test. The higher tempera-
ture, 22°C, seemed to accelerate the accumulation of dye-oil-mucus,
general narcotization, and death. The lower temperature, 4°C,
tended to enhance the stability of the oil emulsion and slow the
narcotization of the clam.
If the clam's gut is full of detritus and silt there is little visual
accumulation. The most rapid accumulation of oil seems to be cor-
related with a partially empty gut. When the dyed oil is in the
stomach, it may be in mucus fragments or as minute droplets. Occa-
sionally, the style tip may be tinted red. Next, the digestive divertic-
ula gradually become tinted from the stomach outward toward the
limit of the digestive diverticula where the tissue becomes undiffer-
endated gonad.
The ability of the clam to accumulate oil, concentrate it in
mucus, and release it, may have deleterious ecological side effects.
While the clam is accumulating oil within its own tissues, it may also
bind and concentrate any oil entering its pallial cavity. Some of this
is accumulated and some ejected from the clam as concentrated
oil-mucus. This may occur at high-oil concentrations even while the
clam is dying. At low-oil concentrations, the effect would be more
insidious. Filter-feeding bivalves must filter large volumes of water
to sustain their nourishment and respiration. Even very low concen-
trations of oil in the water column can be concentrated by the clam
in its tissues and mucus. Essentially, the clams serve as bioconcentra-
tors, accumulating oil in their tissues and rejecting some in the form
of concentrated oil-mucus. This situation is greatly aggravated in the
case of coastal spills when dispersants and sinking agents are used.
The effect of sinking oil or dispersing it is primarily cosmetic and
increases the potential for oil accumulation by filter and detritus
feeders.
The ability of the clam to accumulate and concentrate oil has
other side effects. Eventually, the effects of the accumulated oil can
cause death. The oil in the decaying clam flesh is deposited into the
sediments at depths of 2-14 centimeters. Several studies including
Blumer et al. [2], Blumer and Sass [3], and Farrington and Quinn
[11] have shown that oil may exist in sediments in an undegraded
state for extended periods of time with the potential for release into
the environment again. The release of oil-mucus rejecta by the clam
also has the potential for further contamination. The mucus strings
may be worked into the sediment or utilized as food by a variety of
detritus feeders and benthic scavengers, resulting in their contamina-
tion. Scavengers such as green crabs and brown shrimp are common
prey for fish. The clams themselves are a common food for many
food and game fish such as striped bass [26], black drum, and
flounder. Trevallion et al. [34] reported that the siphons of clam
were a major source of food for plaice. Many crabs, including those
used for human consumption and those utilized by marine predators
such as striped bass, also feed on clams [30]. By these means, low
concentrations of oil may rapidly be disseminated through the
marine sediments and food chains.
ACKNOWLEDGMENT
The analyses for this investigation were performed at the analyti-
cal facility of the U.S. Environmental Protection Agency (EPA),
Industrial Waste Treatment Research Laboratory in Edison, New
Jersey. Use of these facilities was provided by an EPA program that
supports graduate level research of the environment.
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2. Blumer, M.; Sass, J.; Souza, G.; Sanders, H.; Grassle, F.; and
Hampson, G. 1970. The West Falmouth oil spill. Tech. Kept.
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3. Blumer, M., and Sass, J. 1972. Oil pollution: persistence and
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6. Burns, K.A., and Teal, J.M. 1971. Hydrocarbon incorporation
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7. Cahnman, H.J., and Kuratsune,M. 1957. Determination of poly-
cyclic aromatic hydrocarbons in oysters collected in polluted
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8. Currie, A. 1974. Oil.pollution in the Cromarty Firth. Marine
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9. Dame, R.F. 1972. The ecological energies of growth, respiration
and assimilation in the intertidal oyster Crassostrea virginica.
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10. Ehrhardt, M. 1972. Petroleum hydrocarbons in oysters from
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ments and clams (Mercenaria mercenaria). Estuar. & Coastal
Mar. Sci. 1:71-79.
12. Forrester, W.D. 1971. Distribution of suspended oil particles
following the grounding of the tanker Arrow. J. Mar. Res.
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fuel oils in sea water. J. Fish. Res. Bd. Can. 30:1611-18.
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17. J^rgensen, C.B. 1966. Biology of suspension feeding. New
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18. Kellog, J.L. 1915. Ciliary mechanisms of lamellibranchs with
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20. Levy, E.M., and Walton, A. 1973. Dispersed and particulate
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distribution of oil pollutants in Halifax Harbor 10 June to 20
August 1971. fish. Res. Bd. Can. Tech. Rept. 284.
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plankton from the eastern Mediterranean. Marine Pollution
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24. Morton, J.E. 1968. Molluscs. London: Hutchinson & Co., Ltd.
25. Newcombe, C.L. 1936. Validity of concentric rings of Mya
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26. Nichols, P.R. 1966. The striped bass. U.S. Dept. of the Interior,
Fish & Wildlife Serv. Fish Leafl. 592.
27. Pfitzenmeycr, H.T. 1965. Annual cycle of gametogenesis of the
soft shell clam. Mya arenaria, at Solomon, Maryland. Chesa-
peake Sci. 6(l):52-59.
28. Prosser, C.L., and Brown. F.A., Jr. 1961. Comparative animal
physiology. 2nd ed. Philadelphia: W.B. Saunders Co.
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edulis L. and Branchidontes demissus plicatulus Lamarck as a
function of size and temperature. Comp. Biochem. Physiol.
7:89-101.
30. Rees, G.H. 1963. Edible crabs of the United States. U.S. Dept.
of the Interior, Fish & Wildlife Serv. Fish Leafl. 550.
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and benthic animals from shallow depths in Chedabucto Bay,
N.S.y. Fish. Res. Bd. Can. 29:1347-1350.
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CONFERENCE ON PREVENTION AND CONTROL OF OIL POLLUTION
32. Silver, R.R. 1974. Oil spill closes beach along 24 miles of North
Shore. N. Y. Times, July 18, 1974, p. 39.
33. Stegeman, J.J., and Teal, J.M. 1973. Accumulation, release and
retention of petroleum hydrocarbons by the oyster Crassos-
trea virginica.Marine Biology 22(l):37-44.
34. Trevallion, A.; Edwards, R.R.C.;and Steele, J.H. 1970. Dynam-
ics of a benthic bivalve. In Marine Food Chains, ed. J.H.
Steele, pp. 285-295. Edinburgh: Oliver & Boyd.
35. Walne, P.R. 1972. The influence of current speed, body size
and water temperature on the filtration rate of five species of
bivalves. /. Mar. Biol Assoc., U.K. 52:345-374.
36. Zafiriou, O.C. 1973. Petroleum hydrocarbons in Narragansett
Bay. II. Chemical and Isotopic Analysis. Estuar. & Coastal
Mar. Sci. 1:81-87.
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My subject is - The Ultrasonic Dispersion, Source Identification, and
Quantitative Analysis of Petroleum Oils In Water*
I would like to discuss three projects that are now underway in our
laboratory. We are developing methods for identifying and quantitating
small amounts of petroleum oils in water, and a procedure for preparing
stable oil in water dispersions containing known amounts of oil. The
identification method will correlate milligram and sub-milligram amounts
of water dispersed oils with undispersed oils, for the purpose of source
identification; the quantisation method will estimate part-per-billion
amounts of petroleum oils in water, in the presence of non-hydrocarbons;
and the oil in water dispersion method is needed by biologists for oil
toxicity evaluations and by chemists for evaluation of oil quantitation
methodology. Our work on these projects is still in progress and our
conclusions are therefore tentative. In fact, the present discussion
and paper deal only with the rather limited segments of each project that
have actually been completed.
Source identification of waterborne oils is accomplished in three
steps. The oils are first separated from water. The weathered and neat
oils to be compared are then submitted to distillation or to some other
vaporization procedure, which induces comparable volatility losses in
the oils. The non-volatiles are then compared by techniques such as gas
chromatography, infrared, and fluorescence spectroscopy. We used a gas
chromatographic procedure of the American Society for Testing and Materials
(ASTM) to compare the oils. But, we developed a procedure for inducing
volatility losses in milligram and sub-milligram amounts of oils, that
simulated volatility losses resulting from an ASTM distillation method
that requires approximately 40 gm quantities of oil. My present discus-
sion of oil identification is restricted to our vaporization procedure.
^Presented by Michael Gruenfeld, September 8, 1975, at the ICES Workshop
on Petroleum Hydrocarbons, in Aberdeen, Scotland. Mr. Gruenfeld is a
Supervisory Chemist with the U.S. Environmental Protection Agency,
Industrial Environmental Research Laboratory-Ci, Edison, NO 08817
-l-
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- 2
Oil quantitation is the second subject of my discussion. We are de-
veloping an IR spectroscopic method for estimating part-per-billion amounts
of petroleum oils in water, even in the presence of non-hydrocarbons. We
are evaluating as part of this method a rapid silica gel adsorption tech-
nique for separating petroleums from non-hydrocarbons, directly in carbon
tetrachloride solution. My present discussion of oil quantitation deals
only with this adsorption procedure.
Preparation of stable oil in water dispersions having a known oil con-
tent is my third subject for discussion. A commercial ultrasonification
device is used to prepare oil in water concentrates that readily mix fur-
ther with water. We also checked whether ultrasonification causes spectral
or chromatographic changes in oils.
My discussion therefore emphasizes the procedures and results of the
oil vaporization, silica gel adsorption, and ultrasonic dispersion techni-
ques.
We evaluated vaporization techniques by preparing oil in water disper-
sions and then extracting them with Freon 113. Dilutions were then made
to yield solutions containing 0.5 or 30 mg oil. (Slide 1) These were trans
ferred to 150 ml beakers and stripped to 1-2 ml, with a steam table and a
filtered air stream. (Slide 2) The concentrates were then transferred to
small glass vials (10 x 30 mm). Vials containing 30 mg oil were sus-
pended in water at 40 C. The filtered air stream was used to remove
final solvent traces, and this condition was maintained for 10 additional
minutes. Vials containing 0.5 mg oil were maintained at room tempera-
ture, and the air was turned off just before total solvent removal. Final
evaporation then occurred spontaneously. These vials remained open at
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- 3
room temperature for 10 additional minutes. Finally, small amounts of car-
bon tetrachloride (10 - 20 ill) were added to each vial for gas chromato-
graphic injection.
The neat reference oils were treated somewhat differently. Seventy
milligram oil was added directly to the vials, which were then suspended
In water for 15 minutes, at 40°C, while exposed to the air stream. The
oils were then injected onto the gas chromatographic instrument without
further dilution.
, . „ vaporization , . T n ,. ,
(Slide 3) Our techniques, as I previously mentioned, in-
duced losses in small amounts of oil that approximate losses induced by an
ASTM distillation procedure for large amounts of oil. This slide illustrates
the results of the ASTM procedure. The upper chromatogram is South Louisiana
Crude oil before distillation; the lower chromatogram results from use of
the ASTM distillation. The numbers refer to n-alkanes, while pristane and
phytane are abbreviated as Pr and Ph. The ASTM procedure causes major loss
of components below the C-,-, n-alkane. (Slide 4) Our procedure for inducing
vaporization losses in 70 mg neat oils yields similar results. The upper
chromatogram is South Louisiana oil after ASTM distillation; the lower
chromatogram is the neat oil after our procedure. (Slide 5) The lower chro-
matogram here results from our treatment of 30 mg oil that was extracted
from water. The upper chromatogram is the 70 mg portion of neat oil.
(Slide 6) The upper chromatogram here is again the 70 mg portion of neat
oil; the lower chromatogram results from our treatment of 0.5 mg oil that
was extracted from water. The lower chromatogram shows evidence of gas
chromatographic column degeneration, but adequate resolution of pristane
and phytane from the normal alkanes was achieved with a new column. I
should emphasize that although oils that actually weathered in the water
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- 4 -
column have not yet been evaluated, our techniques are usable for identi-
fying small amounts of oil that occur as slicks and shoreline residues.
Next I will discuss the silica gel adsorption technique that was
evaluated in the oil quantitation project. Silica gel was tested for sep-
arating petroleum oils from animal and vegetable oils directly in carbon
tetrachloride solution. We contrasted the sorption capacity of silica gel
with its solution contact time, and degree of activation. Carbon tetra-
chloride solutions of two petroleum oils at 20 mg/100 ml, and vegetable,
olive and cod liver oils at 100 mg/100 ml were prepared in 100 ml volumet-
ric flasks. 3.0 gram portions of activated and partly deactivated silica
gel were added to the flasks. (Slide 7) The solutions were vigorously
stirred for five and ten minute intervals with a magnetic stirrer. (Slide
8) A plot was prepared contrasting oil removal, with silica gel deactivation
and solution stirring time. Maximum separation of petroleum from non-
petroleum oils resulted from fully activated silica gel and ten minute
stirring. No loss of petroleum oils occurred. Decreased activation, and
reduced stirring yielded adverse results.
I will now briefly describe the preparation of oil in water dispersions
[Slide 9) The probe of an ultrasonic instrument was inserted into a 50 ml
graduated cyclinder containing water and an accurately weighed amount of
oil. Maximum dispersion energy was applied for two minutes, while keeping
the cylinder in an ice bath. Cooling prevented oil vapor losses during
ultrasonification, which causes substantial heating. Dispersion stability
diminished with increasing oil viscosity. A non-viscous crude oil yielded
stable 1% (10,000 PPM) dispersions that were stable for days; but a more
viscous oil yielded less stable and less concentrated dispersions. This
technique permitted oil recoveries in the range 96 - 100%. A check for
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- 5 -
ultrasonically induced changes showed no substansive chromatographic or
spectral alterations of the oils.
In closing, I would like to repeat that this discussion and paper
deal with only limited portions of three projects. Our work is still
underway, and we hope to provide final results in the near future.
THANK YOU -
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Slide 1
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Slide 2
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Slide 3
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Slide 4
-------
ETENTION TIME (min.
Slide 5
-------
30 "^RETENTION TIM
Slide 6
-------
Slide 7
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110 r-
100$
A
A
i 90
Key
A South Louisiana Crude Oil
A No.2 Fuel Oil
O Vegetable Oil 10 min. stirring
• Vegetable Oil 5 min. stirring
• Cod Liver Oil 10 min. stirring
O Olive Oil 10 min.
3 5
PERCENT DEACTIVATION
Slide 8
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Slide 9
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The Effect of a No. 2 Fuel Oil and a South
Louisiana Crude Oil on the Behavior of the Soft
Shell Clam, My a arenaria L.
Dennis M. Stainken
Rutgers University
Dept. Zoo/. & Physiology
Newark, N.J.
Present Address: U.S. Environmental Protection Agency
Industrial Environmental Research Laboratory-Ci
Oil & Hazardous Materials Spills Branch
Edison, N.J. 08317
Introduction
The toxic effects other than lethality of oils has often
been treated as a secondary problem in bioassays. According to
STIRLING (1975), there is a need to enlarge the concept of the
routine bioassay test to include quantitative measurements of the
effects of pollutants on behavior, physiology and metabolism.
The inherent interspecific and intraspecific physiological dif-
ferences of test species and various test conditions make direct
comparison of bioassay results difficult. VAUGHAN (1973) and
BEAN, et_ a]^. (1974) discussed these problems in detail.
Few studies have characterized the behavioral effects of
oils on bivalves or examined the effects of temperature simultan-
eously. WILSON (1974) noted that bivalve molluscs have been
avoided for toxicity tests because it is difficult to establish
a simple criteria of effect. This study was therefore performed
to determine the behavioral effects of a No. 2 fuel oil and a
South Louisiana crude oil in bioassay tests conducted at winter
temperatures. The winter temperatures were chosen because spills
are more likely to occur during the inclement winter weather.
The oils were added in an emulsified form to simulate a po-
tential naturally occurring condition. FORRESTER (1971), FOSTER,
et_ al_. (1971), GORDON, et_ a^. (1973) and RANTER (1974) have re-
corded the formation of oil emulsions in sea water by various
mechanisms. In the event of an oil spill during the colder
months, it is probable that much of the oil would be dispersed
and emulsified in the water column through turbulent wave action.
Many of these emulsions tend to be relatively stable and a mech-
ansim for the accumulation of emulsified oil by bivalves has
been reported (STAINKEN, 1975).
Materials and Method
Behavioral observations were recorded during bioassay tests.
The tactile responses of the clams were examined by lightly tap-
ping the shell with a glass stirring rod. Tests were conducted
according to the "Standard Dispersant Effectiveness and Toxicity
Tests" published by the U.S. Environmental Protection Agency
(MCCARTHY, 1973). An experimental concentration at which 50% of
the experimental animals survived (LC5g) was determined during a
96 hr. exposure period. The method employed requires the use of
a standard toxicant. Benzene was used initially but in subse-
quent tests phenol was utilized because benzene emulsions were
not stable.
724
Bulletin of Environmental Contamination & Toxicology,
Vol. 16, No. 6 © 1976 by Springer-Verlag New York Inc.
-------
Oil-in-water emulsions were ultrasonically prepared accord-
ing to a procedure developed by GRUENFELD and BEHM (1973).
Twenty thousand parts per million of Southern Louisiana Crude
oil and No. 2 fuel oil were prepared and dilutions were made to
achieve desired concentrations. The No. 2 fuel oil and Southern
Louisiana were supplied by the U.S. Environmental Protection
Agency, Industrial Waste Treatment Research Laboratory, Edison,
N.J. The fuel oil was composed of 14% aromatics and 86% non-
aromatics. Benzene was also sonified to yield a stock emulsion
of 10,000 ppm. A 10,000 ppm stock solution of phenol was pre-
pared.
All toxicants except benzene were used at 4°C and 14 C.
Benzene was tested at 14°C. The oils were added at concentra-
tions from 50-800 ppm. The test concentrations of the oils
were later increased to 1,600 ppm.
Clams for the experiments were collected from the Princes
Bay - Sequine Point Area of Staten Island, N.Y. from December,
1973 - February, 1974. Clams were collected at water tempera-
tures and salinity closely approximating test conditions. Young
clams with a mean shell length of 25 mm or less were utilized.
Prior to each experiment, clams were acclimated 24-48 hours in
1600 ml of artificial sea water (salinity - 26%0). Fifteen clams
(15-24 mm) were used at each oil concentration. Five clams were
placed in each aerated container. Five clams per container were
also used for standard toxicant testing (benzene or phenol) at
each concentration.
Results
Behavioural observations were made in each LC5Q test. A
noticeable reaction was not observed in clams exposed to benzene.
The clams merely contracted upon tactile stimulation.
Clams exhibited an identical response pattern to oil exposure
in all tests. Initially, at low oil concentrations (50 ppm)
mucus was given off by the clam out the pedal opening and siphon.
Higher oil concentrations resulted in proportionally higher mucus
secretion. As more mucus was secreted, the clams increasingly
shunted more out the pedal opening. All effects appeared to be
both time and dose related, although crude oil effects were never
as severe as the effect of fuel oil. Both oils depressed muscular
contraction. A decrease in irritability and contractibility of
the siphon was noticeable at 50 ppm. At concentrations greater
than 100 ppm, the pedal opening musculature rapidly became to-
tally relaxed and did not contract . The adductor muscles simul-
taneously lost the ability to contract rapidly. At concentra-
tions greater than 400 ppm, the adductors began to relax in less
than 15-20 sec and the animal consequently 'gaped'. It appeared
that the anterior adductor was affected first, then the posterior.
725
-------
The general relaxation of the musculature (adductors, siphon,
pedal aperture) occurred at all oil concentrations. The inten-
sity varied with time and dose.
The large amounts of mucus secreted at the 400 ppm con-
centrations appeared to be clogging the cavity between the valves
by the end of the tests. The clams also appeared too weak to
expel the mucus.
The response pattern of mucus secretion and muscular narcoti-
zation occurred in all tests with oil and seemed to be enhanced
at 14°C compared to responses at 4°C.
The response of clams to phenol was not completely similar to
that of oil. Mucus secretions were never as heavy in response to
phenol, but phenol appeared to narcotize the musculature quicker.
Phenol exposed clams reacted differently than oil exposed clams.
The muscles remained at one length, turgid and lost their irrit-
ability. Adductor muscles remained half or sometimes fully con-
tracted and the pedal aperture remained partially open. The
muscles of the phenol exposed group rapidly became turgid at death.
The' tactile response of control clams remained normal during the
experiments and mucus secretion was not evident.
The static bioassay data derived from the tests were inconclu-
sive. The LCcjQ values were computed on semilog paper. Results
of the tests are in Table I. Tests numbers 1A and IB were run in
natural filtered sea water to determine whether testing in natural
sea water or artificial sea water would have an effect. An ef-
fect was not found and all other tests were conducted in arti-
ficial sea water. Scattered mortality was observed in the major-
ity of tests but in most cases there was insufficient mortality
after 96 hours to calculate a LC^Q. Mortality was not found in
the controls. Death was defined as a total lack of muscle re-
sponse.
At 14°C, two 96 hour LC^g values for No. 2 fuel were obtained.
These were 475 ppm (Test HA) and 535 ppm (Test IIIA). Compari-
son by a t test revealed no significant difference and a mean
LCjg value of 505 ppm was calculated. Some of the tests were
continued 3 days beyond the 96 hour period, to see if there was
a time effect. At 14°C, test IIIA and HIP were continued. An
LC5Q (7 day) was found for No. 2 fuel oil to be less than 100
ppm, compared to test IIIA 1X59 (96 hour) of 535 ppm. The LCjQ
(7 day) of phenol in test IIIB dropped to 535 ppm.
At 4°C, test IVA, IVP, VIA and VIP were also continued for
3 days (total 7 days exposure). In test IVA, a LC$Q could not
be found. In test IVP, the 7 day semilog plot had the shape of
a backwards 'S' and two values were derived, 80 and 225 ppm
phenol. In test VIP, the LC5Q (7 day) for phenol was 450 ppm.
It is probable that more mortality would have been encountered
if the tests were continued beyond seven days.
726
-------
TABLE I. Calculated toxicity (LC ) during a 96 hour exposure
period.
14°C
(IA) So. Louisiana Crude
cone: 50,100,200,4'00,800 ppm
LC5Q = none
(IIA) i2 Fuel Oil
cone: 50,100,200,400,800 ppm
LCcjg = 475 ppm
(IIIA) //2 Fuel Oil
cone: 100,200,400,800,1600 ppm
LC5Q = 535 ppm
(IB) Benzene
10,20,30,40,50 ppm
LC__ = none
(IIB) Benzene
50,60,70,80,90,100 ppm
LC^Q = none
(HIP) Phenol
50,100,200,400,800 ppm
LC5Q = 565 ppm
4°C
(IVA) So. Louisiana Crude
cone: 100,200,400,800,1600 ppm
LC;-,-, = none
(VA) //2 Fuel Oil
cone: 50,100,200,400,800 ppm
LC... = none
(VIA) //2 Fuel Oil
cone: 100,200,400,800,1600 ppm
LC_ = none
(IVP) Phenol
50,100,200,400,800 ppm
cn
= 365 ppm
(VP) Phenol
10,20,30,40,50 ppm
LC_. = none
(VIP) Phenol
50,100,200,400,800 ppm
LC5Q = 535 ppm
The Southern Louisiana Crude oil appeared to have a few
acute toxic effects within the test parameters.
In the tests with No. 2 fuel oil, there seemed to be a
temperature effect. At 4°C, a LC5Q was not found in any tests
(except VIA, 7 days), while at 14°C a mean LC5Q of 505 ppm was
found in the two tests with No. 2 fuel oil. A temperature
effect was not apparent in clams exposed to phenol.
727
-------
Discussion
The behaviour effects found for M. arenaria were repeat -
able for both crude and refined oil. The increasingly greater
concentrations of oil elicited greater mucus secretion and de-
creased tactile response. SWEDMARK, e_t al_. (1973) reported the
effect of crude oil and oil emulsions on bivalves and found a
similar decrease in tactile response. The general behavior se-
quence they reported was: increased activity; successively im-
ppaired activity; immobilization and death. The observations of
this report are similar to their observations.
The copious production of mucus by M. arenaria had several
effects. It imposed a steady drain on the energy reserves of
the clams. The continual production of mucus clogged the gills
and mantle cavity and would disrupt normal feeding mechanisms.
The clogging mucus must be expelled by the clam by increased con-
traction of the adductors and mantle musculature which puts an
additional strain on the metabolic rate. The increased metabolic
demands for mucus production and excretion and the disruption of
normal physiological and biochemical processes occurred at much
lower concentrations of oil exposure than the LC^g indicates.
A problem encountered in comparing the results of this study
was the lack of definition of death in bivalves in published re-
ports. Terms such as "moribund" are found in the literature but
the criteria of death was not defined. The definition of death
in this report was the total lack of muscular response, though
this was often difficult to determine. Stimulation of the mus-
culature by pinching or light rapping with a stirring rod often
elicited muscular twitches though the clam appeared "dead".
The bioassay LC.-Q values obtained for M. arenaria are
greater in concentration (ppm) than those reported for other
species exposed to No. 2 fuel oil (VAUGHAN, 1973; ANDERSON,
et_ ajL_. , 1974). However, the values are lesser than those re-
ported for many species exposed to crude oils. According to
the ranking system of SPRAGUE and CARSON (1970), the No. 2 fuel
tested in this report can be classified as moderately toxic to
M. arenaria. An additional factor noted in the bioassay test
was the time of exposure. Increasing the period of exposure from
96 hours to seven days decreased the LC5Q values obtained.
Similar increases in mortality of molluscs during longer exposure
periods were reported by SPRAGUE and CARSON (1970), KANTER et al.,
(1971) and KASYMOV and ALIEV (1973).
Future bioassay work with molluscs, particularly bivalves,
should be determined over a 7 day exposure period. However, be-
havioral observations appear to have advantages as a toxic
criterion over lethality, and shorter exposure periods can be
employed.
728
-------
Acknowledgement
The analyses for this investigation were performed at the
analytical facility of the U.S. Environmental Protection Agency
(EPA), Industrial Waste Treatment Research Laboratory in Edison,
New Jersey. Use of these facilities was provided by an EPA pro-
gram that supports graduate level research of the environment.
References
ANDERSON, J.W., J.M. NEFF, B.A. COX, H.E. TATEM and
G.M. HIGHTOWER: Mar. Biol. 27_, 75 (1974).
BEAN, R.M., J.R. VANDERHORST, and P. WILKINSON: Interdisciplinary
study of the toxicity of petroleum to marine organisms.
Battelle, Pacific Northwest Laboratories, Richland,
Washington (1974).
FORRESTER, W.D.: J. of Mar. Res. 29_:2, 151 (1971).
FOSTER, M., A.C. CHARTERS, and M. NEUSHUL: Environ. Pollut. _2_, 97
(1971).
GORDON, D.C. Jr. P.O. REISER, and N.J. PROUSE: J. Fish. Res. Bd.
Can. 3Q_, 1611 (1973).
GRUENFELD, M. and F. BEHM: Anal. Quality Contr. Newsl, U.S.E.P.A.
16_, 6 (1973).
KANTER, R., D. STRAUGHAN, and W.N. JESSEE: Proc. Joint Conf.
Prevent. & Contr. Oil Spills, Wash., D.C., A.P.I, 485 (1971).
KANTER, R.: U. So. Cal., Sea Grant Prog. Publ. No. USC-SG-4-74
(1974).
KASYMOV, A.G. and A.D. ALIEV: Water, Air & Soil Pollut. _2_, 235
(1973).
MCCARTHY, L.T. jr., i. WILDER, and j.s. DORRLER: U.S.E.P.A. Publ.
No. EPA-R2-73-201 (1973).
SPRAGUE, J.B. and W.G. CARSON: Fish. Res. Bd. Can. Tech. Rept.
201 (1970).
SWEDMARK, M. , A. GRANMO, and S.KOLLBERG: Water Res. ]_, 1649 (1973)
STAINKEN, D.M.: Proc. Joint Conf. Prevent. & Contr. Oil Spills,
San Francisco, Calif., A.P.I. (1975).
STIRLING, E.A.: Mar. Pollut. Bull. 6_:8, 122 (1975).
WILSON, K.W.: In: Ecological aspects of toxicity testing of oils
and dispersants. (ed. L.R. Beynon, E.B. Cowell), John Wiley
& Sons, New York. 11 (1974).
VAUGHAN, B.E.: A.P.I. Pub. No. 4191 (1973).
729
-------
A Descriptive Evaluation of the Effects
of No. 2 Fuel Oil on the Tissues of
the Soft Shell Clam, Mya arenaria L.
Dennis M. Stainken
U.S. Environmental Protection Agency
Industrial Environmental Research Laboratory-Ci
Oil & Hazardous Materials Spills Branch
Edison, N.J. 08817
Introduction
Bivalve molluscs have been found to accumulate petroleum and
petrochemical derivatives. BLUMER, e_t^ al_ (1970) described hydro-
carbon uptake by oysters and scallops following a fuel oil spill.
ZITKO (1971), LEE, £t al. (1972), STEGEMAN and TEAL (1973),
VAUGHAN (1973), and NEFF and ANDERSON (1975) have reported the
occurrence and uptake of petroleum hydrocarbons by bivalve mol-
luscs. A mechanism by which soft shell clams may accumulate oil
was described by STAINKEN (1975).
The effects of petroleum oils and derivatives on bivalves
are varied. Some of the effects reported have described altera-
tions in oxygen consumption, carbon budgets, larval development,
behavior, filtration rates, mortality and biochemical effects.
The histological effects of petrochemicals are also varied.
Deleterious effects of oil on bivalve tissue structure have been
reported by LAROCHE (1972), CLARK, et_ ajL. (1974) and GARDNER,
e_t _al. (1975). Histological aberrations in bivalves have been
reported by BARRY, &t_ al. (1971), JEFFRIES (1972), and BARRY and
YEVICH (1975). The aberrations were believed to be due to pollu-
tion effects. In contrast, VAUGHAN (1973) found few effects of
oils on bivalves.
Reports on the effects of petroleum oils are often conflict-
ing. Some of the studies were field studies and exposure concen-
trations were unknown. The reported effects of petroleum oil
exposure have ranged from extensive to relatively none. Bivalves
in the environment are frequently exposed to single spill or
chronic discharges. This study was therefore performed to exper-
imentally determine the effects of subacute concentrations of
No. 2 fuel oil on the soft shell clam.
A No. 2 fuel oil was chosen for study because it is commonly
shipped in coastal waters, used in coastal industrial installa-
tions, and has already been involved in a well documented spill
(BLUMER, e_t_ al. 1970) . A winter temperature (A°C) was chosen
because spills are more likely to occur during the inclement
winter weather. In the event of a spill during the colder months,
it is probable that much of the oil would be dispersed and emulsi-
fied in the water column through turbulent wave action. The
clams were therefore exposed 28 days to oil initially added in an
emulsified form to simulate a potential naturally occurring condi-
tion of chronic exposure.
730
Bulletin of Environmental Contamination & Toxicology,
Vol. 16, No. 6 © 1976 by Springer-Verlag New York Inc.
-------
Materials and Method
The No. 2 fuel oil was supplied by the U. S. Environmental
Protection Agency, Industrial Waste Treatment Laboratory,
Edison, N. J. The specific gravity of the oil was 2.40 centi-
stokes. The oil was composed of 14% aromatics and 86% nonaroma-
tics according to ASTM method No. D2549-68. Oil-in-water emul-
sions were ultrasonically prepared according to a procedure
developed by GRUENFELD and BEHM (1973).
Clams for the experiments were collected from Sequine Point,
Staten Island, N. Y. Young clams with a mean shell length of
25 mm were utilized because young bivalves tend to have greater
filtration rates than those of older bivalves. The clams were
acclimated to the experimental conditions for a duration of 6
days before the emulsions were added.
An exposure period of 28 days to No. 2 fuel oil emulsions
having concentrations of 10, 50 and 100 ppm was utilized. Four
20 gallon aquaria containing 60 liters of filtered sea water/aquaria
(salinity = 20%0)were employed. The sea water was collected from
Sandy Hook Bay and filtered through a coarse plankton net. One
aquaria served as a control and each of the remaining aquaria
received either 10, 50 or 100 ppm of oil emulsion. The water was
continuously aerated and the temperature was maintained at 4 C.
Sampling for hydrocarbon content of water and clams was performed
every 7 days. The hydrocarbon content of the water was determined
by the method of GRUENFELD (1972). Complete results from these
experiments will be published at a later date.
At the beginning of the experiment, just before addition of
the emulsions (Time 0), five clams were fixed in Davidson's fixa-
tive (SHAW and BATTLE, 1957). At the end of the 28 day exposure
period, ten clams from each concentration and the control tank
were removed for histological examination. The clams sampled
represented 10% of the experimental population. Five of the ten
clams randomly removed from each concentration were fixed in
Davidson's fixative and five were fixed in cold 10% acetate buf-
fered neutral formalin, pH 7. The two fixatives were employed to
determine whether alterations in tissue structure were fixation
artifacts. The results indicated that artifacts did not occur.
All fixation was done in a refrigerator. After 24 hours,
clams fixed in Davidson's were transferred to cold 70% ethanol.
All fixed material was stored in a refrigerator until further
processing. Prior to embedding, the formalin fixed animals were
washed 14 hours in running tap water. All tissues were dehydrated
and brought to paraffin utilizing an Autotechnicon. Final infil-
tration was accomplished employing a vacuum infiltrator for 15
minutes at 13-15 inches Hg. The paraffin embedded clams were
oriented to cut beginning at the anterior pedal opening. Serial
cross sections were cut at 6 and 7 microns to the level of the
731
-------
heart and kidney. Sections were mounted according to the procedure
of LUNA (1968). The sections were stained with Harris-Lillie
hematoxylin (Fisher Scientific) and 0.5% Eosin Y in 95% ethanol.
Several cross sections of the visceral mass, stomach and
pallium of each clam from each experimental group were stained
for mucosubstances and necrotic tissue. Mucosubstances were
stained using the aldehyde fuchsin-alcian blue method of LUNA
(1968) and azure A/eosin B was employed for necrotic tissues
(GRIMSTONE and SKAER, 1972).
One section of the visceral mass and stomach from each clam
from each experimental group was stained by a modification of
McManus's method for glycogen, the Periodic Acid - Schiff reaction
or PAS (LUNA, 1968). The modifications were made from procedures
described by LUNA (1968) and LILLIE (1965). To check the speci-
ficity of the PAS reaction for glycogen, sections were treated in
an amylase solution (Diastase, Sigma chemical Co.) at a concentra-
tion of 0.1 g/100 ml in distilled water, pH 6.8 for one hour.
Results
Throughout the oil exposure period, all clams in the control
and oil exposed groups seemed to remain in good condition. After
the addition of the oil emulsions, the concentrations of oil in
the water column decreased rapidly. The actual hydrocarbon con-
centrations are listed in Table 1.
Table 1. Hydrocarbon concentration (ppm) in the water column
during the 28 day exposure period.
Tank // Time 0* Week 1 Week 2 Week 3 Week 4
Control
10
50
100
ppm
ppm
ppm
1
2
3
4
0
4.
43.
60.
5
72 •
71
0
1.31
1.04
1.52
0
0.
0.
0.
56
71
78
0
0.
0.
0.
37
37
32
0
0
0.
0.
29
46
* The Time 0 sample measurement was made two hours after
addition of the emulsified oil.
Several factors were probably responsible for the gradual deple-
tion of oil from the water. Much of the oil was apparently
removed from the water column by the mucociliary feeding and
ejection mechanisms of the clams. Large masses of mucus were
732
-------
ejected from the clams and were accumulated on the cooling coils.
Subsequent chemical analysis revealed a large content of oil in
the mucus. After 28 days, a sample of the mucus from the 100 ppm
aquaria was found to contain 833 micrograms of hydrocarbons.
Mass spectrometric analysis demonstrated that these hydrocarbons
were mostly dimethyl and trimethyl naphthalenes and paraffins in
the C-14 and C-15 regions (STAINKEN, 1975).
Harris's Hematoxylin was used to examine the general mor-
phology of clams. Radical tissue aberrations were not observed.
A gradation of tissue effects was apparent. The clams exposed to
100 ppm exhibited the largest number of anomalies from the con-
trols. The pallial muscle appeared edematous in four clams,
similar to that described by PAULEY and SPARKS (1965, 1966).
There were more leukocytes in the pallial blood sinuses of 100 ppm
exposed clams than controls, with occasional leukocyte nests as
described by PAULEY and CHENG (1968) occurring in the pallial
blood sinuses. The leukocytes often formed a band underlying the
mantle epithelium similar to that illustrated by DES VOIGNE and
SPARKS (1968). In some clams, the anterior adductor muscle was
midly edematous and infiltrated with leukocytes. The area between
the mantle membrane and the cell layer next to the shell was also
edematous and contained many leukocytes in some clams. In one
clam, they formed a plug in the hemocoel of part of the foot. In
seven clams, the style sac, intestine and diverticula appeared
very vacuolar and the diverticula appeared much reduced in size.
There was a small loss of chromatophilic material at the top of
the gill filaments in several clams exposed to 100 ppm oil.
At 50 ppm, the effects were less marked, except in the di-
verticula and intestine. The diverticula of seven clams were
shrunken in size. The diverticula epithelium appeared almost
cuboidal instead of the normal columnar epithelium. Portions of
the intestinal mucosa appeared to be sloughing into the lumen
which was not prevalent in the controls. The intestine, style sac
and diverticula were abnormally vacuolar in appearance. In one
clam, a few diverticula near the gonad appeared necrotic and
undergoing resorbtion. The pallial muscle was edematous in one
clam. Several clams had more leukocytes and leukocyte nests in
the blood sinuses below the inner pallial epithelium than the
controls. Only one clam had more than normal leukocytes and
leukocyte nests between the mantle epithelium and the cell layer
next to the shell. In one clam, a portion of the gill had a
pavement of leukocytes along the lining of the blood sinuses next
to the central water tube.
The 10 ppm clams showed fewer effects of oil exposure than the
100 or 50 ppm clams. The diverticula were reduced in size and the
diverticula, stomach and intestine were vacuolated in appearance.
In two clams, a moderate number of leukocytes were observable in
the pallial blood sinuses.
733
-------
Cross sections of the clam visceral mass and pallium were stained
with azure A/eosin B to demonstrate necrotic tissues. Major his-
tological differences were not found between controls and oil
exposed clams. The general effect of holding the clams for four
weeks was an increase in vacuolization of the digestive diverticu-
lar and intestinal cells. The vacuolization was present in all
groups. The diverticular cells of the Time 0 clams were distinct
and contained few vacuoles. After four weeks, the diverticular
cells of all clams appeared to contain many vacuoles and the cell
membranes were often indistinct. However, this effect was exacer-
bated in the oil exposed clams compared to controls (Figure 1-3).
Figure 1. Section of the digestive diverticula. lOOx. Stained
Azure A/eoson B. Time 0.
734
-------
Figure 2. Section of the digestive diverticula. lOOx. Stained
Azure A/eosin B. Control.
Figure 3. Section of the digestive diverticula. lOOx. Stained
Azure A/eosin B. 100 ppm exposed.
735
-------
An aldehyde Fuchsin - Alcian Blue 8GX stain was used to stain
mucosubstances. Histological differences were not found between
exposed and control groups along the intestines and the periphery
of the visceral mass. There was a decrease in mucoid cells and
staining intensities from two of the 100 ,ppm clams. The same
effect occurred in one 50 ppm clam.
Clam sections were stained for glycogen according to the PAS
technique. There was a gradation of effects of oil exposure in
the digestive diverticular and intestinal cells. The 100 ppm clams
had the least amount of PAS positive material (glycogen). Six of
the 100 ppm clams had observable differences from the control
clams. Generally, the diverticular cells decreased in size and
contained much less PAS positive material. The diverticular cells
of all 100 ppm clams appeared very vacuolar with few glycogen
deposits. Several cells almost appeared amylase treated. The
intestinal cells and basement membranes also contained less PAS
positive material. Most of the diverticular cells appeared to be
devoid of cytoplasm. The stomach mucosa of two of the 100 ppm
clams contained less PAS positive material than did the controls.
The gill filament tips and margins appeared to have a decrease in
PAS positive material.
Generally, a pattern of cellular glycogen depletion was ob-
served in 50 ppm clams similar to that described for 100 ppm clams.
The diverticular cells were reduced in size, vacuolar in appearance
and contained less PAS positive material than did the controls.
The cells also appeared to be depleted of cytoplasm. The intestin-
al mucosa appeared to be sloughed into the lumen. The stomach
mucosa of the 50 ppm clams also showed a depletion of PAS positive
material as compared to controls.
Most digestive cells in the 10 ppm clams appeared normal.
However, several clams diverticular and intestinal cells were
depleted of PAS positive material and were more vacuolar in appear-
ance than were the controls.
In the control clams, the bulk of the diverticular cells were
full, round, and most of the cytoplasm was red. In the 10 ppm,
50 ppm and 100 ppm clams, the diverticular cells were more vac-
uolar and contained less cytoplasm than did those of the control
group.
Discussion
Petroleum hydrocarbons are generally assumed to be carcino-
genic, particularly the polycyclic aromatics. Reviews of the
general carcinogenic effects of oil have been published by
HEIDELBERGER (1970) and ZOBELL (1971). CLARK, et_ al. (1974)
reported alterations in tissue structure of oysters and mussels
exposed to outboard motor effluent. LAROCHE (1972), BARRY and
YEVICH (1975) have reported a high incidence of gonadal tumors
in soft shell clams exposed to oils. Hyperplastic germ cell
736
-------
tumors were also present in the gills. BARRY, j2t_ al_. (1971) re-
ported a high incidence of hyperplasia of the gills and kidneys in
soft shell clams from areas believed polluted. In contrast,
VAUGHAN (1973) did not find evidence of histopathological change
in oysters exposed to No. 2 fuel oil. There were some nonpatho-
logical changes evident in the epithelial layer of the inner
mantle lobe, and it was suggested that oil restricted feeding
activity.
The results of this study with Mya arenaria revealed that
radical tissue changes did not occur after exposure to No. 2 fuel
oil. It is possible, however, that either the very low concentra-
tion of oil present in the water column was not suffiecient to
alter tissue structure (i.e. neoplasms), or the exposure time was
not long enough. The hydrocarbon concentrations in the water
column of each tank measured during the last 3 weeks of exposure
varied from 1.52 to 0.29 ppm.
The general effects of subacute oil exposure can be charac-
terized as a depletion of glycogen and generalized leukocytosis
particularly evident in the blood sinuses of the pallium and
mantle membrane. There was also an increase in vacuolization
of the diverticula, stomach and intestines. The histological
effects in Mya arenaria appeared to be dose dependent. The clams
exposed to the initial 100 ppm oil emulsion had more frequent
and noticeable histological differences from the controls. The
depletion of glycogen and vacuolization may have been due to a
suppression of feeding and consequent use of body reserved coupled
with an altered respiratory rate. The increased vacuolization of
oil-exposed clams may also represent inclusion and intracellular
compartmentalization of hydrocarbons. The leukocytosis of the
mantle blood sinuses beneath the inner epithelium probably repre-
sents an inflammation reaction with a migration of leukocytes
into the affected areas.
Acknowledgement
The analyses for this investigation were performed at the
analytical facility of the U. S. Environmental Protection Agency
(EPA), Industrial Waste Treatment Research Laboratory in Edison,
N. J. Use of these facilities was provided by an EPA program that
supports graduate level research of the environment.
737
-------
References
BARRY, M.M., and P.P. YEVICH: Mar. Pollut. 'Bull. 6_, 171 (1975).
BARRY, M.M., P.P. YEVICH, and N.H. THAYER: J. Invert. Path. 17, 7
(1971).
BLUMER, M., G. SOUZA, and J. SASS: Mar. Biol. 5_, 195 (1970).
CLARK, R.C. Jr., J.S. FINLEY, and G.G. GIBSON: Environ. Sci. &
Tech. 8, 1009 (1974).
DES VOIGNE, D.M., and A.K. SPARKS: J. Invert. Path. _12_, 53 (1968).
GARDNER, G.R., P.P. YEVICH, and P.F. ROGEROSON: Proc. Conf. on
Prevent. & Contr. Oil Pollut., San Francisco, Calif., A.P.I.,
473 (1975).
GRIMSTONE, A.V., and R.J. SKAER: A guidebook to microscopical
methods. N.Y.: McGraw Hill Book Co. 1965.
GRUENFELD,. M. : Anal. Qual. Contr. Newsl. , U.S.E.P.A., _15_, 5 (1972).
GRUENFELD, M., and F. BEHM: Anal. Qual. Contr. Newsl., U.S.E.P.A.,
16, 6 (1973).
HEIDELBERGER, C.: Eur. J. Cancer j^, 161 (1970).
JEFFRIES, H.P.: J. Invert. Path. 20, 242 (1972).
LAROCHE, G.: Proc. Natl. Conf. on Contr. of Hazardous Material
Spills, U.S.E.P.A., 199 (1972).
LEE, R.F., R. SAUERHEBER, and A.A. BENSON: Science _T7_Z» 344 (1972).
LILLIE, R.D.: Histopathologic technic and practical histochemistry.
3rd ed. N.Y. : McGraw Hill Book Co. 1965.
LUNA, L.G.: Manual of histologic staining methods of the Armed
Forces institute of Pathology. 3rd ed. N.Y.: McGraw Hill Book
Co. 1968.
NEFF, J.M., and J.W. ANDERSON: Proc. Joint Conf. Prevent. & Contr.
Oil Spills, San Francisco, Calif., A.P.I., 467 (1975).
PAULEY, G.B., and T.K. CHENG: J. Invert. Path. JU, 504 (1968).
PAULEY, G.B., and A.K. SPARKS: J. Invert. Path. _7, 248 (1965).
PAULEY, G.B., and A.K. SPARKS: J. Fish. Res. Bd. Can. J23_, 1913
(1966).
STAINKEN, D.M.: Proc. Joint. Conf. Prevent. & Contr. Oil Spills,
San Francisco, Calif., A.P.I., 463 (1975).
STEGEMAN, J.J., and J.M. TEAL: Mar. Biol. 2_2, 37 (1973).
SHAW, B.L., and H.I. BATTLE: Can. J. Zool. 35, 325 (1957).
VAUGHAN, B.E.: A.P.I. Pub. No. 4191 (1973).
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Wash., D.C., A.P.I., 441 (1971).
738
-------
UNITED STATES ENVIRONMENIAL PRO'T ECTION AGENCY
INDUSTRIAL ENVIRONMENTAL RESEARCH LABORATORY-Ci
LDISON NLW JbRStY O8817
June 16, 1976
This bibliography of petroleum oil analysis methods
addresses major publications during 1974 and 1975.
Its availability was announced in EPA's Analytical
Quality Control Newsletter No. 29, April, 1976.
The bibliography is rather incomplete, however,
and information regarding obvious omissions would
certainly be appreciated.
Michael Gruenfeld
Chief, Chemistry Staff
-------
Adams, C. E. (1974): "A Method for the Separation of Oil from an Aqueous Oil-
Detergent Solution Prior to IR Analysis", Government Reports Announcements,
74(25): Technical Report NOLTR-74-102
Ahmed, S. M., et al. (1974): "Sampling Errors in the Quantitation of Petroleum
in Boston Harbor Water", Anal. Chem.. 46:1858
American Petroleum Institute (1974): "A Second Oil Pollution Survey of the
Southeast Florida Coast", API Publication No. 4231
Anbar, M., Scolnick, M. E., and Scott, A. C. (1974): "Identification of Mineral
Oils by Field lonization Mass Spectrometry", In the Proceedings of the
Marine Pollution Monitoring Symposium, NBS Special Publication 409:229
Anonymous (1974): "Analytical Techniques Seek to Fingerprint Oil Spills",
Chem. & Eng. News, 52:30
Anonymous (1974): "'Fingerprints' Trace Spill Culprits", Chemical Week, 114:40
Atanus, H. (1974): "TLC Finds Hexane Solubles", Water and Wastes Engineering,
J_l:26-28
Bean, R. M. (1974): "Suspensions of Crude Oils in Sea Water: Rapid Methods of
Characterizing Light Hydrocarbon Solutes", In the Proceedings of the Marine
Pollution Monitoring Symposium, NBS Special Publication 409:127
Belcher, R. S. (1974): "Determination of Mineral Oil in Water", In: Examination
of Waters: Evaluation of Methods for Selected Characteristics; Australian
Water Resources Council Technical Paper No. 8:79-83
Bieri, R. H., et al. (1974): "Identification of Hydrocarbons in an Extract From
Estuarine Water Accommodated No. 2 Fuel Oil", NBS Special Publication
409:149
Bird, C. W., and Lynch, J. M. (1974): "Formation of Hydrocarbons by Micro-
Organisms", Chem. Soc. Rev., 3^3°9
Blaylock, J. W., Bean, R. M., and Wildung, R. E. (1974): "Determination of
Hydrocarbon Types in Lake and Coastal Sediments", NBS Special Publication
409:217
Boehm, P. D., and Quinn, J. G. (1974): "The Solubility Behavior of No. 2 Fuel
Oil in Sea Water'1, Marine Pollution Bulletin, _5:101-105
Bogatie, C. F. (1974): "Rapid Identification of Oil and Grease Spills from Pulp
and Paper Mills by Infrared Spectroscopy", Tappi, 57:130-134
Brown, C. W., Lynch, P. F., Ahmadjian, M. (1974): "Monitoring Narragansett Bay
Oil Spills by Infrared Spectroscopy", Environmental Science and Technology,
18:669-670
Brown, C. W., Lynch, P. F., and Ahmadjian, M. (1974): "Novel Method for Sampling
Oil Spills and for Measuring Infrared Spectra of Oil Samples", Anal. Chem..
46:183-184
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Brown, R. A., Elliot, J. J., and Searl, T. D. (1974): "Measurement and Charac-
terization of Nonvolatile Hydrocarbons in Ocean Water", NBS Special Publi-
cation 409:131
Brown, R. A., et al. (1974): "Measurement and Interpretation of Nonvolatile
Hydrocarbons in the Ocean. Part I. Measurements in Atlantic, Mediter-
ranean, Gulf of Mexico, and Persian Gulf", U. S. Department of Commerce,
Washington, D. C.
Bruce, H. E., and Cram, S. P. (1974): "Sampling Marine Organisms and Sediments
for High Precision Gas Chromatographic Analysis of Aromatic Hydrocarbons",
NBS Special Publication 409:181
Budininkas, P., and Remus, G. A. (1974): "Development of Classification Scale
for Characterizing Bilge Waters Used in Evaluating Oil Removal Techniques",
USCG-D-75-74. Contract DOT-CG-32521-A
Chernatskaya, A. N. (1974): "Determination Using Modern Methods of Impurities
Polluting the Waste Waters from Petroleum Refineries", Khimiya Tekhnolo-
giya Topliv i Masel, 9^:24
Clark, R. C. Jr. (1974): "Methods for Establishing Levels of Petroleum Contam-
ination in Organisms and Sediment as Related to Marine Pollution Monitoring",
NBS Special Publication 409:189
Clark, R. C. Jr., and Finley, J. S. (1974): "Analytical Techniques for Isolating
and Quantifying Petroleum Paraffin Hydrocarbons in Marine Organisms", NBS
Special Publication 409:209
Cretney, W. J., and Wong, C. S. (1974): "Fluorescence Monitoring Study of Ocean
Weather Station "p"", NBS Special Publication 409:175
Domostroeva, N. G. (1974): "Determination of the Content of Petroleum Products
in Water by an Optical Acoustical Method", Izmeritel'naya Tekhnika, ^3:66
Ehrhardt, M., and Heineman, J. (1974): "Hydrocarbons in Blue Mussels from the
Kiel Bight", NBS Special Publication 409:221
Farrington, J. W. (1974): "Some Problems Associated with the Collection of
Marine Samples and Analysis of Hydrocarbons", Paper Presented at: Confer-
ence/Workshop on Marine Environmental Implications of Offshore Drilling in
the Eastern Gulf of Mexico
Farrington, J. W., et al. (1974): "Analysis of Hydrocarbons in Marine Organisms:
Results of IDOE Intercalibration Exercises", NBS Special Publication 409:163
Feldman, M. H., and Cawlfield, D. E. (1974): "Marine Environmental Monitoring:
Trace Elements in Persistent Tar Ball Oil Residues", NBS Special Publication
409:237
Garza, M. E., and Muth, J. (1974): "Characterization of Crude, Semirefined and
Refined Oils by Gas-Liquid Chromatography", Environmental jacience and Tech-
nology, 8^:249-255
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Giger, W., and Blumer, M. (1974): "Polycyclic Aromatic Hydrocarbons in the
Environment: Isolation and Characterization by Chromatography, Visible,
Ultraviolet, and Mass Spectrometry", Anal. Chem., 46 :1663
Giger, W., Reinhard, M., Schaffner, C., and Stuiran, W. (1974): "Petroleum-
Derived and Indigenous Hydrocarbons in Recent Sediments of Lake Zug,
Switzerland", Environmental Science and Technology, £:454-455
Gordon, D. C., and Keizer, P. D. (1974): "Estimation of Petroleum Hydrocarbons
in Seawater by Fluorescence Spectroscopy: Improved Sampling and Analytical
Methods", Technical Report No. 481 Marine Ecology Lab. Bedford Institute
of Oceanography, Dartmouth,Nova Scotia
Gordon, D. C. Jr., and Keizer, P. D. (1974): "Hydrocarbon Concentrations in
Seawater Along the Halifax-Bermuda Section: Lessons Learned Regarding
Sampling and Some Results", In the Proceedings of the Marine Pollution
Monitoring Symposium, NBS Special Publication 409:113
Gordon, D. C. Jr., Keizer, P- D., and Dale, J. (1974): "Estimates Using
Fluorescence Spectroscopy of the Present State of Petroleum Hydrocarbon
Contamination in the Water Column of the Northwest Atlantic Ocean1',
Marine Chemistry. 2^:251-261
Hellman, H. (1974) : "Differentiation Between Hydrocarbons of Biogenous and
Petrol Origin by Way of Fluorescence Spectroscopy", Zeits Anal. Chem. (Ger),
272:30
Hellmann, H., and Zehle, H. (1974): "On Which Conditions are Identifications of
Mineral Oils on Water Surfaces Possible?", Zeits Anal. Chem. (Ger), 269:353
Hertz, H. S., et al. (1974): "Methods for Trace Organic Analysis in Sediments
and Marine Organisms", NBS Special Publication 409:197
Hornig, A. W. (1974): "Identification, Estimation and Monitoring of Petroleum
in Marine Waters by Luminescence Methods", NBS Special Publication 409:135
Hunter, I., Guard, H. E., and DiSalvo, L. H. (1974): "Determination of Hydro-
carbons in Marine Organisms and Sediments by Thin Layer Chromatography",
NBS Special Publication 409:213
Ilordi, A. M. (1974): "Identification of Crude Oil Leaks at Sea", La Rivista dei
Combustibili, 28:367-371
Iliffe, T. M., and Calder, J. A. (1974): ''Dissolved Hydrocarbons in the Eastern
Gulf of Mexico Loop Current and the Caribbean Sea", Deep Sea Res., 21:481
Jeffrey, L. M., et al. (1974): "Pelagic Tar in the Gulf of Mexico and Caribbean
Sea", NBS Special Publication 409:233
Jeltes, R. (1974): "Fingerprinting Techniques as Aides in the Analysis of
Composite Chemical Pollutants in the Environment", Jour. Chromatog. Sci.,
12:599
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Jeltes, R. (1974): "Prompt Detection and Tracing of Oils and Other Detrimental
Chemicals in the Environment", Water Res. (G.B.), 8_:977
Johnson, J. D., and Gram, H. R. (1974): "Discrimination of Waste Oils by Micro
Emission Spectro-Chemical Analysis", Available from the National Technical
Information Service, Springfield, Virginia: Report No. CG-D-21-75
Kawahara, F. K. (1974): "Recent Developments in the Identification of Asphalts
and Other Petroleum Products", NBS Special Publication 409:145
Kawahara, F. K., Santner, J. F., and Julian, E. C. (1974): "Characterization of
Heavy Residual Fuel Oils and Asphalts by Infrared Spectrophotometry Using
Statistical Discriminant Function Analysis", Anal. Chem.. 46:266-273
Koelle, W. (1974): "Mineral Oil Loading of Lake of Constance Sediments", Kern-
forschungszentrum Karlsruhe (Berlin) , KFK1969UF:8
Lamontagne, R. A., et al. (1974): "Cj-C^ Hydrocarbons in the North and South
Pacific", Tellus, 26:71
Ledet, E. J., and Laseter, J. L. (1974): "Alkanes at the Air-Sea Interface from
Offshore Louisiana and Florida", Science, 186:261
Lee, C. C., Craig, W. K., and Smith, P. J. (1974): "Water-Soluble Hydrocarbons
from Crude Oil", Bulletin of Environmental Contamination and Toxicology,
J_2:212-217
Lewis, B. W., et al. (1974): "Hydrocarbons Identified in Extracts from Estuarine
Water Accomodated No. 2 Fuel Oil by Gas Chromatography-Mass Spectrometry",
NASA, Hampton, Virginia
Lordi, R., Manci, C., and Petronio, B. M. (1974): "Studies on Industrial Waters
Containing Oil Emulsions", Inquinamento, 16:31
LysyJ,I-» and Russel, E. C. (1974): "Dissolution of Petroleum-Derived Products
in Water", Water Resources (G.B.), 8^:863
Majori, L., et al. (1974): "Marine Pollution by Hydrocarbons in the Northern
Adriatic Sea", Rev. Int. Oceanog. Med., 31:137
Mallevialle, J. (1974): "Measurement of Hydrocarbons in Water: Application to
Cases of Surface Water Pollution", Water Research, j}: 1071-1075
Masimi, M., et al. (1974): "Hydrocarbon Components of Floating Oil Pollutants of
Sea Water", Bull. Jap. Soc. Sci. Fish, 40:111
Mayo, D. W,, et al. (1974): "Long Term Weathering Characteristids of Iranian
Crude Oil: The Wreck of the "Northern Gulf"", NBS Special Publication 409:201
McAuliffe, C. D. (1974): "Determination of Cj^-C^Q Hydrocarbons in Water", In the
Proceedings of the Marine Pollution Monitoring Symposium, NBS Special Publi-
cation 409:121
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McGlynn, J. A. (1974): "A Review of Techniques for the Characterization and
Identification of Oil Spillages", In: Examination of Waters: Evaluation
of Methods for Selected Characteristics; Australian Water Resources
Council Technical Paper No. 8:85-89
Medeiros, G. C., and Farrington, J. W. (1974): "IDOE-5 Intercalibration Sample:
Results of Analysis After Sixteen Months Storage", NBS Special Publication
409:167
Mitra, G. D., et al. (1974): "Gas Chromatographic Analysis of Complex Hydro-
carbon Mixtures", Journal Chromatography, 91;633
Mommessin, P. R. , and Raia, J. C. (1974): "Chemical and Physical Characterization
of Tar Samples from the Marine Environment", U.S. Coast Guard, Washington, D.C
Novotny, J. (1974): "Applications of Gas Chromatography for Analyses of Water
Polluted by Petroleum Products", Vodni Hospodarstvi, 24:45
Osgood, J. 0. (1974): "Hydrocarbon Dispersion in Ground Water: Significance and
Characteristics", Ground Water, 12:427
Polak, J., and Lu, B. C.-Y. (1974): "Determination of the Total Amount of Volatile
but Slightly Soluble, Organic Materials Dissolved in Water from Oil and Oil
Products", Anal. Chim. Acta., 68:231
Pozdnyshev, G. N. , et al. (1974): "Extraction Separation of Petroleums into Oils,
Tars and Asphalts", Khimiya Tekhnologiya Topliv i Masel, 10:54
Rashid, M. A. (1974): "Degradation of Bunker C Oil Under Different Coastal Envi-
ronments of Chedabucto Bay, Nova Scotia", Estuarine & Coastal Mar. Sci., ^:137
Ray, S. M., Oja, R. K., Jeffrey, L. M., and Presley, B. J. (1974): "A Quantitative
and Qualitative Survey of Oils and Tars Stranded on Galveston Island Beaches",
Available from the National Technical Information Service, Springfield,
Virginia: Report No. CG-D-10-75
Rijks, J. A., et al. (1974): "Characterization of Hydrocarbons in Complex Mixtures
by Two-Dimensional Precision Gas Chromatography", Journal Chromatography,
9^:603
Sackett, W. M., and Brooks, J. M. (1974): "Use of Low Molecular-Weight-Hydrocarbon
Concentrations as Indicators of Marine Pollution", NBS Special Publication
409:171
Sleeter, T. D., et al. (1974): "Quantitative Sampling of Pelagic Tar in the North
Atlantic, 1973", Deep Sea Res., 21:773
Straughan, D. (1974): "Field Sampling Methods and Techniques for Marine Organisms
and Sediments", NBS Special Publication 409:183
Sutton, C., and Calder, J. A. (1974): "Solubility of Higher-Molecular-Weight
n-Paraffins in Distilled Water and Seawater", Environmental Science and Tech-
nology, 8:654-657
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Suzuki, R., Yamaguchi, N., and Matsumoto, R. (1974): "Determination of Trace
Amounts of Dispersed Oil in Waste Water by Solvent Extraction - Infrared
Analysis", Japan Analyst. 23; 1296
Swinnerton, J. W. , Lamontagne, R. A. (1974): "Oceanic Distribution of Low-
Molecular-Weight Hydrocarbons-Baseline Measurements", Environmental
Science and Technology, j^:657-663
Tu-Ching, T. (1974): "The Infrared Studies of Santa Barbara Channel Oil Spill",
Available from University Microfilms, Inc., Ann Arbor, Michigan, (Ph.D.
Thesis)
U. S. Coast Guard (1974): "Oil Spill Identification System-Interim Report",
Available from the National Technical Information Service, Springfield,
Virginia: Report No. CG-D-41-75
vonHellman, H., and Holeczek, M. (1974): "Kohlenwasserstoffe in Quellwassern-
Olverschmutzung oder Naturstoffe?", Tenside Detergents, 11:197
Warner, J. S. (1974): "Quantitative Determination of Hydrocarbons in Marine
Organisms", NBS Special Publication 409:195
Wasik, S. P. (1974): "Determination of Hydrocarbons in Sea Water Using an
Electrolytic Stripping Cell", Jour^ Chromatog. Sci. , 12:845
Whitham, B. T. (1974): "Marine Pollution by Oil, Characterization of Pollutants,
Sampling, Analysis, and Interpretation", by Institute of Petroleum, Oil
Pollution Analysis Committee, Applied Science Publishers Ltd., Essex, England
Whittle, K., Mackie, P. R., and Hardy, R. (1974): "Hydrocarbons in the Marine
Ecosystem", South African Journal of Science, 70:141
Zeller, M. V. (1974): "Infrared Analysis of Oil in Water Using the Model 100",
Perkin-Elmer Infrared Bulletin 22
Zeller, M. V. (1974): "Oil in Water: Use of Non-Toxic Solvent and Importance of
Acidification", Perkin-Elmer Infrared Bulletin 41
*
Zsolnay, A. (1974): "Determination of Aromatic and Total Hydrocarbon Content in
Submicrogram and Microgram Quantities in Aqueous Systems by Means of High
Performance Liquid Chromatography", In the Proceedings of the Marine Pollu-
tion Monitoring Symposium, NBS Special Publication 409:119
Zsolnay, A. (1974): "Determination of Total Hydrocarbons in Sea Water at the
Microgram Level with a Flow Calorimeter", Journal Chromatography, 90:79
-------
ASTM D3325-74T (1975): "Tentative Method for Preservation of Waterborne Oil
Samples", ASTM Standards, 31;565-567
ASTM D3325-74T (1975): "Tentative Method of Teat for Preparation of Sample
for Identification of Waterborne Oils", ASTM Standards, 31;561-564
ASTM D3327-74T (1975): "Tentative Methods of Analysis for Selected Elements
in Waterborne Oils", ASTM Standards. 31;568-576
ASTM D3328-74T (1975): "Tentative Methods of Test for Comparison of Petroleum
Oils by Gas Chromatography", ASTM Standards. 31:577-583
Clark, H. A., and Jurs, P. C. (1975): "Qualitative Determination of Petroleum
Sample Type from Gas Chromatograms Using Pattern Recognition Techniques",
Anal. Chem., 47^374-378
Cramer, C. D. (1975): "Detection and Characterization of Animal/Vegetable and
Petroleum Oil in Municipal Wastewater by Thin Layer Chromatography", Dis-
tributed during 1975 D-19.10 ASTM meeting; Available from Nalco Chemical
Company, 2901 Butterfield Rd., Oak Brook, 111. 60521
Davis, C. E., Krc, A. E., Szakasits, J. J., and Hodgson, R. L. (1975): "Multi-
element True Boiling Point Gas Chromatography for Monitoring Oil Pollution",
Joint Conference on Prevention and Control of Oil Pollution, San Francisco*:
93-97
Davis, S. J., and Gibbs, C. F. (1975): "The Effect of Weathering on a Crude Oil
Residue Exposed at Sea", Water Research, 9.:275-285
Dell'Acqua, R., Egan, J. A., and Bush, B. (1975): "Identification of Petroleum
Products in Natural Water by Gas Chromatography", Environmental Science and
Technology, 2=38-41
Duewer, D. L., Kowalski, B. R., and Schatzki, T. F. (1975): "Source Identifica-
tion of Oil Spills by Pattern Recognition Analysis of Natural Elemental
Composition", Anal. Chem., 47:1573-1583
Farrington, J. V. , and Medeiros, G. C. (1975): "Evaluation of Some Methods of
Analysis for Petroleum Hydrocarbons in Marine Organisms", Joint Conference
on Prevention and Control of Oil Pollution, San Francisco*: 115-123
Frank, U. (1975): "Identification of Petroleum Oils by Fluorescence Spectroscopy",
Joint Conference on Prevention and Control of Oil Pollution, San Francisco*:
87-93
Gruenfeld, M. (1975): "Quantitative Analysis of Petroleum Oil Pollutants by
Infrared Spectrophotometry", ASTM STP-573:290
Harrison, R. M., Perry, R., and Wellings, R. A. (1975): "Polynuclear Aromatic
Hydrocarbons in Raw, Potable, and Waste Waters", Water Research, 9^: 331-346
Harrison, W., Winnik, M. A., Kwong, P. T. Y., and Mackay, D. (1975): "Disappear-
ance of Aromatic and Aliphatic Components from Small Sea Surface Slicks",
Environmental Science and Technology, 9^:231-234
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Regnier, Z. K., and Scott, B. F. (1975): "Evaporation Rates of Oil Components",
Environmental ScJence and Technology, 9_: 469-472
Smiley, C. T., Montgomery, D. S., and Sawatzky, H. (1975): "A Gas Liquid - Gas
Solid Chromatographic Method for the Identification of Sources of Oil
Pollution", ASTM STP-573:271
Smith, W. E., Napier, B., and Home, 0. J. (1975): "Characterization of
Petroleum Pitches Used for Coke Production", Symposium on Petroleum Derived
Carbon Presented Before the Division of Petroleum Chemistry, Inc. American
Chemical Society Philadelphia Meeting, April 6rll, 1975: 369-375
Spiker, E. C., and Rubin, M. (1975): "Petroleum Pollutants in Surface and
Graundwater as Indicated by the Carbon-lA Activity of Dissolved Organic
Carbon", Science, 187:61-64
Walker, J. 0., Colwell, R. R., Hamming, M. C., and Ford, H. T. (1975): "Extrac-
tion of Petroleum Hydrocarbons from Oil-Contaminated Sediments", Bulletin
of Environmental Contamination and Toxicology, 13:245-248
Warner, J. S. (1975): "Determination of Sulfur-Containing Petroleum Components
in Marine Samples", Joint Conference on Prevention and Control of Oil
Pollution, San Francisco*: 97-103
Weiss, F. T. (1975): "Activities of Standardization in the Identification of
Waterborne Oils", Prepared for the National Bureau of Standards Workshop
on Standard Reference Materials for Offshore Drilling-Petroleum; Available
from Shell Development Co., P- 0. Box 481, Houston, Texas 77001
Yu, T. S., and Coleman, W. H. (1975): "A Quantitative Method for Determining
Apparent Oil Concentration in Water Containing Detergents", Naval Ship
Research and Development Center, Report TM-28-75-10
**Bentz, A. P. (1976): "Oil Spill Identification", Anal. Chem., 48:454A
**John, P., Soutar, I. (1976): "Identification of Crude Oils by Synchronous
Excitation Spectrofluorimetry", Anal. Chem., 48:520
**Mackay, D., Shiu, W. Y. (1976): "Aqueous Solubilities of Weathered Northern
Crude Oils", Bulletin of Environmental Contamination and Toxicology,
15:101
*Available from the American Petroleum Institute, 1801 K Street, N. W. ,
Washington, D. C. 20006
**1976 search in progress
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Hargrave, B. T., and Phillips, G. A. (1975): "Estimates of Oil in Aquatic
Sediments by Fluorescence Spectroscopy", Environmental Pollution, ji:
193-215
Hunt, G., Horton, D., Levine, J., Mayo, D., Donovan, D., Shelly, W. , Jiang, L. ,
Grove, R. , and Johnson, R. (1975): "The Evaluation and Development of
Passive Tagging Procedures for the Identification of Crude Oil Spilled
on Water", Joint Conference on Prevention and Control of Oil Pollution,
San Francisco*: 129-143
Hunter, L. (1975): "Quantitation of Environmental Hydrocarbons by Thin-Layer
Chromatography, Gravimetry/Densitometry Comparison", Environmental Science
and_ Technology, 9^:241-246
Jackson, B. W., Judges, R. W., and Powell, J. L. (1975): "Characterization
of Australian Crudes and Condensates by Gas Chromatographic Analysis",
Environmental Science and Technology, 9^:656-660
Ladner, L., and Hagstrom, A. (1975): "Oil Spill Protection in the Baltic Sea",
Journal Water Pollution Control Federation, 47:796-809
Lynch, P. F., Tang, S., and Brown, C. W. (1975): "Application of Cryogenic
Infrared Spectrometry to the Identification of Petroleum", Anal. Chem.,
47:1696-1699
MacKay, D., Shiu, W. Y., and Wolkoff, A. W. (1975): "Gas Chromatographic
Determination of Low Concentrations of Hydrocarbons in Water by Vapor
Phase Extraction", ASTM STP-573:251
Miles, D. H., Coign, M. J., and Brown, L. R. (1975): "The Estimation of the
Amount of Empire Mix Crude Oil in Mullet, Shrimp, and Oysters by Liquid
Chromatography", Joint Conference on Prevention and Control of Oil
Pollution, San Francisco*: 149-154
Mommessin, P. R., and Raia, J. C. (1975): "Chemical and Physical Character-
ization of Tar Samples from the Marine Environment", Presented at the
1975 Joint Conference on Prevention and Control of Oil Pollution, San
Francisco*: 155-167
Neff, J. M., and Anderson, J. W. (1975): "An Ultraviolet Spectrophotometric
Method for the Determination of Naphthalene and Alkylnaphthalenes in
the Tissues of Oil-Contaminated Marine Animals", Bulletin of Environmental
Contamination and Toxicology, _L4:122-128
Pancirov, R. J., and Brown, R. A. (1975): "Analytical Methods for Polynuclear
Aromatic Hydrocarbons in Crude Oils, Heating Oils, and Marine Tissues",
Joint Conference on Prevention and Control of Oil Pollution, San Francisco*;
103-115
Pym, J. G., Ray, J. E., Smith, G. W., and Whitehead, E. V. (1975): "Petroleum
Triterpane Fingerprinting of Crude Oils", Anal. Chem. , 4_7_: 1617-1622
*USGPO: 1977 — 757-056/6451 Region 5-11
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