&EPA
United States
Environmental Protection
Agency
Industrial Environmental Research
Laboratory
Cincinnati OH 45268
Research and Development
Publications on the
Analysis of Spilled
Hazardous and
Toxic Chemicals
and Petroleum Oils
-------
PREFACE
THIS COLLECTION OF OIL AND HAZARDOUS CHEMICAL POLLUTION RELATED REPORTS
DESCRIBES NEWLY DEVELOPED ANALYTICAL TECHNIQUES AND ENVIRONMENTAL
STUDIES OF THE OIL AND HAZARDOUS MATERIALS SPILLS BRANCH AT EDISON, NEW
JERSEY, THIS MATERIAL IS REPRINTED FROM JOURNALS, NEWSLETTERS, AND
CONFERENCE PROCEEDINGS, WHICH ARE REFERENCED,
COVER ILLUSTRATION FEATURES A HIGHLY INSTRUMENTED MOBILE EMERGENCY RESPONSE
LABORATORY, FOR FURTHER INFORMATION SEE THE ARTICLE ON PAGE 23.
-------
PUBLICATIONS ON THE ANALYSIS OF
SPILLfD HAZARDOUS AND TOXIC CHEMICALS
AND PETROLEUM OILS
OIL AND HAZARDOUS MATERIALS SPILLS BRANCH
INDUSTRIAL ENVIRONMENTAL RESEARCH LABORATORY
U, S, ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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CONTENTS
I. HAZARDOUS CHEMICALS
A. FLUORESCENCE ANALYSES
Reference
AQC Newsletter #34
July 1977
AQC Newsletter #36
January 1978
EPA Quality Assurance
Newsletter Vol.1 No. 1,
April 1978
Title
Quantitation of Hazardous
Materials.
by U. Frank, J. Munn, and
N. Pangaro
Comparison of Synchronous and
Single Wavelength Excitation
Fluorescence Spectroscopy
by U. Frank
Quantification of Mixed
Hazardous Materials
by U. Frank, P. Ward, and
J. L. Callender
Rapid Quantification of
Hazardous Materials in Sediments
by Synchronous Excitation
Fluorescence Spectroscopy
By U. Frank and D. Remeta
Page
2
Proceedings of 1978 National
Conference on Control of
Hazardous Material Spills,
Miami Beach, Florida
April 11-13, 1978
Purification of Isopropanol for
Fluorescence Analyses
by S. Frysinger, D. Remeta and
N. Pangaro
Use of Synchronous Excitation
Fluorescence Spectroscopy for
In Situ Quantifications of
Hazardous Materials in Water
by U. Frank, and M. Gruenfeld
5-9
iii
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I. HAZARDOUS CHEMICALS
B. OTHER ANALYSES
Reference
Title
Page
AQC Newsletter #20
January 1974
AQC Newsletter #25
April 1975
AQC Newsletter #29
April 1976
AQC Newsletter #32
January 1977
AQC Newsletter #35
October 1977
AQC Newsletter #36
January 1978
EPA Quality Assurance
Newsletter Vol 1. No. 2,
July 1978
Stability of Stored Chromographic 11
and Spectral Data
by F. Behm and M. Gruenfeld
Determination of EDTA by NMR 12
by U. Frank and F. laconianni
Kepone Analyses 13
by U. Frank and J. Lafornara
Mobile Laboratory 13
by U. Frank, R. Frederick, and
M. Urban
Mobile Laboratory 14
by M. Urban and R. Losche
Analysis of Hazardous Materials 15
in Water by NMR
by U. Frank
Interference by Sodium Sulfate 15
Impurities
by R. Losche, M. Urban and
M. Gruenfeld
Evaluation of An Emission 16
Spectroscope
by R. Losche and M. Gruenfeld
Removal of Sulfur from 17
Sediment Extracts
by D. Stainken
Sediment Hydrocarbon 17
Quantification
by D. Stainken
Interaction of m-Cresol with 18
Plastic Tubing
by M. Royer, S. Frysinger,
M. Gruenfeld and G. Fraser
IV
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I. HAZARDOUS CHEMICALS
Reference
B. OTHER ANALYSES
Title
Page
EPA Quality Assurance
Newsletter Vol. 1, No.
October 1978
- Analysis of Polynuclear
Aromatic Hydrocarbons in
Estuarine Water
by D. Stainken and U. Frank
19
Chemical Marketing
Reporter
April 3, 1978
Proceedings of 1978 National
Conference on Control of
Hazardous Material Spills
April 11-13, 1978
Industrial .Water Engineering
September, 1978
Mobile Lab Johnny on the Spot 20-22
In EPA's Toxic Spill Operations
Development and Use of a Mobile 23-26
Chemical Laboratory for Hazardous
Material Spill Response Activities
by M. Urban and R. Losche
EPA's Mobile Lab and Treatment 27-32
System Responds to Hazardous
Spills
by M. Gruenfeld, F. Freestone
and I. Wilder
-------
II. Petroleum Oils
A. FLUORESCENCE ANALYSES
Reference
AQC Newsletter #13
April 1972
AQC Newsletter #15
October 1972
AQC Newsletter #20
January 1974
AQC Newsletter #21
April 1974
AQC Newsletter #22
July 1974
AQC Newsletter #24
January 1975
AQC Newsletter #31
October 1976
AQC Newsletter #32
January 1977
AQC Newsletter #33
April 1977
Title
Analysis for Crankcase
Oil in Water by
Fluorescence Spectrophotometry
by U. Frank
Passive Tagging of Oil by
Fluorescence Spectrophotometry
by U. Frank
Passive Tagging Of Oils by
Fluorescence Spectrometry
by U. Frank
An Improved Solvent for
Fluorescence Analyses of Oils
by U. Frank
Reclaiming a Waste Solvent
by U. Frank
Effect of Fluorescence Quenching
on Oil Identification
by U. Frank
Identification of Petroleum Oils
By Fluoresence Spectroscopy
by U. Frank
Synchronous Excitation
Fluorescence Spectroscopy
by U. Frank and L. Pernell
Synchronous Excitation
Fluorescence Spectroscopy
by U. Frank and J. Munn
Synchronous Excitation
Fluorescence Spectroscopy
by U. Frank and M. Gruenfeld
Adsorption of Fluorescing
Oil Components Into
Silica Gel
by U. Frank
Page
34
35
36
37
37
38
39
40
41
42
42
vi
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II. Petroleum Oils
A. FLUORESCENCE ANALYSES
Reference
AQC Newsletter #34
July 1977
EPA Quality Assurance
Newsletter, Vol.1. No.
July 1978
Title
Substitute Deozonator for
Fluorescence Spectrometers
by R. Frederick
Oil Quantitation by
Fluorescence Spectrescopy
by U. Frank and J. Susman
Rapid Quantification
of Petroleum Oils in Sediments
by U. Frank
Page
42
43
44
Proceedings of 1975 Conference - Identification of Petroleum Oils
on Prevention and Control of By Fluorescence Spectroscopy
Oil Pollution, San Francisco, by U. Frank
Ca., March 25-27, 1975
45-49
Presented at the 1977
Pittsburgh Conference On
Analytical Chemistry and
Applied Spectroscopy
Feb. 28 - March 4, 1977
Cleveland, Ohio
Bibiliography Of Recent
Methods For The Fluorescence
Analysis of Petroleum Oils
- Determination of Petroleum
Oils in Sediments By
Fluorescence Spectroscopy
and NMR
by U. Frank and M. Gruenfeld
- by U. Frank
50
Toxicological and Environmental- A Review of Fluorescence
Chemistry Reviews, Vol. 2, Spectroscopic Methods for
Issue 3, oil Spill Source Identification
December 1978 by U. Frank
51
216-238
vii
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II. Petroleum Oils
B. OTHER ANALYSES
Reference
Title
Page
AQC Newsletter #12
January 1972
AQC Newsletter #15
October 1972
AQC Newsletter #16
January 1973
AQC Newsletter #17
April 1973
AQC Newsletter #18
July 1973
Extraction of Oil
From Water For
Quantitative Analysis By IR
by M. Gruenfeld
Preparation Of Heavy
Oils For Infrared Analysis
by M. Gruenfeld
Storage And Transport
Of Oil Containing Samples
In Plastic Bottles
by B.F. Dudenbostel
and M. Gruenfeld
Quantitative Analysis
Of Oils By IR
by M. Gruenfeld
A TLC Method To Facilitate
The Quantitation Of Oil By IR
by U. Frank
Ultrasonification For Preparing
Stable Oil In Water Dispersions
by M. Gruenfeld and F. Behm
Plastic Bottles For Storing
and Transporting Oil Containing
Samples
by B.F. Dudenbostel and
M. Gruenfeld
Use Of Gas Chromatographic
Peak Height Ratios For Passive
Tagging Of Petroleum Oils
by B.F. Dudenbostel
Storage and Transport Of Oils
In Solvents For Quantitative
Analysis By IR
by M. Gruenfeld and J. Puglis
55
56
57
57
58
59
60
61
61
Vlil
-------
II. Petroleum Oils
B. OTHER ANALYSES
Reference
AQC Newsletter #18
July 1973
AQC Newsletter #20
January 1974
AQC Newsletter #21
April 1974
AQC Newsletter #24
AQC Newsletter #26
July 1975
Title
Solvent Extraction Of Oil
From Water
by U. Frank
Glassware Cleaning For The
Quantitation of Oil In Water
by J. Puglish and M. Gruenfeld
Calculation Of Absorbance
From IR Ordinate Expansion
Measurements
by M. Gruenfeld
Sulfur Interference
In U.V. Analysis
by J. Lafornara and
M. Gruenfeld
Removal Of Charred
Oil Deposits From Glassware
by H. Jeleniewiski and U. Frank
Solvent For Oil
Analysis
by M. Gruenfeld
Evaluation Of A Portable
IR Spectrophotometer
by M. Gruenfeld and
U. Frank
Identification Of Milligram
Quantities Of Petrolum Oils
by M. Gruenfeld and R. Frederick
Separation Of Petroleum and
Non-Petroleum Oils
by M. Gruenfeld
Page
62
62
62
63
63
64
64
65
66
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II. Petroleum Oils
B. OTHER ANALYSES
Reference
AQC Newsletter #29
April 1976
Title
Replicate Oil Chromatograms
by M. Gruenfeld
Page
67
AQC Newsletter #30
July 1976
AQC Newsletter #36
January 1978
Proceedings of the 1973
Conference on Prevention
and Control of Oil Spills,
Washington, B.C.
March 13-15, 1973
Environmental Science
and Technology, Vol. 7, No. 7,
July 1973
Presented at
National Bureau of
Standards Conference on
"Standard Reference"
Materials for Offshore
Drilling - Petroleum
Santa Barbara, Calif.
October 1975
Water Quality Parameters
ASTM-STP 573
American Society For Testing
and Materials, 1975
Determination Of Oil
In Sediment By NMR
by U. Frank and M. Gruenfeld
Differentiation Of A Synthetic
Lubrication Oil From Several
Petroleum Derived Automotive
Lubricating Oils
by R. Frederick and N. Pangaro
Identification of Oil
Pollutants: A Review
Of Some Recent Methods
by M. Gruenfeld
Extraction Of Dispersed
Oils From Water For
Quantitative Analysis
By Infrared Spectrophometry
by M. Gruenfeld
Review of Methods and
Standards Used For Oil
in Brine Analysis By
Contracting Laboratories
On The Louisiana Coast
by U. Frank
Quantitative Analysis
of Petroleum Oil
Pollutants by Infrared
Spectrophotometry
by M. Gruenfeld
68
69
70-84
85-88
89-97
98-116
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II. Petroleum Oils
B. OTHER ANALYSES
Reference
Title
Page
Journal Of Water
Pollution Control
Federation,
February 1977
Proceedings of the 1977
Oil Spill Conference
(Prevention, Behavior,
Control, Cleanup), New
Orleans, La.,
March 8-10, 1977
Rapp. P-V. Reun. Cons.
Int. Explor. Her, 171:
33-38, 1977
Presented at ICES
Workshop on Petroleum
Hydrocarbons, Aberdeen,
Scotland
September 8, 1975
Proceedings of the 1979
Oil Spill Conference
(Prevention, Behavior,
Control, Cleanup), Los
Angeles, Ca., March 19-22, 1979.
Petroleum Hydrocarbons 117-127
From Effluents:
Detection in Marine Environment
by J. T. Tanacredi
A Review Of Some Commonly Used 128-132
Parameters For The Determination
Of Oil Pollution
by M. Gruenfeld and U. Frank
The Ultrasonic Dispersion, 133-138
Source Identification, and
Quantitative Analysis Of
Petroleum Oils In Water
by M. Gruenfeld and
R. Frederick
(Oral Presentation) 139-152
The Ultrasonic Dispersion
Source Identification, and
Quantitative Analysis Of
Petroleum Oils In Water
by M. Gruenfeld
Bibliography of Petroleum 153-162
Oil Analysis Methods
1974 - 1975
Methods For the Source Ident- 207-215
ification and Quantification of
Oil Pollution
By U. Frank, D. Stainken, and
M. Gruenfeld
-------
II. Petroleum Oils
C. ENVIRONMENTAL STUDIES
Reference
Title
Page
AQC Newsletter #13
April 1972
Proceedings of 1975,
Conference on Prevention
and Control Of Oil Pollution,
San Francisco, Ca
March 25,27, 1975
Fate and Effects Of
Petroleum Hydrocarbons
In Marine Organisms
and Ecosystems, Proceedings
of a Symposium, Seattle, Wa.,
November 10-12 , 1976
Bulletin of Environmental
Contamination and Toxicalogy
1976, Vol. 16, No. 6
Bulletin Of Environmental
and Toxicology, Vol. 16, No. 6
December 1976
Proceedings Of the Symposium
on "Recovery Potential Of
Oiled Marine Northern
Environments",
Halifax, Nova Scotia,
October 10-14, 1977
Bulletin of NJ Acadamy of
Science Vol.24, No. 1.
Comparison Of Hydrocarbons 164
In Marine Organisms From
Unpolluted Water With
Petroleum Oils
by B.F. Dudenbostel
Preliminary Observations On 165-170
The Mode Of Accumulation Of
No. 2 Fuel Oil By The Soft Shell
Clam, Mya arenaria by D. Stainken
The Accumulation and Depuration 171-184
Of No.2 Fuel Oil By The Soft
Shell Clam, Mya arenaria L.
by D. Stainken
The Effect Of a No. 2 Fuel Oil 185-190
and a South Louisiana Crude Oil
On The Behavior Of The Soft Shell
Clam, Mya arenaria L.
by D. Stainken
A Descriptive Evaluation Of The 191-199
Effects Of No. 2 Fuel Oil On
The Tissues Of The Soft Shell
Clam, Mya arenaria L.
by D. Stainken
Effects Of Uptake and Discharge 200-205
Of Petroleum Hydrocarbons On
The Respiration Of The Soft
Shell Clam, Mya arenaria
by D. Stainken
Occurrence of Extractable Hydro- 239-244
carbons in Sediments From Raritan
Bay, New Jersey
by D. Stainken
xii
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I, HAZARDOUS CHEMICALS
A, FLUORESCENCE ANALYSES
-------
(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #34, July 1977.
Quantitation of Hazardous Materials
The use of synchronous excitation fluorescence spectroscopy for
the analysis of hazardous materials (40 CFR, Part 116) has been
described in AQC Newsletters Nos. 31 and 32. We are now extend-
ing this technique to non-fluorescent materials by using deri-
vatization techniques. Fluorescamine (available from Fisher
Scientific Company as Roche Fluran) was used for in-situ quanti-
tations of butylamine and ethylenediamine in water. The deri-
vatization reaction proceeded rapidly in aqueous media at room
temperature. Preliminary findings indicate nearly 100% recoveries
and detection limits in the ppb range. (U. Frank, Coml. 201-321-
6626, FTS 340-6626/J. Munn/N. Pangaro, Coml. 201-321-6628, FTS
340-6628)
Comparison of Synchronous and Single Wavelength
Excitation Fluorescence Spectroscopy
Synchronous excitation and conventional single wavelength excit-
ation fluorescence spectroscopy were compared for in-situ water
analyses of aromatic compounds that contaminated a town's water
reservoir. Fluorescence analyses were performed on site, on
board our Mobile Spills Laboratory. Single wavelength excitation
yielded spectra that were totally obscured by Rayleigh-Tyndall
scatter as a consequence of colloidal particulates in the water
samples. Synchronous excitation (described in AQC Newsletters
Nos. 31 and 32) yielded spectra that were free of scatter inter-
ferences and that contained only one sharp peak and a complex
envelope. The peak's fluorescence intensity served to monitor
the extent of the reservoir's contamination, and also served to
determine the feasibility of water detoxification. A total of
250 in-situ water measurements were performed by this method in
four days. (U. Frank, Coml. 201-321-6626, FTS 340-6626)
* Inquiries regarding this newsletter should be directed to:
U.S. Environmental Protection Agency
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268
-------
(*) ANALYTICAL DUALITY CONTROL NEWSLKTTLR //36, January 1978
Quantification of Mixed Hazardous Materials
Synchronous excitation (SE) fluorescence spectroscopy for the in
situ quantification of Hazardous Materials in water samples con-
taining only the individual toxicants, was previously described
in AQC Newsletters 31, 32 and 34. A current effort in our labo-
ratory addresses the in situ quc\ntif ication of specific toxicants
in water samples containing mixtures of toxicants. Preliminary
findings indicate that well resolved fluorescence spectra of in-
dividual toxicants in mixtures are obtained by the SE procedure,
when these toxicants differ in their number of aromatic rings.
For example, phenol and naphthenic acid yield two well resolved
fluorescence peaks which are readily quantified. Conversely,
aniline, phenol and toluene are all single ring compounds, which
as a mixture yield only one fluorescence peak. Quantification
of each toxicant in this mixture was accomplished by suppressing
the fluorescence of phenol and aniline through pH adjustment.
Observation of the behavior of the three materials demonstrated
that all three toxicants fluoresced at pH 7, aniline and toluene
fluoresced at pH 11, while phenol and toluene fluoresced at pH 2.
Quantification of the individual toxicants was successfully
accomplished by performing these pH adjustments, and by using
three simultaneous equations. (U. Frank, Coml. 201-321-6626,
FTS 340-6626, P. Ward/J.L. Callender, Coml. 201-321-6628, FTS
340-6628)
* See page 2.
-------
(*)EPA QUALITY ASSURANCE NEWSLETTER, Volume 1 No.l, April 1978.
Rapid Quantification of Hazardous Materials in Sediments by
Synchronous Excitation Fluorescence Spectroscopy ~ *rne use of
synchronous excitation fluorescence spectroscopy for the in
situ quantification of hazardous materials (40 CFR 116) in
water was previously described in AQC Newsletters 31-34 and
36. We have now extended this technique to the quantification
of hazardous materials in sediments. Phenol, resorcinol, and
styrene were spiked into fluvial sediments at concentrations of
10 mg/Kg. Analyses were performed by adding 2-propanol to the
spiked sediments and vigorously agitating for 15 minutes. The
propanol extract was then filtered and analyzed by the synchro-
nous excitation technique. Recoveries in the range 80-100%
were obtained. (U. Frank, FTS 340-6626, Coml. 201-321-6676/D.
Remeta, FTS 340-6628, Coml. 201-321-6628)
Purification of Isopropanol for Fluorescence Analyses - While
quantifying hazardous materials in sediments by Synchronous
Excitation (SE) fluorescence spectroscopy, an interfering
impurity was encountered in the solvent isopropanol. Use of
the SE technique was previously described in AQC Newsletters
31-34 and 36. The impurity yielded a single peak at 290 nm,
when scanning the excitation monochromator through 270 nm.
This impurity was found in both Burdick and Jackson distilled-
in-glass isopropanol and in Fisher certified 99 Mol % pure
isopropanol. This impurity was also apparent when using the
Single Wavelength Excitation technique. Attempts to remove
the contaminant by distillation were unsuccessful. This prob-
lem was satisfactorily resolved by percolating the solvent
through a 17 mm (i.d.) chromatographic column containing a
425 mm height of granular activated carbon and using a solvent
flow of 6-10 ml/min. Subsequent fluorescence analyses re-
vealed no further interference. (S. Frysinger/D. Reraeta/N.
Pangaro, FTS 340-6628, Coml. 201-321-6628)
* See page 2.
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REPRINTED FROM: Proceedings of 1978 National Conference on Control of
Hazardous Material Spills, Miami Beach, Florida,
April 11-13, 1978, pp 119-123. Proceedings available
from API Washington D.C.
Use of Synchronous
Excitation Fluorescence
Spectroscopy for In Situ
Quantifications of Hazardous
Materials in Water Uwc Frank and Michael Gruenfeld
U.S. Environmental Protection Agency
Edison, New Jersey
INTRODUCTION
Spilled hazardous materials often cause substantial eco-
nomic damage and also threaten human life. Consequently,
their rapid quantification in environmental samples is high-
ly desirable. Most analytical methods address the quantifi-
cation of toxicants in co'mplex aqueous matrices such as in-
dustrial and municipal effluents. These methods require te-
dious and time consuming procedures involving solvent ex-
traction, evaporation and concentration steps and some-
times chromatographic cleanup steps.
Hazardous materials are often spilled into bodies of
water that yield less complex sample matrices and can there-
fore be more rapidly and directly analyzed. Few available
methods, however, specifically address the quantification of
toxicants in spill samples. The following discussion there-
fore describes an unusually rapid, sensitive and novel
method for the quantification of fluorescent hazardous
materials directly in water. This method avoids time con-
suming extraction, concentration and cleanup steps, and
can be used to rapidly and accurately quantify fluorescent
hazardous materials in aqueous samples.
Ultraviolet fluorescence spectroscopy is a useful analyt-
ical technique for the measurement of many hazardous
materials. It is exceptionally sensitive and often more speci-
fic than other spectroscopic methods such as ultraviolet,
visible, ancl infrared absorption techniques. Kullbom, era/1,
used fluorescence spectroscopy to analyze for phenols and
lignins in water. Other investigators2"6 used it to measure
petroleum and non-petroleum hydrocarbons in water. Some
of the latter investigators2>3)S>6 extracted the toxicants
from water with solvents prior to fluorescence measure-
ment. Kullbom, et a/1 and Frank4, measured toxicants di-
rectly in water. All of these investigators used conventional
single wavelength excitation (SWE) techniques.
SWE fluorescence spectroscopy is not satisfactory for
in-situ measurements of hazardous materials in water.
Spectra derived from use of the SWE technique are dis-
torted by Rayleigh-Tyndall and Raman scatter radiations,
especially when these spectra are obtained from samples
containing only mg/1 of toxicants. Consequently, the
authors describe, in this paper, the use of "Synchronous
Excitation" (SE) fluorescence spectroscopy for rapid in-situ
quantifications of hazardous materials in water. While other
investigators7^8 have demonstrated that the SE fluores-
cence technique yields substantially improved spectral reso-
lution when applied to spill source identifications of petro-
leum oil pollutants, the authors demonstrate that this tech-
nique also yields spectra free of Rayleigh-Tyndall and Raman
scatter when used for in-situ quantifications of hazardous
materials in water.
Differences between SWE procedures and the SE tech-
nique and the advantages of the latter for in-situ quantifica-
tions of hazardous materials in water are discussed within
the context of a three dimensional coordinate system. This
system was previously described by Frank9, and Frank and
Gruenfeld1 °. Three interdependent variables that are inher-
ent to fluorescence spectroscopy are used as axes: (^exci-
tation wavelength (x), (2) emission wavelength (y) and (3)
fluorescence intensity (z). Within this context, the fluores-
cence properties of hazardous materials are viewed as "total
fluorescence spectra" and the SE and SWE fluorescence
approaches are readily compared.
A three dimensional total spectrum of a compound that
resembles a mountainous region is shown in Figure 1. The
two mountain peaks (solid lines) are in fact the fluores-
cence maxima of this compound. SWE spectra are two
dimensional slices through the three dimensional spectrum,
parallel to the (y) axis. According to Lloyd7, such spectra
provide only limited useful information. SE fluorescence
spectra, however, are slices oriented at a 45° angle to the
(x) and (y) axes and often yield more useful information.
The present study of SE fluorescence spectroscopy for
in-situ quantifications of hazardous materials in water ad-
dresses representative materials that fluoresce and that are
designated as hazardous by 40 CFRPart 116. These mater-
ials were also shown by a recent EPA study11 to be the
most frequently spilled toxicants. In fact, 80% of the organ-
ic hazardous materials shown by this EPA study to be fre-
quently spilled, fluoresce strongly and can be quantified
directly in water by the SE procedure. This discussion of
these analyses also addresses spectral interferences, con-
centration quenching phenomena and optimum wavelength
intervals set between excitation and emission monochro-
mators. Measurements were performed using synthetically
prepared samples consisting of hazardous materials in
waters collected from areas of heavy marine traffic and high
spill potential.
-------
Use of Spectroscopy
FLUORESCENCE
INTENSITY
TOTAL SPECTRUM
SYNCHRONOUS EXCITATION (SE)
SPECTRUM
SINGLE WAVE LENGTH
EXCITATION(SWE)
SPECTRUM
EXCITATION
WAVELENGTH
EMISSION
WAVELENGTH
Figure 1: Comparison of SE, SWE and Total Fluorescence
Spectra Within the Context of the Three Dimensional Co-
ordinate System
Experimental
Apparatus: A Perkin-Elmer model MPF-3 Fluorescence
Spectrometer, and 10 mm pathlength quartz cell were used.
Materials: High purity reagents were used.
Procedure: Accurate aqueous stock solutions were pre-
pared. Water soluble liquids were dispensed directly by
using appropriate syringes; solids were weighed; water in-
soluble materials were dissolved in 2-propanol, prior to
water addition. Further dilution of the stock solutions were
performed with water to yield final concentrations that
were below each material's solubility limit. SE fluorescence
spectra were obtained by simultaneously scanning the in-
strument's excitation and emission monochromators. The
excitation rnonochromator was set ahead of the emission
monochromator by an "optimum" wavelength interval.
Wavelength intervals in the range 20-100 nm were evaluated
for each toxicant.
Some SWE spectra of the above solutions were also ob-
tained for comparison purposes. These solutions were
excited at single wavelengths, which yielded maximum
emission intensities for each compound. The emission
spectra were scanned in the range 220-500 nm.
Results and Discussion
The ability of SE fluorescence Spectroscopy to avoid
Rayleigh-Tyndall and Raman scatter distortions of spectral
profiles is demonstrated within the context of the three di-
mensional coordinate system. The combined effect of these
scattering phenomena can be viewed as resembling a three
dimensional "interference wedge" that bisects total fluores-
cence spectra (Figure 2). Although this interference wedge
causes detrimental spectral distortions when using SWE
techniques, it can be avoided by using the SE procedures
(Figure 3). Rayleigh-Tyndall (reflected) scatter radiation is
especially troublesome when measuring natural waters that
contain suspended particulates. Spectra obtained with the
SE and SWE procedures are contrasted in Figure 4; a syn-
thetically prepared sample of phenol in brackish marsh
water containing suspended particulates was measured. This
example illustrates the obvious advantage of the SE tech-
nique, i.e., its elimination of scatter interferences.
FLUORESCENCE
2 INTENSITY
RAYLEIGH/RAMAN
SCATTERING WEDGE
TOTAL SPECTRUM
EXCITATION
WAVELENGTH
Y
EMISSION
WAVELENGTH
\ v
Figure 2: A Three Dimensional Presentation of a Rayleigh-
Tyndall/Raman Radiation "Scattering Wedge", Superim-
posed on a Total Fluorescence Spectrum of a Hazardous
Material
FLUORESCENCE
INTENSITY
SE SPECTRUM
SWE SPECTRUM
- RAYLEIGH/RAMAN
SCATTERING WEDGE
EXCITATION
WAVELENGTH
Y
EMISSION
WAVELENGTH
Figure 3: SE and SWE Fluorescence Spectra, and a Rayleigh-
Tyndall/Raman "Scattering Wedge" (This Demonstrates
that the Scattering Wedge is Readily Avoided by Use of the
SE Technique)
-------
Use of Spectroscopy
PHENOL IN WATER
WATER BLANK
SWE SPECTRA
'
9
;.
-
.
SE SPECTRA
270
EXCITATION
WAVELENGTHS (ntn)
Figure 4: SE and SWE Spectra of Phenol "Spiked" into
Water Obtained from a Marsh in Sayreville, New Jersey
(The Water Contains Suspended Colloidal Particulates.
Phenol Concentration is 1 mcl/1)
Raman scatter interferences emanating from water are
less pronounced than Rayleigh-Tyndall scatter. They be-
come apparent, however, when using high instrument sen-
sitivities for measuring low toxicant concentrations. The
SWE technique fails to avoid this scatter problem, while the
SE procedure avoids it readily. Elimination of scatter inter-
ferences by the SE procedure also minimizes the need for
blank or background sample analyses. SWE techniques re-
quire blank sample measurements whenever instrument
parameters are changed resulting in a large number of addi-
tional and time consuming analyses.
Elimination of scatter interferences by using the SE pro-
cedure also permits increased sensitivity. While actual de-
tection limits obtained with the SE procedure are deter-
mined by a fluorescence instrument's design, SWE tech-
niques detection limits are severely influenced by scatter
interferences. Detection limits of several hazardous materials
in distilled water, determined by the SE technique, are
listed in Table I.
The effect of indigenous fluorescent species in natural
waters, on analysis results is illustrated in Figure 5. The SE
fluorescence spectrum of aniline in Raritan River water is
shown in Figure 5a while the SE fluorescence spectrum of
aniline in Arthur Kill River water is shown in Figure 5b.
These rivers represent heavily trafficked and polluted
bodies of water in New Jersey. The SE fluorescence spec-
trum of aniline in distilled water is shown in Figure 5c. The
spectrum of the latter figure was used as standard for quan-
tifying aniline in Figure 5a and 5b; recoveries were 96% and
94%, respectively. In effect, the aniline in distilled water
solution was used as the standard for quantifying the ani-
line content of synthetically prepared samples containing
this hazardous material in two polluted rivers waters.
Table I: Detection Limits for Several Hazardous Materials
in Water
Hazardous Material
Aniline
0-Ctesol
Dodecyl Benzene Sulfonic Acid
Naphthalene
Naphthenic Acids
Phenol
Quinoline
Resorcinol
Styrene
Toluene
Xylene
Xylenol
Detection Limit (ppm)
<
0.1
0.1
0.02
0.1
0.01
0,005
0.02
0.005
0.1
0.1
0.1
The fluorescence intensity versus concentration plot of
aniline in water is illustrated in Figure 6. Linearity extends
to slightly above 20 mg/1. Measurements of solutions having
concentrations below this value should be free of concen-
tration quenching phenomena. The deleterious effects of
quenching become progressively worse at higher concentra-
tions. Fluorescence intensities may actually decrease with
increasing concentration when aniline in water concentra-
tions exceed 200 mg/1. Deviations from linearity, therefore
must be considered, if grossly erroneous hazardous material
quantifications are to be avoided. Excessively high concen-
trations yield spectral shifts to longer wavelengths, in addi-
tion to non-linear plotted results. These properties are use-
ful for establishing optimum concentrations for the quanti-
fication of hazardous materials.
The effect of various wavelength interval adjustments
between emission and excitation monochromators, when
using synthetically prepared samples of o-cresol in water is
illustrated in Figure 7. Polluted water from a stream adjoin-
ing an oil refinery was used in these samples. The different
wavelength intervals clearly determined the degree to which
indigenous fluorescent materials in this water distorted the
spectrum of o-cresol. An evaluation of the "optimum wave-
length interval" is necessary for each type of sample analy-
sis of a hazardous material in water. The optimum wave-
length interval in Figure 7 is 40 nm. This interval sacrifices
-------
Use of Spectroscopy
a small amount of sensitivity in exchange for adequate
spectral resolution from indigenous fluorescent species.
Optimum wavelength intervals between emission and exci-
tation monochromators were determined for several hazard-
pus materials in a number of polluted waters (Table II).
— WATER BLANK
|
J
A
EXCITATION WAVELENGTHS |nm|
Figure 5: SE Spectra of Aniline "Spiked" into River Waters
(A) and (B) and Distilled Water (C). Aniline in Water Con-
centration is 1 mcl/1. The Waters used to Obtain (A) and (B)
were Collected from the Raritan and Arthur Kill Rivers in
New Jersey, Respectively. Both Rivers are Subject to Heavy
Marine Traffic and Chronic Pollution from Petroleum and
Industrial Chemicals
100
180
EXCITATION WAVELENGTHS (nml
Figure 7: SE Spectra Obtained at Three Different Wave-
length Intervals Between the Emission and Excitation Mono-
chromators. The Spectra Derive from 1 mcl/1 of o-cresol
Spiked into Water Collected from the Woodbridge Creek in
Sewaren, New Jersey. Dashed lines Represent Water Blanks.
Table II: Optimum Wavelength Intervals for Quantification
of Several Hazardous Materials in Water
Hazardous Material
Aniline
Benzene
Benzole Acid
0-Cresol
Dodecyl Benzene Sulfonic Acid
Ethyl Benzene
Naphthalene
Naphthenlc Acids
Phenol
Quinoline
Resorcinol
Strichnine
Styrene
Toluene
Xylene
Xylenol
Optimum Wavelength Interval (nm)
20
35
15
40
50
20
45
60
25
60
60
40
60
15
15
JO
60 100 140
CONCENTRATION (mcl/1)
Figure 6: Concentration Versus Fluorescence Intensity Plot
of Aniline in Distilled Water
SUMMARY
Use of SE fluorescence spectroscopy was evaluated for
in-situ quantifications of hazardous materials in water. This
method is discussed and contrasted with SWE methods
within the context of a three dimensional coordinate
system. The relative impact of Rayleigh-Tyndall and Raman
scatter interferences on quantifications by SE and SWE
methods are contrasted and the advantage of the SE tech-
nique is illustrated. In-situ quantifications of several hazard-
ous materials in representative polluted waters were per-
formed, good recovery capabilities were demonstrated and
analysis parameters are provided. The SE fluorescence pro-
-------
Use of Spectroscopy
cedure is shown to facilitate accurate quantifications of
fluorescent hazardous materials directly in water, thereby
avoiding the tedious and time consuming solvent extraction
steps that are normally associated with quantifications of
organic toxicants in water.
ACKNOWLEDGMENTS
The Authors wish to express grateful appreciation to Mr.
Nick Pangaro, Ms. Pamela Ward, Ms. Jocelyn Callender and
Mr. John Munn, for the data acquisition that has. made
these findings possible.
REFERENCES
1. Kullbom, S.D., Smith, H.F.and Flandreau, P.S. Fluores-
cence Spectroscopy in the Study and Control of Water
Pollution. Abstracts of the Pittsburgh Conference on
Analytical Chemistry and Applied Spectroscopy Paper
No. 288, Cleveland, Ohio, (1970).
2. Goldberg, M.C., and Devonald, D.H. Fluorescence
Spectroscopy: A Technique for Characterizing Surface
Films./. Res. U.S. Geol. Survey 1 (6): (1973)709-717.
3. Gordon, D.C., Jr., and Keizer, P.D. Estimation of Petro-
leum Hydrocarbons in Seawater by Fluorescence Spec-
troscopy: Improved Sampling and Analytical Methods;
Fisheries and Marine Services Technical Report No. 481.
Environment Canada, (1974).
4. Frank, U. A Method for Quantitating Oil Directly in
Water by Fluorescence Spectrophotometry. Analyt.
Quality Contr. Newsl, 18:9-10. U.S. Environmental Pro-
tection Agency, Cincinnati, Ohio, (1973).
5. Keizer, P.D., and Gordon, D.C., Jr. Detection of Trace
Amounts of Oil in Sea Water by Fluorescence Spectro-
scopy. Journal of the Fisheries Research Board of
Canada 30(8), (1973), 1039-1046.
6. Zitko, V., and Tibbo, S.N. Fish Kill by an Intermediate
Oil From Coke Ovens. Bulletin of Environmental Con-
tamination and Toxicology 6(1), (1971), 24-25.
7. Lloyd, J.B.F. The Nature and Evidental Value of the
Luminescence of Automobile Engine Oil and Related
Materials-I. Synchronous Excitation of Fluorescence
Emissions./. Fores. Sci. Soc. 11 (1971), 83-94.
8. John, P., and Soutar, I. Identification of Crude Oils by
Synchronous Excitation Spectrofluorimetry. Anal
Chem. 48(3), (1976), 520-524.
9. Frank, U. Identification of Petroleum Oils by Fluores-
cence Spectroscopy. Proceedings of Joint Conference on
Prevention and Control of Oil Pollution, San Francisco,
California: American Petroleum Institute, (1975) 87-91.
10. Frank, U., and Gruenfeld, M. Determination of Petro-
leum Oils in Sediments by Fluorescence Spectroscopy
and NMR. Abstracts of the Pittsburgh Conference on
Analytical Chemistry and Applied Spectroscopy, Paper
No. 400, Cleveland, Ohio, (1971).
11. Buckley, J.L. and Weiner, S.A. Hazardous Material Spills:
A Documentation and Analysis of Historical Data. EPA
Contract No. 68-03-0317, Industrial Environmental
Research Laboratory, U.S. Environmental Protection
Agency, Cincinnati, Ohio (1976).
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I, HAZARDOUS CHEMICALS
B, OTHER ANALYSES
10
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(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #20, January 1974.
STABILITY OF STORED CHRQMATOGRAPHIC AND SPECTRAL DATA
A severe fading problem of chromatograms and spectra recorded in
red ink has been encountered. This appears to be inherent to
several commercial red inks and manifests itself only after pro-
longed storage of such data output. These chromatograms and
spectra were used in our laboratory for display purposes, and
were generally found to fade beyond recognition within 16 months.
Similar data recorded in black ink exhibited no image degrada-
tion. The commercial sources of the red inks involved cannot be
positively identified because such records were not maintained.
A cursory test was developed that is thought to have predictive
value about the image stability of inks. Normal thickness lines
of several red, black, and blue inks were exposed to the fumes
of full strength hydrochloric acid, for 30 seconds. Two of the
four red inks exhibited severe fading, the one blue ink tested
exhibited some fading, while the two black inks tested did not
fade. It is believed that these test results correlate well with
observation of stored spectra, especially because the two red
inks that faded in this test are thought to be the ones that
faded during data storage. These observations underscore the
need for care in the choice of inks.
(F. Behm/M. Gruenfeld, FTS 201-648-3542, Coml. 201-548-3347)
* See page 2.
11
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(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #25, April 1975.
Determination of EDTA by NMR
The possible discharge of sodium ethylenediaminetetraacetate
in some industrial waste effluents is being evaluated by the
Region II Edison Laboratory. This compound is present as the
tetrasodium salt (Na.EDTA) in some sommercial formulations of
boiler cleaners, and is used as a chelating agent. As a first
step in determining the concentration of Na.EDTA in waste eff-
luents, distilled water samples were prepared from a commercial
formulation and analyzed by ASTM Method D 3113-72T. Erroneously
low results were obtained, however, and these were ascribed to
the presence of interfering additives. The samples were then
analyzed with a 60 MHz Nuclear Magnetic Resonance (NMR) Spectrom-
eter and the results agreed very closely with the declared form-
ulation. The accuracy of the NMR determination was also confirmed
by using a pure reference standard. Na.EDTA exhibited two sharp
singlets at 3.1 and 2.6 ppm, relative to sodium 3-trimethylsilyl-
propane sulfonate, and integration of these peaks yielded the
expected ratio 2:1. The 3.1 ppm peak was used for quantitating
the EDTA salt/ and methanol was selected as the internal standard.
The NMR detection limit of Na.EDTA in the distilled water samples
of the formulation was 0.5% (w/v). We are now testing a number
of concentration procedures in anticipation of actual industrial
wastewater samples. (U. Frank/F. laconianni, FTS 340-6626, Coml.
201-321-6626)
* See page 2.
12
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<*) ANALYTICAL QUALITY CONTROL NEWSLETTER #29, April 1976.
Kepone Analyses
At the request of the special EPA Kepone Task Force, established
by the Office of the Administrator, Washington, D.C., our labora-
tory conducted analyses to measure the removal of Kepone Pesti-
cide from water that had been contaminated in the washdown and
dismantling of the Life Sciences Products Plant in Hopewell,
Virginia. Our "Mobile Hazardous Spills Treatment System", which
utilizes three activated carbon columns about 7 ft high x 7 ft
in diameter, was used for the on-site removal of 99.996% of
Kepone from the washdown water. Samples deriving from clean up
operations of 22 railroad tank cars containing 220,000 gallons
of contaminated water were concurrently analyzed by extracting
one liter portions with one 40 ml and two 30 ml aliquots of
benzene in glass stoppered graduated cylinders. A minimum of
1 part per billion (pg/l) of Kepone was detected by electron
capture gas chromatography without concentrating the extracts.
Column influent concentrations ranged from 100 to 3 ppm (mg/1);
treated effluent concentrations ranged from 6 to 0.2 wg/1 of
Kepone. This data was verified by two other laboratories that
analyzed rejplicate samples and used diethyl ether as the ex-
traction solvent. Procedures and results of these interlabora-
tory analyses are available on request. (U. Frank, FTS 342-
7510, Coml. 201-548-3347, J. Lafornara, FTS 342-7523)
Mobile Laboratory
Several ongoing extramural efforts at Edison involve the develop-
ment of large scale hazardous material clean up systems that are
needed to provide flexibile emergency response capability in
the event of accidental spills of hazardous materials. Optimum
utilization of these systems necessarily requires minimum
analytical turn around time in order to monitor treatment effec-
tiveness. At the present time, samples are usually mailed or
otherwise transported to laboratories that are distant from the
spill site, and this often causes intolerable delays. Conse-
quently, we constructed a mobile laboratory to support the
treatment systems in the field, during emergency response situ-
ations. The laboratory was constructed in a 35 ft semi-trailer,
equipped with heating and air conditioning systems. Laboratory
construction was accomplished largely through in-house efforts,
using laboratory furniture and accessories that were available
within our facility. The mobile laboratory is fully equipped
for solvent storage, and contains a fume hood, explosion proof
refrigerator, computerized gas chromatograph, and an infrared
spectrometer. An atomic absorption spectrometer will soon be
added. Other associated equipment includes a hazardous spill
detection field kit, pH meter, centrifuge, and a sanitary facil-
ity, distilled water supply, and a solvent disposal facility.
This mobile laboratory was built in about four months, at a cost
of approximately $5,000. This figure includes both salaries and
hardware, but does not include the cost of analytical instruments
Testing of the laboratory is now in progress, prior to actual
use. (U. Frank, FTS 342-7510, Coml. 201-548-3347, R. Frederick,
FTS 342-7524, M. Urban, FTS 342-7517)
13
* See page 2.
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(*)ANALYTICAL QUALITY CONTROL NEWSLETTER #32, January 1977.
MOBILE LABORATORY
Our mobile laboratory (described in AQC Newsletter No. 29, April
1976) just completed 6 weeks of field operation in Haverford,
Pennsylvania. The laboratory was used to monitor the effecti-
veness of pentachlorophenol (PCP) removal operations from ground
water, using a mobile activated carbon treatment system. Con-
centrations of PCP in water exceeded 20 mg/1 (ppm) before treat-
ment, and were less than 1 mcg/1 (ppb), after treatment. Appro-
ximately 150 sample analyses were performed in the mobile lab-
oratory, on site, using gas chromatography. Concurrent oil
analyses were also performed using infrared spectroscopy. A
method by Chau, A.S.Y. and Coburn, J.A., JAOAC, 57 (2), 389 (1974),
was used for the PCP analyses. (M. Urban/R, Losche, FTS 340-
6628, Coml. 210-321-6628.)
* See page 2.
14
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(*) ANALYTICAL QUALITY CONTROL NEWSLETTER // 35, October 1977.
Analysis of Hazardous Materials in Water by NMR
A preliminary evaluation of the use of NMR for the analysis of
ppm levels of hazardous materials.in water has been performed.
Fifty milligram portions of activated carbon retained in a
funnel over glass wool were used to adsorb ppm levels of cyclo-
hexane and xylene from one liter of water. Following this
"filtration" step, the carbon was rinsed with 2 ml of deuterium
oxide and transferred to an NMR tube containing 1.0 ml carbon
tetrachloride. The presence of powdered carbon in the NMR
tube caused little spectral interference; only slight peak
broadening was observed. Recoveries were in the range 85 -
90 percent. Evaluation of this method for other hazardous
materials listed in 40 CFR Part 116 is now underway. (U. Frank,
Coml. 201-321-6626, FTS 340-6626)
Interference by Sodium Sulfate Impurities
While analyzing water samples for PCBs at sub-part per billion
levels (0.05) by GC-ECD, interferences were encountered that
were traced to sodium sulfate. Sodium sulfate was used for
dehydrating the extraction solvent prior to concentration.
Impurities in sodium sulfate yielded extraneous peaks with
retention times that overlapped the PCS range. Repeated rins-
ings of sodium sulfate with 15 percent methylene chloride in
hexane failed to eliminate the interferences. This problem
was successfully resolved by the method of Garrison, (AQC News-
letter No. 14, July 1972), by maintaining sodium sulfate at
600 C for two hours prior to its use for dehydration. (R.
Losche/M. Urban, Coml. 201-321-6628, FTS 340-6628/M. Gruenfeld,
Coml. 201-321-6625, FTS 340-6625)
* See page 2.
15
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(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #36, January 1978.
Evaluation of an Emission Spectroscope
The evaluation of a low cost emission spectroscope capable of
rapid identifications of metals is described. This unit, a
Vreeland Spectroscope, Model 7 (Spectrex Company, Redwood City,
California) costs approximately $3,000 and incorporates a novel
method for rapid spectral interpretation. Matching of spectral
lines is accomplished by comparison of sample spectra with spec-
tral reference lines that are incorporated into a movable reference
film in the instrument. Spectral interpretation and instrument
use are simple and require little experience. Our anticipated
use of this instrument is aboard the Mobile Spills Laboratory de-
scribed in AQC Newsletters 29 and 32. Instrument dimensions are
approximately 16" H X 18" W X 24" L, and it weighs approximately
35 Ibs. Synthetic water and sediment samples containing mix-
tures of the following metals were analyzed: V, Cr, Ni, Co, Ti,
Pb, Ba, and Hg. The instrument yielded reliable identifications
at high metal concentrations (0.5%), but was less useful at low
concentrations (below 0.1%). These samples required preconcen-
tration prior to measurement. The emission spectroscope will be
used in conjunction with the Atomic Absorption Spectrometer while
responding to spills of hazardous substances containing toxic
metals. It will be used for monitoring source samples or grossly
contaminated environmental samples to identify their metal con-
tent. Quantification of metals identified in this manner will
be accomplished by Atomic Absorption Spectroscopy. (R. Losche,
Coml. 201-321-6628, FTS 340-6628/M. Gruenfeld, Coml. 201-321-
6625, FTS 340-6625)
* See page 2.
16
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(*) EPA QUALITY ASSURANCE NEWSLETTER, Volume 1 No.2, July 1978.
Removal of Sulfur from Sediment Extracts - Estuarine sediments
were recently analyzed for PCB content according to EPA's
manual, "Analysis of Pesticide Residues in Human and Environ-
mental Samples". Removal of the sulfur in the hexane extracts
was accomplished by modifying a more complex column procedure
detailed in the EPA report, "Correlation of Oils and Oil Pro-
ducts by Gas Chromatography", (EPA-600/2-77-163). Each extract
concentrate (2 ml) was eluted through a short stem disposable
Pasteur pipette (9 in. length) packed with copper. This was
prepared by placing a glass wool plug at the base of the
pipette, and adding 2-2.5 cm of copper metal (electrolytic
dust). This was activated with one column volume of 1 N HCL
followed by a rinse of two column volumes of hexane. Samples
were then passed through the columns to remove sulfur. The
bright copper turned black as the sulfur was removed, and the
solvent flow was accelerated as necessary, by using positive
pressure from a pipette bulb. (D. Stainken, Coml. 201-321-
6628, FTS 340-6628).
Sediment Hydrocarbon Quantification - While quantifying vola-
tiles and extractable hydrocarbons in estuarine sediments, a
statistical relationship was found between the silt-clay frac-
tion of the sediments and percent volatiles. Generally, the
percent volatiles of the sediments ranged from 0.85 to 11.39,
and increased as the silt-clay fractions of the sediments in-
creased. Percent volatiles were determined by heating pre-
dried sediments in a muffle furnace at 500-800 c for 24 hours.
Modifying a procedure by Giger et al, (1974, Environ. Sci. &
Technol. 8_, 454) , extractable hydrocarbons in dried sediments
were measured after tumbling the ground sediments in flasks
containing Freon®113 solvent. The extracts were quantified
by IR spectroscopy following silica gel treatment. This
procedure is described by Gruenfeld and Frederick (Publication
announced in AQC Newsletter 36). A statistical relationship
was also found between the percent volatiles and micrograms
of extractable hydrocarbons. Values of extracted hydrocarbons
ranged from 2.2 to 1098 ug/g of dry sediment. Generally,
the amount of hydrocarbons extracted increased as the amount
of volatiles increased. GC analysis of selected samples ex-
hibited a large unresolved complex mixture with few well re-
solved peaks. The maximum peak in many of the chromatograms
had a retention time that matched the n~C alkane. An n-C__
sewage derived hydrocarbon has been reported from English
sediments, and it is possible that this n~c23 compound derives
from sewage. Analysis of these samples by synchronous ex-
citation fluorescence spectroscopy (described in AQC News-
letters 31-34 and 36) indicated the frequent presence of 1-6
ring polynuclear aromatic hydrocarbons (PAH). Sediments with
large amounts of volatiles, and silt-clay fractions, are there-
fore expected to contain large amounts of extractable hydro-
carbons, possibly necessitating several extractions. The
hydrocarbons may be PAH or other complex mixtures. (D.
Stainken, Coml. 201-321-6628, FTS 340-6628)
* See page 2.
17
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Interaction of m-Cresol with Plastic Tubing - While using pilot
scale water decontamination equipment to evaluate the effi-
ciency of activated carbons for removing organic hazardous'mate-
rials from water, we examined the interaction of one of the
materials, m-cresol, with two kinds of plastic tubing, m-cre-
sol solutions in water/ at 100 ppm, were exposed to clear in-
dustrial grade PVC tubing, and to bev-a-line VHT tubing (Ther-
moplastic Scientifics, Inc., 57 Stirling Road, Warren, New
Jersey 07060). Contact between the m-cresol solution and the
tubing was accomplished by: (1) maintaining an unbroken solution
flow at'approximately 15 ml/min. through 9 foot lengths of
each tubing; (2) maintaining the solution flow, containing air
gaps, through 19 feet of the clear PVC tubing, and (3) perform-
ing a static test whereby portions of solution were sealed
for two hours in one foot lengths of both types of tubing.
These tests yield progressively longer contact times between
the m-cresol solution and the plastic materials. The bev-a-
line tubing was of special interest because it is less rigid than
Teflon®tubing, and according to product literature it strongly
resists chemical interaction. Our test findings confirm this
for m-cresol. While m-cresol was progressively lost to the
clear industrial grade PVC tubing in amounts proportional to
increasing contact time, no measurable loss to the bev-a-line
VHT tubing occurred. Test results were as follows: test (1)
yielded no measurable loss of m-cresol to either tubing mate-
rials; test (2) yielded a lowering of m-cresol from approx-
imately 100 ppm to approximately 93 ppm by the clear PVC
tubing; while test (3) yielded a lowering of m-cresol from
approximately 100 ppm to approximately 60 ppm by the clear
PVC tubing, but no lowering by .the bev-a-line tubing. {M.
Royer/S. Frysinger/M. Gruenfeld/G. Fraser, Coral. 201-321-
6627, FTS 340-6627)
18
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(*) EPA QUALITY ASSURANCE NEWSLETTER Volume 1, No. 3, October 1978
Analysis of Polynuclear Aromatic Hydrocarbons in Estuarine -
Water - Raritan Bay bottom waters, sediments, and clams were
surveyed to determine the quantities of polynuclear aromatic
compounds (PNA's) present. The estuarine bottom water samples
were recently analyzed for PNA content using synchronous
excitation (SE) fluorescence spectroscopy (Quality Assurance
Newsletter, Volume 1, Number 1, April 1978). Use of the SE
technique yielded spectra of PNA mixtures that were resolved
according to the number of aromatic rings. Our procedure in-
volved the extraction of one liter water samples with two 50 ml
portions of cyclohexane (glass distilled). Emulsions were
alleviated by adding five ml of concentracted HC1 prior to
extraction. One to three grams of anhydrous sodium sulfate
(precleaned by baking three hours at 450 C) was added to each
extract. SE spectra were then obtained using a 25 nm interval
with the excitation monochromator leading the emission mono-
chromator. SE spectra of PNA standards at concentrations of
one mg/1 in cyclohexane were similarly obtained for comparison
purposes. Confirmation of the samples' PNA content was ob-
tained by HPLC analyses of sample extract concentrates.
Appropriate HPLC fractions were collected and analyzed by SE
and single wavelength excitation fluorescence spectroscopy.
Results indicated that PNA compounds in the water were in
the low ppb range. The identity of some of the compounds de-
tected included isomers of methyl naphthalenes, pyrene, and
benz-a-pyrene. The SE fluorescence method is currently being
used to analyze sediments and bivalve tissue extracts for
their PNA content. (D. Stainken, Coml. 201-321-6628, FTS 340-
6628/U. Frank, Coml. 201-321-6626, FTS 340-6626)
* See page 2.
19
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REPRINTED FROM:
Chemical Marketing Reporter, April 3,
1978, pp 61 & 64.
Mobile Lab Johnny-on-the-Spot
In EPA's Toxic Spill Operations
Two summers ago, residents of
Haverford,Pa..noticed thaf'oil slicks"
were forming in a nearby stream
called Naylors Run. The pollution
seemed most evident at a number of
places where storm drains and springs
emptied into the Delaware River trib-
utary. Alarmed, they reported the
problem to the regional office of the
Environmental Protection Agency.
Normally.KPA would have obtained sam-
ples of the stream's water and sent them
to a large laboratory equipped to carry out
the sophisticated analyses necessary to de-
tect chemicals which may be toxic in con-
centrations as low as a few parts per mil-
lion.
That time-consuming process was avoid-
ed, however, by bringing EPA's mobile lab-
oratory Into action. An addition of only a
few years to the agency's physical assets,
the laboratory consists of a close-packed
array of delicate detection and measuring
Instruments housed in the back of a semi-
trailer truck.
•LAB ARRIVES QUICKLY
It took little time for the laboratory,
manned by two chemists, to be driven to
Haverford from Its base with EPA's Oil and
Hazardous Materials Spills Branch in Edi-
son, N.J. Once .on the scene, where strong
chemical odors hung above the stream and
dead and dying fish had begun to dot the
surface, the team set to work quickly. A
combination of field investigation and labo-
ratory analysis pinpointed the source of the
pollution at a wood-treating operation
where a 5% solution of pentachlorophenol
(PCP) suspended in fuel oil was used as a
preservative. Tests by a Perkin-Elmer in-
frared spectrophotometer and a gas chro-
ma tog r a ph confirmed that the Naylors Run
oil slicks contained large amounts of PCP.
EPA investigators later discovered that
the wood-preservative plant site was satu-
rated with the chemical. Wastes from the
operation had been injected by the compa-
ny into a twenly-five-foot deep well for ap-
proximately thirty years, and when .the
well could hold no more, P0P-containing oil
had seeped into an underground culvert,
leaked into storm drains and emptied into
Naylors Hun. The location of the pollution
made the agency particularly apprehen-
sive, because the stream ultimately flows
through the suburbs of Philadelphia and
into Tinicum National Wildlife Refuge, the
last freshwater tidal marsh in Pennsylva-
nia.
PCP, a fungicide and pesticide, is mixed
with fuel oil before being applied to wood as
a preservative. It is commonly used on
farm buildings and other outdoor struc-
tures, much like creosote. It has also been
linked to many human health problems, in-
cluding lung, liver and kidney damage. A
year ago, PCP was blamed for the death of
a number of dairy cows in Michigan, but
subsequent testing was unable to prove that
the chemical, present in large quantities in
the dead animals, was the cause.
At Naylor's run, the EPA laboratory
team determined that the concentration of
PCP In the oil was greater than 10,000 parts
per million (ppm) and in the stream water
was 0.5 ppm. Concentrations of 0.2 ppm are
lethal to fish.
POLLUTION ATTACKED
The laboratory was accompanied to
Haverford by another arm of EPA's mobile
strike force - a.hazardous spill treatment
system consisting of trailer-mounted acti-
vated carbon columns and prefillration,
flocculation and sedimentation tanks.
Through well-drilling and trenching to
collect oil-contaminated groundwater,
skimming of the oil in tanks and the use of
the activated carbon columns, the Oil and
Hazardous Materials Spills Branch and
EPA's Region III response team cut the
PCP concentrations in the water to below 1
part per billion (ppb). It took three months
to process 210,000 gallons from the stream.
The laboratory chemists monitored the effi-
ciency of the clean-up effort throughout
with their instruments.
The two mobile units have been used In
a number of detection and clean-up opera-
tions.
"Once we become involved in a toxic
chemical spill," says Michael Gruenfeld,
director of the Oil and Hazardous Materials
Spills Branch, "because of the urgency of
the situation it is not always logistically
feasible to request analytical support front
permanent laboratory facilities. If we were
to depend on a centralized laboratory, or
olhor laboratory, it would mean tak-
ing samples, transporting them .to the labo-
ratory and waiting for answers. By the
time we received the analysis, the priori-
ties for the spill clean-up might have
changed.
"Additionally", he explains, "laborato-
ries normally do not do this specific type of
20
-------
work, nor would they want to interrupt
their normal flow to handle it. Therefore,
we often send experts into the field to do
these dedicated analyses."
The panelled inside of the mobile labora-
tory bristles with the machines of precise
chemical analysis. The mainstays arc the
Perkin-Elmer atomic absorption spectro-
photometer, infrared and flourescense
spectrophotomcters and a gas chromalo-
graph.
The atomic absorption instrument can
analyze metallic components of a sample in
concentrations as low as ppb. The technolo-
gy of the EPA machine, which uses a heat-
ed graphite atomizer to reduce the sam-
ples, is three times more delicate a detec-
tion device than the earlier generation of
atomic absorption spectrophotometers,
which worked on what is called the "flame
mode."
"The fluorescense instrument," Mr.
Gruenfcld says, "has an extremely high
sensitivity for materials that fluorcsce
under ultraviolet light. It is valuable for
aqueous analyses at a site and possesses
the advantage over gas chromatograph
analysis in not needing a solvent in sample
preparation. A sample can be used directly
from the source. It also has the ability to
measure the presence and levels of fluores-
cing toxicants, even if they remain un-
known. The identity of contaminating mate-
rial can then be adduced from corre-
sponding evidence. In some cases, the in-
strument has been able to measure reduc-
tions in levels of unknown aromatic toxi-
cants, following water treatment, while
other labs were still trying to identify the
contaminants."
The infrared spectrophotometer analy-
zes petroleum oils that are used as vehicles
for toxicants. The device can also identify
unknown materials through "fingerprim"
spectra. The classified spectra are stored
in the Edison library of spectra, and the
mobile lab has at times called into the li-
brary for telefacsimile transfer of a spec-
trum's distinguishing characteristics.
The gas chromatograph separates com-
plex mixtures into their components. The
lab can also carry out total organic carbon
analysis (TOC), a process in which the total
organic content of water is rapidly meas-
ured without extraction.
For the most part, the rolling lab has
dealt with bodies of water than have been
contaminated and that threaten drinking
water supplies or aquatic life.
For the purposes of broad distinction,
OHM divides spills and their subsequent
clean-up problems into three categories.
"The different environments where
water-threatening or contaminating spills
occur can be divided into spills on soils, into
stream or into large unconfined bodies of
water," says Frank Freestone, head of the
hazardous materials spill group at Edison.
"Water bodies present different problems
depending on their size. A small stream or
pond, for instance, in an impoundablo, or
damablc, body of water. A spill could be
isolated from the remainder of the stream,
permitting clean-up to proceed relatively
easily.
"Large water courses are unconfinable.
The treatment approach must be different
for such large bodies of water.
"Our function has been to develop large-
scale equipment to return the environment
as closely as possible to its prespill state,"
Mr. Freestone adds. "Our analytical work
is in support of this primary engineering re-
sponsibility. We have to provide answers as
to how well the equipment words, and also,
research in how to improve the engineer-
ing."
VANDALS CAUSE SPILL
The hazardous materials detectives got
another chance to test their sleuthing ma-
chinery last June. Vandals in Kernersvillc,
N.C. crept onto the grounds of a chemical
wastes disposal company there one night
and opened the valves of six storage tanks.
Over 30,000 gallons of mixed wastes gushed
into the town's reservoir.
It didn't take long for the townspeople to
realize something was wrong. Vapors drove
a thousand residents from their homes
and dead fish began to float to the surface.
Mistakenly, local authorities believed an oil
spill had occurred. When they treated the
water accordingly, there was no effect.
At that point, the EPA mobile laboratory
was called in. Fish were dying in increas-
ing numbers, fleeing the spreading chemi-
cals to the mouths of the inlets of the reser-
voir. Within seventy-two hours, the toxic
chemicals overtook the fish and killed
20,000 of them, about 90 percent of the
aquatic life of the reservoir. Town officials
reacted by closing down the water source.
For the EPA chemists, the problem was
a tough one. The toxicants were unknown
and thoroughly mixed, meaning proper
identification would be a lengthy process.
The scientists ran samples of the polluted
water through a pilot-scale carbon adsorp-
tion system. That, tied in with a bioassay
system, revealed that the water going into
the carbon columns was toxic, but the
water coming out was not. The discovery
suggested to the EPA team the most likely
way to clean up the reservoir - carbon ad-
sorption.
"The fluorescense device was very valu-
able in this situation," Mr. Gruenfeld says.
"By running samples with the fluorescense
spectrometer, we were able to isolate dis-
tinctive profiles that were not present in
samples taken before the spill occurred.
"It was intial use of the mobile treat-
ment system that first pointed to the need
for a mobile laboratory when it was noted
that although oil spills are visible enough
to locate and isolate, chemical spills most
often are not. With the critical aid of the
laboratory, we are able to deal with an
acute public safety hazard*, such as chlo-
rine, as" well as with hazards of a chronic
nature like PCB's, or low-level pesticide
releases, on-a nationwide 24-hour-response
basis. In effect, the mobile laboratory is an
extension of the base laboratory, bringing
to the field all the sophisticated technology
that is required, in more immediate and
timely way."
21
-------
TREATMENT SYSTEM: EPA'* trailer-mounted hazardous materials treatment
with activated
orescence spectrophotometer.
22
-------
REPRINTED FROM: Proceedings of National Conference on Control of Hazardous
Material Spills, Miami Beach, Florida, April 11-13, 1978,
pp 311-314. Available from API Washington B.C.
Development and Use of a
Mobile Chemical Laboratory
for Hazardous Material Spill
Response Activities
Michael Urban and Richard Losche
U.S. Environmental Protection Agency
Edison, New Jersey
INTRODUCTION
As part of a continuing effort to provide prompt re-
sponse to spills of oil and hazardous materials, EPA's Oil
and Hazardous Materials Spills Branch at Edison, New
Jersey, developed a Mobile Spills Laboratory to perform
chemical analyses in remote field locations. The laboratory
was specifically designed to provide on-site analytical ser-
vices during hazardous material spill response and cleanup
operations. It was constructed to aid in the cleanup of spill
impacted areas by eliminating the delay associated with the
transport of samples to distant laboratories for analysis. Im-
mediate laboratory data is often urgently needed to make
decisions regarding spill cleanup efforts and to monitor
cleanup efficiency.
The laboratory was constructed within a 35-foot van
semi-trailer, obtained as an excess government property
item (Figure 1); the van is equipped with heating and air
conditioning systems. Laboratory construction was accom-
plished largely through the efforts of Branch personnel
using available laboratory furniture and accessories; the in-
terior construction required four months and cost approx-
imately $5,000. This figure includes salaries and hardware
but excludes analytical instruments and supplies.
Capabilities
In order to provide optimum laboratory services during
any potential spill situation and to assure analysis of virtu-
ally all organic and inorganic hazardous substances (pesti-
cides, PCB's, heavy metals, etc.), the Mobile Spills Labora-
tory was equipped with a spectrum of sophisticated analyt-
Figure 1: Exterior View of the EPA Mobile Spills Laboratory Operating in a Remote Field Location
23
-------
Mobile Chemical Lab
Figure 2: Interior View of the Mobile Spills Laboratory. Instruments and Equipment Include a Fume Hood, Telefac-
similie, Fluorescence and Infrared Spectrophotometers, Gas Chromatograph, and an Atomic Absorption Spectrometer
ical instruments (Figures 2, 3 & 4) including (1) two gas
chromatographs, one computerized and one portable, both
with flame ionization and electron capture detectors, (2) a
computerized atomic absorption spectrophotometer with a
graphite furnace accessory and automatic sampling system,
(3) infrared and fluorescence Spectrophotometers and (4)
an emission spectrograph. Safety considerations necessita-
ted the inclusion of a fume hood, vented solvent locker, ex-
plosion-proof refrigerator, running water (100 gal.), safety
shower, eyewash, fire blanket, fire extinguishers and a
modification of the existing air conditioning and heating
systems, to allow a once through pass of air.
Other dedicated equipment include a telefacsimile,
which permits transmission of hard copy data (spectra,
chromatograms, methods of chemical analysis, etc.) via tele-
phone lines, a pH meter, balances, hot plates, magnetic
stirrers, desiccator, steambath and all necessary glassware,
solvents, reagents, and supporting equipment needed for
fully independent operation in remote field locations.
The laboratory was also equipped to carry a pilot scale
water treatment system consisting of four three-inch I.D.
by four foot deep pyrex columns. These can be charged
with activated carbon, ion exchange resins, or other mass
transfer agents to conduct treatment feasibility studies,
before initiating full scale cleanup operations.
Case Histories
The following summary describes the use of the Mobile
Spills Laboratory during several repsonse actions to hazard-
ous material spill incidents. Included is an overview of the
type of incident encountered and an outline of analytical
support provided. Additional detailed descriptions of the
incidents are provided by other authors1 >3.
Haverford, Pennsylvania
During November 1976, at the request of the U.S. EPA's
Region III Environmental Emergency Branch, the Mobile
Spills Laboratory was sent to Haverford, Pennsylvania. On-
site analytical assistance was required in cleanup operations
involving groundwater contaminated with pentachloro-
phenol (PCP) and oil. The contaminants had been leaching
into a stream called Naylor's Run, killing higher aquatic life
forms. The pollution originated from a wood preserving
company which, prior to 1963, disposed of its wastes by
means of shallow well injection on its property. Through
the years, the waste, which consisted of PCP in fuel oil, had
found its way into storm drainage pipes and ultimately into
Naylor's Run.
Cleanup efforts consisted of drilling several wells on the
company's property from which oil and contaminated
water could be removed. An intercepting trench was also
dug at the point where the contaminant had been entering
Naylor's Run, permitting its collection and subsequent re-
moval for treatment. Following the removal of the oil and
water mixture from the trench and wells, the oil was gravity
separated and incinerated. The water was purified by car-
bon adsorption, by the Mobile Physical Chemical Treat-
ment System (MPCTS)4. The Mobile Spills Laboratory con-
tinuously conducted numerous sample analyses for PCP and
oil to monitor the effectiveness of each stage of treatment
by the MPCTS. Electron capture-gas chromatrography and
24
-------
Mobile Chemical Lab
Figure 3: The Use of a Computerized Gas Chromatograph Aboard the Mobile Spills Laboratory for the Analysis of
Spilled Organic Hazardous Substances
Figure 4: The Use of a Computerized Atomic Absorption Spectrometer Aboard the Mobile Spills Laboratory tor the
Analysis of Toxic Metals
25
-------
Mobile Chemical Lab
infrared spectroscopy were used for the PCP and oil
analyses.
After treatment, the water was held in five 3,000 gal.
storage tanks for analysis prior to discharge. PCP concentra-
tions in the effluent were consistently below 1 ppb. Analy-
sis required less than one hour thereby permitting storage
of the treated water until its purity was determined while
continuing processing. In addition to support provided dur-
ing this cleanup, the laboratory was utilized daily to moni-
tor PCP levels in two sections of Naylor's Run. More than
125 analyses were performed on-site over a period of five
weeks in support of these efforts.
Ditrmer, Missouri
During April 1977, the Mobile Spills Laboratory was dis-
patched to Dittmer, Missouri, at the request of the U.S.
EPA's Region VII Emergency Response Section. On-site
chemical analyses were required to support cleanup opera-
tions involving an oil and mixed chemical spill. The spill
occurred when rainwater caused the contents of a waste
chemical disposal pit to overflow and contaminate a nearby
stream. PCB's (Aroclor 1260) were identified as major
components of the waste.
Cleanup efforts included diversion of the stream around
the contaminated area, excavation of the material from the
pit, disposal of this material in an approved landfill, treat-
ment of the stream water by the MPCTS, removal of con-
taminated vegetation and debris from the streambed and
installation of a field-improvised activated carbon water
treatment device for continued long term stream water de-
contamination. During the commitment of the Mobile
Spills Laboratory to this spill, numerous chemical analyses
were performed, using electron capture-gas chromatography.
This work supported each segment of the cleanup effort.
Measurements included the concentration of PCB's in the
contaminated streambed, at the perimeter of the pit during
excavation and in the stream water before and after treat-
ment by both the MPCTS and the field improvised treat-
ment device. Approximately 75 PCB analyses were per-
formed during the laboratory's one-month involvement in
this spill.
Kernersville, North Carolina
In June 1977, an oil and mixed chemical spill contami-
nated the water supply reservoir of Kernersville, North
Carolina. After the pollutants entered the reservoir, a total
fishkill resulted. The Mobile Spills Laboratory, together
with an EPA Region IV Mobile Bioassay Unit, were de-
ployed to the reservoir to monitor'pilot plant studies of the
ability of activated carbon to detoxify this water.
Numerous chemical analyses were performed aboard the
Mobile Spills Laboratory, using Synchronous Excitation
Fluorescence Spectroscopy5"7. Although the absolute con-
centrations of each component in the toxic mixture could
not be readily calculated by this technique, a semi-quantita-
tive fingerprint of the total mixture in water was obtained.
Reductions in concentration of the toxic mixture following
activated carbon treatment were measured; these data cor-
related well with the bioassay data. During a period of 12
days, more than 250 analyses were performed.
SUMMARY
The Mobile Spills Laboratory has proven itself to be an
excellent means for providing reliable analytical services in
remote field locations during hazardous material emergency
response actions. It can be readied for transport in a few
hours to any part of the United States, and operates with
total independence of other support. Telephone communi-
cations are maintained with a fixed laboratory facility in
Edison, New Jersey. Data output, calculations, reports, and
published and specially modified methods of analysis are
transmitted back and forth by telefacsimile. Addition of a
gas chromatograph-mass spectrometer to the complement
of instruments aboard the Mobile Spills Laboratory is now
planned. This will provide the laboratory with the on-site
capability to identify virtually any unknown organic haz-
ardous substance.
ACKNOWLEDGMENTS
The Authors wish to express grateful appreciation to Mr.
Michael Gruenfeld, Dr. Joseph Lafornara and Dr. Dennis
Stainken for their advice and assistance in preparing this
manuscript.
REFERENCES
1. Massey, T., F.J. Freestone, "Assessment and Control of
a Surface and Groundwater Contamination Incident,
Haverford, Pennsylvania, November-December 1977".
These "Proceedings"
2. Gilmer, H., FJ. Freestone, "Cleanup of an Oil and
Mixed Chemical Spill at Dittmer, Missouri, April-May
1977". These "Proceedings"
3. Stonebreaker, J., F.J. Freestone, Witt Peltier, "Cleanup
of an Oil and Mixed Chemical Waste Spill at Kernersville,
North Carolina, June 1977". These "Proceedings"
4. Lafornara, J.P., "Cleanup After Spills of Toxic Sub-
stances", Journal Water Pollution Control Federation, in
press.
5. Frank, U., M. Gruenfeld, "Use of Synchronous Excita-
tion Fluorescence Spectroscopy for In Situ Quantitative
Analysis of Spilled Hazardous Materials". These "Pro-
ceedings"
6. Frank, U., J. Munn, "Synchronous Excitation Fluores-
cence Spectroscopy", Analyt, Quality Contr. Newsl,
U.S. Environmental Protection Agency, Cincinnati, Ohio
33:34, 1977
7. Frank U., "Comparison of Synchronous and Single Wave-
length Excitation Fluorescence Spectroscopy", Analyt.
Quality Contr. Newsl., U.S. Environmental Protection
Agency, Cincinnati, Ohio 34:4, 1977
26
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EPA'S MOBILE LAB AND TREATMENT
SYSTEM RESPONDS TO
HAZARDOUS SPILLS *
By Michael Gruenfeld, Frank Freestone and Ira Wilder, U.S. Environmental Protection Agency
Industrial Environmental Research Laboratory-Cincinnati Oil and Hazardous Materials Spills Branch
INDUSTRIAL WATER ENGINEERING,
September, 1978, PP 18-23
A view of the Mobile Physical-Chemical Treatment
System. The unit, together with a mobile laboratory
containing analytical instruments, responds to
hazardous materials spills for urgent identification
and cleanup of toxic water contaminants.
A laboratory-on-wheels, contain-
highly sophisticated analytical
"struments, is working in conjunc-
u°n with a trailer-mounted mobile
-mical treatment system in emer-
1CV cleanup responses to spills of
azardous materials into streams and
111 across the nation.
The mobile laboratory, managed
°y the Oil and Hazardous Materials
"« Branch of the U.S. Environ-
mental Protection Agency (EPA),
Alison, New Jersey, and manned by
0 chemists, is contained within a
'-toot-long semi-trailer1. It is
readily transported for service
throughout the United States, to-
gether with the treatment unit con-
sisting of trailer-mounted activated
carbon columns, plus prefiltration,
flocculation and sedimentation
tanks'.
The mobile treatment unit so far
has treated about a dozen major con-
tamination incidents, the latest four
making use of the mobile laboratory.
It was successful in removing be-
tween 90 and 99.9% of the toxic sub-
stances. In most cases, the spills have
threatened drinking water supplies
and aquatic life with such toxic
chemicals as dinitrobutylphenol,
PCB, toxaphene, chlordane,
Kepone, pentachlorophenol and
other aromatic and chlorinated
organics.
The rapid detection of spills and
their accurate identification, as well
as their marking and tracking, are all
important if effective response to
emergencies is to be made. A key to
the ability of the emergency response
system to work rapidly and accurate-
ly lies in the precision analytical in-
•Mention of trade names or commercial products docs noi constitute an endorsement or recommendation by the U.S. Government.
27
September 1978
-------
struments contained in the mobile
laboratory. Among these are atomic
absorption, infrared and fluor-
escence spectrophotometers by the
Perkin-Elmer Corporation, as well as
a gas chromatograph) by the Hewlett
Packard Corporation, and an emi-
sion spectroscope by the Spectrex
Corporation.
Once the Branch becomes involved
in a toxic chemical spill, because of the
urgency of the situation it is not
always logistically feasible to request
analytical support from permanent
laboratory facilities. If it is to depend
on a centralized laboratory, or other
laboratory, it would mean taking
samples, transporting them to the
laboratory and waiting for answers.
By the time analysis results, are
received the priorities for the spill
cleanup might have changed.
Additionally, laboratories normal-
ly do not do this specific type of
work, nor would they want to inter-
rupt their normal work flow to han-
An adjustment is made in the operation of a
fluorescence spectrophotometer in the mobile
laboratory where analysis for contaminants in a
spill situation is taking place.
A sample is analyzed with a computerized gas
chromatograph.
die it. Therefore, we often send
specially trained personnel into the
field to accomplish these dedicated
analyses.
The atomic absorption instrument
in use in the laboratory is provided
with an automatic sampling system
and a heated graphite atomizer
(HGA). The HGA is used instead of
the more common, and earlier, flame
mode. It permits analysis of metallic
components to approximately three-
fold lower limits (down to ppb's)
than the flame mode. It does this by
combusting, ashing and vaporizing
samples at high temperatures in an
electric furnace. Measurements for
the purpose of quantification are
usually accomplished after using the
emission spectroscope to identify the
metals that are present.
The fluorescence instrument has
an extremely high sensitivity for
materials that fluoresce under
ultraviolet light. It is valuable for
water analyses, and possesses the ad-
vantage over gas chromatograph
analyses in not needing a solvent, in
sample preparation, to improve sen-
sitivity; a sample can be used directly
from the source, it also has the abili-
ty to measure the presence and levels
of fluorescing toxicants, even if they
remain unknown. The identity of
contaminating materials can then be
adduced from corresponding evi-
dence. In some cases, the fluor-
escence instrument has been able to
measure reductions in levels of
unknown aromatic toxicants, follow-
ing water treatment, even before
identification of the contaminants
was accomplished.
The infrared instrument is used for
analysis of petroleum oils that are used
Industrial Water Engineering
28
-------
as venicles for toxicants, and for
identification, through "fingerprint"
spectra, of unknown materials. In
some instances, it lias even provided
identification following transmission
'I the spectra via a telefacsimile ear-
ned in the mobile laboratory.
We anticipate also equipping the
mobile laboratory with a mass spectro-
meter, and communicating informa-
tion to experts throughout the country
by means of the telefacsimile.
In addition to gas chromatog-
raphy, a method used to separate
complex mixtures for the purpose of
quantification and identification, the
laboratory also engages in total or-
ganic carbon analysis (TOC) where-
by the total organic content of water is
rapidly measured, without extraction.
The EPA has published a list of
271 chemicals considered hazardous
The sample tray of an atomic absorption spec-
trophometer equipped with an automated sampler
and heated graphite atomizer is filled by a chemist
in an effort to measure quantities of metallic
elements to be found in the spill of a toxic
substance.
The sample holder of an infrared spec-
trophotometer is filled by a chemist prior to spill-site
analysis of petroleum oils containing toxicants.
to the environment. The emergency
response system is concerned' with
even more than these.
The laboratory is concerned with
ha/ardous chemicals under a wide
variety of circumstances. These in-
clude gases, volatile components, li-
quids and solids involved in situa-
tions or incidents that pose public
health or environmental threats,
sometimes beyond spill situations.
Predominantly, the laboratory has
dealt with bodies of water that have
been contaminated and that threat-
ened drinking water supplies, or
aquatic life. Chemicals that are being
transported by truck, or rail, and are
spilled through accident, also come
under the branch's emergency pur-
view, if the spill is sufficiently large
to present problems of acute toxicity.
Spills are divided into a number of
categories, depending upon what is
spilled and where it takes place.
These each demand different cleanup
treatment.
Spills can be grouped into those on
soils, into streams, or into large un-
confined bodies of water. A small
stream, or a pond, is considered to be
an impoundable, or damable, body
of water in which a spill can be
isolated from the remainder of the
stream, permitting cleanup to pro-
ceed relatively easily. Large water
courses (such as the Ohio River, or
Raritan Bay, off New Jersey, where
there have been hazardous spills), all
categorized as uneonfinable, require
different treatment approaches.
The Edison Branch views its main
function as the development of
equipment for returning the environ-
ment as closely as possible to its pre-
spill state. The chemical analysis
work supports this primary engineer-
ing responsibility, i.e., it provides
answers as to how well the equip-
ment works, and how to improve the
engineering.
Some successful applications of
the emergency of the emergency
response unit have been:
• Oswego, New York, where leak-
age from three lagoons holding
waste chemicals of over a million
gallons was discovered to be
draining into a tributary of Lake
Ontario, threatening to pollute
that body of water,. The EPA
mobile treatment unit arrived on
April 1, 1977, and, after analyses
by the mobile laboratory, started
to filter the waste leakage. By
29
September 1978
-------
TOXIC COMPOUNDS REMOVED FROM WATER USING THE MOBILE TREATMENT UNIT
DNBP
PCB
Toxaphene
Chlordane
Heptachlor
Aid r in
Dieldrin
Kepone
Pentachorophenol
Methylene Chloride
Carbon
Tetrachloride
Benzene
Toluene
Xylenes
Trichloroethane
Trichloroethylene
Phenol
Cresol
Dimethylphenol
Trimethylphenol
Butylphenol
Dimethylaniline
PCB
LOCATION QUANTITY OF WATER INFLUENT EFFLUENT
OF INCIDENT TREATED GAL. CONG, ppb CONG, ppb
Clarksburg, NJ
Seattle, WA
The Plains, VA
Strongstown, PA
Strongstown, PA
Strongstown, PA
Strongstown, PA
Hopewell, VA
Haverford, PA
Oswego, NY
Oswego , NY
Oswego , NY
Oswego, NY
Oswego , NY
Oswego , NY
Oswego , NY
Oswego, NY
Oswego, NY
Oswego, NY
Oswego, NY
Oswego, NY
Oswego, NY
Ditmer, MO
2,000,000
600,000
12,000,000
250,000
100,000
3,000
100,000
3,000
100,000
3,000
100,000
3,000
225,000
215,000
1,000,000
1,000,000
1,000,000
1,000,000
1,000,000
1,000,000
1,000,000
1,000,000
1,000,000
1,000,000
1,000,000
1,000,000
1,000,000
180,000
8
400 ppb
1 ppb
36
13
1,430
6.1
80
8.5
60.5
11
60.5
4,000
20,000
190
1.1
1
120
140
12
21
140
230
1,220
130
300
380
19
< .002
< .075
< .075
1
.35
.43
.06
.1
.19
.15
< .01
< .01
< 1
< 1
51
< .1
< .1
.3
< .1
< .1
.3
< .1
8.1
5.4
10
15
23
< .1
%
REMOVAL
99.9
99.9
92.5
97.2
97.3
99.9
99.0
99.9
97.8
99.8
99.9
99.9
99.9
99.9
73.2
90.9
90
99.8
99.9
99.2
98.6
99.9.
96.5
99.6
92.3
95
94.0
99.5
30
September 1978
-------
November 1977, it had treated
over 1 million gallons of waste
and contaminated runoff waters.
The wastes, consisting of mixed
chemicals and oils containing
PCB's, had been dumped into the
overflowing lagoons by a waste
disposal company that had had its
incinerating facilities shut down
for air and water pollution viola-
tions. At the height of operations,
during the month of November
!977, from 50,000 to 60,000
gallons of water were processed.
• Haverford, Pennsylvania, where,
in the summer of 1976, residents
°f the town reported to the EPA
regional office that a tributary of
the Delaware River, Naylors Run,
was being polluted by toxic "oil
slicks" at a number of places
where storm drains and springs
emptied into the stream4. There
was a strong odor along the
stream's course, and fish were dy-
lng. The source was discovered to
be a wood treating operation
where a 5% solution of pen-
tachlorophenol (PCP) in fuel oil
was used as a preservative. Lab-
oratory analyses by infrared spec-
trophotometer, and by gas chro-
matograph, confirmed that PCP
was present in large amounts in
fhe Naylors Run oil slicks. Further
'nvestigation showed that the
wood-preservative plant site was
saturated with the chemical and
that waste from the operation had
been injected into a 25-foot-deep
well for some 30 years. Seepage
from the well entered an under-
ground culvert, leaked into storm
drains and emptied into Naylors
Run. This stream passes through
Philadelphia suburbs and enters
Tmicum National Wildlife
Refuge, the last freshwater tidal
marsh in Pennsylvania.
,*y ' a fungicide and pesticide, is
aaded to fuel oil before being applied
0 w°°d as a preservative. It has been
mked to many human health prob-
'err>s, including lung, liver and
laney damage. The concentration in
"ie oil was greater than 10,000 parts
P" million (ppm), and in the stream
ater, 0.5 ppm. Concentrations of
u-z PPm are lethal to fish.
Through well drilling and trench-
ng operations for oil-contaminated
sroundwater collection, skimming of
ne oil in tanks, and use of the ac-
, ated carbon columns of the mobile
[reatment unit, the Oil and Hazar-
dous Materials Spills Branch assisted
EPA Region III response personnel
to reduce PCP concentrations in the
water to below 1 part per billion
(ppb). Oil-in-water concentrations
were also reduced from 100 ppm to
less than 1 ppm. Altogether, 215,000
gallons were processed during a
three-month operation. In all stages
of the cleanup, infrared spectroscopy
and gas chromatography were used
to monitor removal efficiency.
• Kernersville, North Carolina,
where, in June, 1977, vandals
opened the valves on six storage
tanks at a chemical wastes
disposal company and emptied
30,000 gallons of mixed wastes in-
to the reservoir that supplied
drinking water,. Vapors from the
wastes were so strong that 1,000
people in the area of the spill had
to be evacuated. Dead fish were
observed in the reservoir and were
reported by town officials. It was
believed at first that an oil spill
had occurred and the water was
treated accordingly. When this did
not clear up the problems, the
Industrial Water Engineering
31
-------
EP\ mobile laboratory and a
pilot scale water treatment unit
were called in. Fish, meanwhile,
continued to die, fleeing the
chemicals to the mouths of
freshwater inlets to the reservoir.
Within 72 hours after the spill, the
toxic chemicals caught up with the
fish, killing about 20,000 of them,
some 90% of the aquatic popula-
tion. At this point, town officials
closed down the reservoir.
Because the toxicants were
unknown and the materials were
grossly mixed, identification could
not immediately be made. A mobile
bioassay system was brought in to
determine the acute toxicity in the in-
fluent to, and effluent from the pilot
scale water treatment unit. It was
discovered that the influent was tox-
ic, but the effluent not, suggesting
cleanup of the reservoir system by
carbon adsorption methods.
The fluorescence spectrophoto-
meter was very valuable in this situ-
ation. By running samples with this
instrument, we were able to isolate
distinctive profiles that were not
present in samples taken before the
spill occurred. The reservoir mate-
rials yielded a profile for the presence
or absence of materials, that worked
hand-in-glove with the bioassay tox-
icity tests, providing excellent corre-
lation for monitoring contamination
levels.
The greatest benefit of the use of
the fluorescence instrument was this
relative indication of toxicant con-
centrations through correlation with
the behavior of bioassay organisms.
Other significant operations in
which the mobile treatment unit has
engaged2 include: Kepone contami-
nation at Hopewell, Va., in January
1976; spillage of dinitrobutylphenol
(DNBP) into a tributary of the Mill-
stone River, a public water supply
for Allentown, N.J., at Clarksburg,
N. J., in August 1974; spillage of 265
gallons of PCB from an electrical
transformer into the Duwamish
Waterway, Seattle, in September
1974; spillage of a pesticide contain-
ing chlordane, heptachlor, aldrin and
dieldrin, to a concentration of 39
ppb, into Carney Run, at Strongs-
town, Pa., in July, 1975.
It was initial use of the mobile
treatment unit that first pointed to
the need for a mobile laboratory,
when it was noted that although oil
spills are visible enough to locate and
isolate, chemical spills are not. The
waters or soils have to be analyzed to
find out the areas of toxic concentra-
tions. With the critical aid of the
laboratory, we are able to deal with
an acute public safety hazard, such
as chlorine, as well as with hazards of
a chronic nature, such as PCB, or
low-level pesticide releases on a na-
tionwide 24-hour-response basis. In
effect, the mobile laboratory is an ex-
tension of our base laboratory at
Edison, bringing to the field all the
sophisticated technology that is re-
quired, but, in more immediate and
timely terms.
The attached table lists some of
the hazardous chemicals, their
amounts, and their removal efficien-
cies achieved at a number of sites.
References
1. Urban, M., and Losche, R.,
"Development and Use of a
Mobile Chemical Laboratory for
Hazardous Material Spill Re-
sponse Activities", Proceedings
of the 1978 National Conference
on Control of Hazardous
Material Spill Response Ac-
tivities", Proceedings of the 1978
National Conference On Control
of Hazardous Material Spills,
Miami Beach, Florida, April
11-13, 1978, pp 311-314.
2. Lafornara, J.P., "Cleanup After
Spills of Toxic Substances", Jour-
nal Water Pollution Control Fed-
eration, in press.
3. Lafornara, J.P., Freestone, F.J.,
and Polito, M., "Spill Clean-up at
a Defunct Industrial Waste Dis-
posal Site", Proceedings of the
1978 National Conference On
Control of Hazardous Material
Spills.
4. Lamp'l, H.J., Massey, T., and
Freestone, F.J., "Assessment and
Control of a Ground-and Surface
Water Contamination Incident,
Haverford, Pennsylvania,
November-December, 1976",
Proceedings of the 1978 National
Conference On Control of Haz-
ardous Material Spills.
5. Stonebreaker, J., Freestone, F.J.,
and Peltier, W.H., "Cleanup of
an Oil and Mixed Chemical Waste
Spill at Kernersville, N.C., June,
1977", Proceedings of the 1978
National Conference On Control
of Hazardous Material Spills.
Michael Gruenfeld obtained his Bachelor
Degree in Chemistry in 1961 from New York
University. He has been with the Environmental
Protection Agency since 1971 and is presently
serving as Chief of the Chemistry Staff of the Oil
and Hazardous Materials Spills Branch. Mr.
Gruenfeld formerly served with the U.S. Food and
Drug Administration where he was responsible for
the analyses of foods, drugs, cosmetics and haz-
ardous household products.
Frank J. Freestone obtained his Bachelor of
Mechanical Engineering Degree from Rutgers
University in 1967, and his Masters Degree-in
Ocean Engineering from the University of Miami in
1969. He has been with the Environmental Protec-
tion Agency since 1971 and is presently serving as
Chief of the Hazardous Materials Spills Staff of
the Oil and Hazardous Materials Spills Branch.
Mr. Freestone formerly served as a commissioned
officer in the U.S. Army, including one year's ser-
vice in Vietnam.
Ira Wilder obtained his Bachelor of Chemical
Engineering Degree in 1954 from the City College
of New York. He has been with the Environmental
Protection Agency since 1970 and is presently ser-
ving as Chief of the Oil and Hazardous Materials
Spills Branch of the Industrial Environmental
Research Laboratory, Edison, New Jersey. The
Branch is responsible for the initiation, planning
and management of research and development
programs relative to the prevention, control and
treatment of Oil and hazardous material spills. Mr.
Wilder formerly served with the Naval Applied
Science Laboratory where he was responsible for
the management of R&D programs relative to li-
quid fuel fire protection for the Navy.
Industrial Water Engineering
32
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II, PETROLEUM OILS
A, FLUORESCENCE ANALYSES
33
-------
("*) ANALYTICAL QUALITY CONTROL NEWSLETTER #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. (U. Frank")
* See page 2.
34
-------
(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #15, October 1972.
PASSIVE TAGGING OF OILS BY FLUORESCENCE SPECTROPHQTOMETRY
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
rag/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 mu 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 tne
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 mj.i 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)
See page 2.
35
-------
(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #20, January 1974.
PASSIVE TAGGING OILS BY FLUORESCENCE 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 and 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 % pure 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
nm. 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 201-548-2510, Coml. 202-548-3347)
* See page 2.
36
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(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #21, April 1974.
AN IMPROVED SOLVENT FOR FLUORESCENCE ANALYSES OF OILS
Increased resolution in the fluorescence spectra of oils is
obtained by using dipropylene glycol instead of cyclohexane,
as the solvent matrix. The effect of this solvent on the
fluorescence spectra of five oils (No. 2 and No. 6 Fuel Oils,
South Louisiana, W. Texas Sour and Bachaquero Crude Oils)
was investigated. Because of some solubility problems, so-
lutions were initially prepared in cyclohexane, from which
1 ml aliquots were diluted to 25 ml with dipropylene glycol.
The final concentrations were 10 mg oil per liter of solvent.
Similar solutions in cyclohexane, were also prepared. Emission
spectra were obtained in the range 220-600 nm, by exciting at
290 nm. The spectral envelopes of the oils in dipropylene
glycol showed distinct and sharp bands, while the envelopes
of the oils in cyclohexane displayed only slight deflections
as shoulders. The price of dipropylene glycol offers an
additional advantage. While cyclohexane that is suitable
for fluorescence analysis costs $47.00 per gallon (3kg), an
equal quantity of dipropylene glycol costs about $7.25.
(U. Frank, FTS 340-6626, Coral. 201-340-6626).
RECLAMING A WASTE SOLVENT
Due to current restrictions on the availability of cyclohexane,
Which is a required solvent for most of our fluorometric analyses
of oils, it was necessary to reclaim the waste solvent. Because
of time constraints only a minimum effort could be expended, and
therefore a simple, one step distillation procedure was tested.
An all glass apparatus was used, consisting of a 2 liter pot in
a heating mantle, a reflux condenser, and a collection flask.
Cyclohexane (99 Mol%Pure) that was contaminated with up to 5%
oil was distilled at a rate of 2 liters per hour. The reclaimed
Product regained its initial purity to fluorescence analysis.
(U. Frank, FTS 340-6626, Coml. 201-321-6626)
* See page 2.
37
-------
(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #22, July 1974.
Effect of Fluorescence Quenching on Otl Identification
raised by several Investigators about the
on fluorescence spectra when used for
oils. Quenching as defined for the
of oil/ occurs at high concentrations and
Questions have been
effects of quenching
passively tagging
fluorescence analysis
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 3i»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.1' Those points corresponded to the following
concentrations:
Oil
*2 Fuel Oil
#6 Fuel ON
Bachaquero Crude
Iran - Gach Crude
CONCENTRATION OF
"ONSET
mg/1
OF QUENCHING"
Ex 290 nm
30
6
6
16
Ex 3UO nm
No Emission
7
18
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. In 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 otls by the fluorescence technique. (U. Frank, FTS 201-
5U8-3510, Coml. 201-5W-33U)
* See page 2.
38
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(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #24, January 1975.
.Identification of Petroleum Oils by Fluorescence Spectroscopy
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
e*citation of the oils at 15 wavelengths, in the range of 220-
500 nanometers (nm), at 20 ran intervals. The emission monocro-
roator is rapidly scanned at each excitation wavelength to obtain
the emission maximum. These maxima are then plotted versus the
e*citation 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. Prank, FTS 340-6626, Coml. 201-321-6626)
See page 2.
39
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(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #31, October 1976.
Synchronous Excitation Fluorescence Spectroscopy
A preliminary evaluation of the utility of synchronous excitation
fluorescence spectroscopy for the quantitative determination of
petroleum 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
interferences 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. Pernell, FTS
342-7517, Coml 201-548-3347)
* See page 2.
40
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(*) ANALYTICAL QUALITY CONTROL NEWSLETTER # 32, January 1977.
Synchronous Excitation Fluorescence Spectroscopy
Use of 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, benzene, 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 mcg/1 (ppb) - 0.1 mg/1 (ppm), using
100 ml cyclohexane for extracting 1 liter water. A preliminary
^valuation 'of interference by commonly present materials in
brackish lake and marsh waters, was also performed. No inter-
ference was found. (U. Frank, FTS 340-6626, Coml. 201-321-6626,
M- Gruenfeld, FTS 340-6625, Coml. 201-321-6625).
* See page 2.
-------
(*\ ANALYTICAL QUALITY CONTROL NEWSLETTER #33, April 1977
Synchronous Excitation Fluorescence Spectroscopy
Synchronous excitation fluorescence spectroscopy (AQC Newsletters
31 and 32) was used for the measurement of residual aromatics in
water. In-situ analyses of benzene, methyl substituted benzenes,
and dimethyl aniline in water samples from an industrial waste
holding pond were performed in order to monitor the effectiveness
of a mobile carbon column treatment unit in an emergency response
situation (AQC Newsletter No. 29) . One of the two peaks in the
fluorescence spectrum was attributed to benzene and methyl sub-
stituted benzenes. The other peak was attributed to dimethyl
aniline. These characterizations were confirmed with standard
compounds, and by GC/MS. (U. Frank, FTS 340-6626, Coml. 201-321-
6626/J. Munn, FTS 340-6628, Coml. 201-321-6628)
Adsorption of Fluorescing Oil Components Onto Silica Gel
Silica gel and alumina adsorption techniques that are used for
the separation of hydrocarbons from other organics, prior to
petroleum oil quantitation by IR (AQC Newsletter No. 26), should
not be used prior to fluorescence measurements. Major fractions
of fluorescing oil components adsorb onto silica gel, causing
substantial spectral distortions and losses of emission inten-
sities. (U. Frank, FTS 340-6626, Coml. 201-321-6626)
Substitute Deozonator for Fluorescence Spectrometers
Following breakdown of the deozonator of our fluorescence spectro-
meter, a hot air blower (2,000 watt, 400°C-540°C) was modified
to serve as a substitute. The air blower was connected to the
outlet hose of the fluorescence instrument's lamp housing, using
a cardboard adaptor. This arrangement was used satisfactorily
for several days, without a preceptable odor of ozone. We be-
lieve that a simple hot air blower (approximate cost $65.00) is
adequate for the commercial deozonator (approximate cost $500.00).
(R. Frederick, FTS 340-6627, Coml. 201-321-6627)
* See page 2,
42
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(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #34, July 1977.
Oil Quantitation by Fluorescence Spectroscopy
ydrocarbon solvent contamination of sample jars precludes the
Quantitation of petroleum oils by infrared spectroscopy. Re-
cn??17'•:5ar3 that were rinsed with n-hexane prior to water sample
Collection were forwarded to our laboratory. Residual hexane
n the water prevented use of our usual IR procedure, but the
analyses were performed successfully by the synchronous excitation
fluorescence method that was outlined in AQC Newsletter No 31
uctober 1976. (U. Frank, Coml. 201-321-6626, FTS 340-6626/
J- Susman, Coml. 201-321-6628, FTS 340-6628)
See page 2.
43
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(*) EPA QUALITY ASSURANCE NEWSLETTER, Volume 1 No.2, July 1978.
Rapid Quantification of Petroleum Oils in Sediments - Wet sedi-
ment extraction combined with Synchronous Excitation (SE)
fluorescence spectroscopy are currently under evaluation as a
rapid method for estimating petroleum oil concentrations in
sediments. This method circumvents time comsuming procedures/
such as soxhlet extraction, sediment drying and saponification
steps. Wet sediments are simply extracted with a solvent mix-
ture consisting of 60 percent isopropanol and 40 percent cyclo-
hexane. The method was tested with several estuarine sediments
spiked with No. 2 and No. 6 fuel oils, and Kuwait and South
Louisiana Crude oils, at concentrations of approximately 10
mg/Kg oil in wet sediment. Five grams of the spiked sediments
were placed into 50 ml, glass-stoppered graduated cylinders
and diluted to 50 ml with the solvent mixture. The cylinders
were then agitated for 15 minutes using a mechanical rotary
mixer and 10 minutes were allowed for settling. Analysis of
the supernatants was performed by the SE fluorescence techn-
nique as described in EPA's Quality Assurance Newsletter,
Vol. 1, No. 1, April 1978, using 25 nm wavelength intervals.
Recoveries obtained were in the range of 100-106 percent. Use
of this procedure for the quantification of weathered oils in
sediments is now under evaluation. (U. Frank, Coml. 201-321-
6626, FTS 340-6626)
* See page 2.
44
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REPRINTED FROM: Proceedings of 1975 Conference on Prevention and Control
of Oil Pollution, San Francisco, California, March 25-27,
1975, pp 87-91. Available from API Washington, B.C.
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.
-------
CONFERENCE ON PREVENTION AND CONTROL OF OIL POLLUTION
IXCITAIION
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 2f>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 photodccomposition, 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. Thruston and Knight [ 1 ], Coakley [2J, and
Freegarde et al. [6] surmised that significant changes in the
intensity and shape of the emission spectrum can occur when oils
are 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
Thruston and Knight 11 ] 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
•— iiiHOuini riomi
{MISSION IPECTIA
EMISSION
WAVELINOTHS
Figure 3. Three-dimensional presentation of the derivation of the
silhouette profile from the oil in figure 1
characterized by the formation of excimers. These are combinations
of excited molecules. Kxcimer 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 excimcr formation.
Excimcr 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 Pcrkin-Elmer model MPF-3 Fluorescence Spectro-
photometer, 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° t 0.5°C.
1 Mention of trade names or commercial products does not
constitute endorsement by the U.S. Government.
46
-------
MONITORING
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 foi 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-
ochromator 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. Photodecom posit ion 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. Sea water 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 particulate 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
70
60
z
o
40
30
10 •
ONSET OF
QUENCHING
10 20 30 40 50 «0 70
CONCENTRATION (•«/!)
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 otts (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
47
-------
CONFERENCE ON PREVENTION AND CONTROL OF OIL POLLUTION
240
UNHEATHERED OIL
WEATHERED OIL
NUMBER 6 FUEL
NUMBER 2 FUEL
S. LOUISIANA CRUDE
L_l I
BACHAQUERO CRUDE
320
400
480 240
320
400 480 240 320
EXCITATION WAVELENGTH (nm)
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
240 280
320 360 400
EXCITATION KAVEIEHGTH !nm)
480 520
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.
48
-------
MONITORING
WEATHERED OIL
WEATHERED OIL
100 ,. UUO TBtCO CRUDE
II
-
. n
LIGHT ARABIAN CRUDE
HEAVY IRAH1AN CRUDE
T1A JUANA 102 CRUOt
VX
240 320 400 4BO 240 320 urn
320 400 480
320 400 480 240 320 400
)ure 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
:
Number 2 Fuel Oil
Number 6 Fuel Oil
Bachaquero Crude
Ira" - Gach Crude
CONCENTRATION (mg/1)
Ex 290 nm Ex 340 nm
n
'.
No Emission
7
:
".
ACKNOWLEDGMENTS
Q,C author is grateful to Mr. Michael Gruenfeld, Supervisory
assjs?mt of 'his laboratory, for many helpful discussions and for his
sjjj f an''e 'n preparing this manuscript, and to Mr. Henry Jeleniew-
the data acquisition that has made these findings possible.
Th*uston, A.D., and Knight, R.W. 1971. Environmental science
* technology, 5 :64.
^-oakley, W.A. 1973. Proceedings of Joint Conference on the
prevention and Control of Oil Spills, p. 215. Washington,
j P'C-: American Petroleum Institute.
aaamec, J.R. 1974. Abstracts of Pittsburgh Conference on
Analytical Chemistry and Applied Spectroscopy. Cleveland,
Ohio.
JJoyd, J.B.F. 1971. J. Forens. Sci. Soc. 1 1 :83.
p'd-P-153.
«fgarde, M.; Hatchard, C.G.; and Parker, C.A. 1971. Lab.
ice, 20(I):35.
' T'' and KasPer. K- 1955- z Elektrochem. 59:976.
ay, J.F., and Latham, D.R. 1972. Analytical Chemistry
<
EXCITATION AT 340nm
EXCITATION AT 290nm
340 380
J L
290
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 Mo! % Pure
cyclohexane is used
9. McKay, J.F., and Latham, D.R. 1973. Analytical Chemistry
10. Parker, C.A. 1959. Analyst 83:446.
11. Frank, U. 1974. EPA Analytical Quality Control Newsletter
21:1 1.
-------
NO. WO
Presented at the
1977 Pittsburgh Conference On
Analytical Chemistry And
Applied Spectroscopy
February 28 - March 4, 1977
Cleveland, Ohio
DETERMINATION OF PETROLEUM OILS IN SEDIMENTS
BY FLUORESCENCE SPECTROSCOPY AND NMR
U. FRANK AND M GRUENFELD, Oil 4 Hazardous Materislj Spllli Branch, Ind. Env
Re~l,ab. Ci, US. Environmental Potection Agency, Edison, N. J. 08817
The use of severs! 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 »s control samples.
The following fluorescence techniques, that are believed to be most commonly used for
oil spill source identification, were examined: (a) Single Wavelength Excitation1•''3'4;
(b) Synchronous Excitation5; and (c) Derived Silhouette Profiles6. 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 highlight* 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 at 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 comprehansive 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 sediicents
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
FIGURE 2
10
8
p.p.
U)
Hatchard, and
Practice, 20 (1)
v-j
/ :•• I N. / I I "* 1. M. Freegard, C.G.
n ' C.A. Parker, Lab.
35-40 (1971)
A.D. Thruston and H.W. Knight, Env.
Sci. and Tech., 5, 64 (1971)
W.A. Coakley, Proc. of Joint Conf.
on Prevent. 6 Contr. of Oil Spills,
Wash., D.C., A.P.I., 215 (1973)
J.R. Jndamec and T.J. Porro, Abstract Pittsburgh Conference (1974)
P. John, and I. Soutar, Anal. Chem., 48, 520 (1976)
II. Frank, Proc. of Joint Conf. on Prevent. 4 Contr. of Oil Spills, San Pranctsro,
CA., A.P.I., 87 (1975)
References:
1.
2.
3.
50
-------
BIBLIOGRAPHY OF REGENT 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 Fingerprinting
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. Wewsl., U.S. Environmental
Protection Agency, Cincinnati, Ohio 13:3.
51
-------
11. Frank, U. 1972. Passive Tagging of Oils by Fluorescence Spectrophoto-
metry. Analyt. Quality Contr. News1., 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, D.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.
52
-------
11• Quantitative Methods
1. Goldberg, M.C., and Devonald, D.H. 1973. Fluorescent Spectroscopy,
A Technique for Characterizing Surface Films. JL 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(1):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.
53
-------
II, PETROLEUM OILS
B, OTHER ANALYSES
-------
(,*) 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 mm and 100 mm path length po-
tassium bromide cells and near infrared silica cells. (M. Gruenfeld)
* See page 2.
-------
(*) 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 EI%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.
No. 2 Fuel Oil
South Louisiana
Crude Oil
Bachaquero Crude Oil
No. 6 Fuel Oil
Carbon Freon
Tetrachloride 113
23.3 21.5
25.3 22.5
2% - 98%
Solvent Mix
21.5
23.1
17.8 ~ 16.5
26.2 ~ 23.7
(M. Gruenfeld, 201-548-3247)
* See page 2.
56
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STORAGE AND TRANSPORT OF OIL 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-
scopic 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
°f 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
f°r 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, 201-548-234?)
QUANTITATIVE ANALYSIS OF OIL BY IR
A Preliminary evaluation of infrared spectrophotometry for
^Uantitating petroleum oils has been completed. No. 2 and
N°. 6 fuel oils and South Louisiana and Bachaquero Crude oils
Were examined. Solutions having 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-
Jous with Freon-113 which is obtainable from E. I. DuPont de
Nemours, Co. Oil absorbances were measured at 2930 cm'1 in
1° mm path lengths silica cells at ordinate expansions 1 and 5.
^bsorbance 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
ff1* were preferred for measurements at ordinate expansion 1 in
;°0 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
Oll in water when using the extraction method described in the
^alytical Quality Control Laboratory Newsletter #12, January
i?72- A final volume of 100 ml was selected to facilitate
Direct correlation with the previous method.
(M. Gruenfeld, 201-548-3347)
57
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(_*) 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, 201-548-3247)
* See page 2.
58
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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 100T) 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
°il 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, 201-548-3347)
59
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(,*) ANALYTICAL QUALITY CONTROL NEWSLETTER #17, April 1973.
Plastic Bottles for Storing and Transporting Oil Containing Samples
In AQC Newsletter No. 15, HALAR plastic bottles were evaluated
for storing and transporting oil containing samples. Vanguard
Plastics, Inc., was cited as the source of supply. We have since
been informed that Vanguard only manufactured a small number of
these bottles for research purposes. They do not now plan to
market HALAR bottles unless orders are placed for large, single-
size lots. Another company, the Nalge Company (75 Panorama Creek
Drive, Rochester, New York 14602), may retail HALAR bottles if
the market warrants. Information from Nalge can be obtained by
contacting Mr. Stephen Hooper (716-586-8800).
(B. F. Dudenboetel/M. Gruenfeldt 201-548-3347)
* See page 2.
60
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(*)ANALYTICAL QUALITY CONTROL NEWSLETTER #18, July 1973.
USE OF GAS CHROMATOGRAPJIIC 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 201-548-5419, 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,
^hile 2 mg/100 ml solutions were prepared in a 2% CC14 - 98%
*reon 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
"ays, at normal room temperature and lighting conditions. Solution
stabilities were monitored by measuring the 2930 cm~l absorbance
^and of oil. All the solutions proved to be stable; less than 1%
Change in absorbance occurred during the test period. This pre-
i:LIninary study suggests that dispersed oils should be promptly
e*tracted from water, prior to prolonged transport or storage.
(M. Gruenfeld/J. Puglis, FTS 201-548-3543, Coml. 201-548-3347)
See page 2.
61
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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?)
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 cnT1, 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 201-548-3543t 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 5X, 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. , F$S 201-548-3543 - Coml. 201-548-334?)
62
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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, 31. (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
°f South Louisiana and Bachaquero Crude, and No. 2 and No. 6 Fuel
°ils. These are viscous and non-viscous crude and processed oils
that are considered to be representative of many petroleum oils.
s°lutions 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 mn and 260 my. The oil
sPectra also exhibit absorption maxima or shoulders at these
aPproximate wavelengths. The spectral profiles of South Louisiana
Cttade and No. 6 Fuel Oils most closely resemble that of elemental
sulfur, but the spectral profile of No. 2 Fuel Oil differs from
i-t significantly. (J. Lafornara/M. Gruenfeld, FTS 201-548,3543,
Coml. 201-548-3347)
REMOVAL OF CHARRED OIL DEPOSITS FROM GLASSWARE•
Curing 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 were constantly reused
an<3, therefore, required frequent cleaning. Scrubbing with steel
w°ol 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
*°r routine use. Complete removal of all visible traces of oil
^posits was achieved by heating the glassware for 10 minutes at
5°0°c, in a muffle furnace. It is thought that this technique
°an be used to remove other carbonaceous deposits from glassware
that can withstand this temperature.
(H. Jelen-ieuski/U. Frank, FTS 201-548-3510, Coml. 201-548-3347)
See page 2.
63
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(*) ANALYTICAL QUALITY CONTROL NEWSLETTER #21, April, 1974.
SOLVENT FOR OIL ANALYSIS
Recurring questions have been raised about the identity and
commercial source of trichlorotrifluorethane, 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-trifluoroethane. 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 340-6625, Coml. 201-
321-6625.)
EVALUATION OF A PORTABLE IR SPECTROPHOTOMETER
A preliminary evaluation of a compact portable infrared
spectrophotometer that is suitable for the quantitative
analysis of oils in the field, is described. This unit, a
Miran I Fixed Filter Infrared Analyzer (Wilks 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 ordinate 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 ordinate scale expansion, and
by using expansion settings 5x and 2Ox. Absorbance versus
concentration curves were generated, and these prove to be
nonlinear. Miran "absorbances" do not match absorbance
readings of a double beam IR instrument. In accordance with
a recommendation by Wilks, 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 made to correct these to
linear plots. At ordinate expansion 2Ox, points where quite
scattered and did not yield a smooth curve. The concentration
range 0.1 - 150 mg/100 ml oil in carbon tetrachloride was
examined in this study, and the lower concentration figures
is considered to be the reasonable detection limit of the
instrument at 20x expansion. This represent^ an oil in
water concentration of 0.1 ppm when 1 liter water samples
are extracted with 100 ml carbon tetrachloride. (M. Gruenfeld
and U. Frank, FTS 340-6625, Coml. 201-321-6625.)
* See page 2.
64
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(*JANALYTICAL 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 1971* Annual Book of ASTM Standards, Part
31, Water. They include a procedure for sample preparation
and laboratory weathering (D3326-7i*T), and for gas
chromatographic correlation (D3328-7»*T). Fairly large volumes
of oil (50 ml) are required by D3326, 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, - trifluorethane (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 40°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 concentration
determinations. Small amounts of CC1^(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 201-51*8-3543, Coml. 201-
5^8-331*7)
* See page 2.
65
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C*) ANALYTICAL QUALITY CONTROL NEWSLETTER #26, July 1975.
Separation of Petroleum and Non-Petroleum Oils
The direct addition of silica gell 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 straighforward. We examined this method as
part of a project to develop a procedure for quantitating 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 deactivation 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 340-6625, Coml. 201-
321-6625.)
* See page 2.
66
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(*) 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, O., 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)
* See page 2.
67
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(.*) 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-up" 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)
See page 2.
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(*)ANALYTICAL QUALITY CONTROL NEWSLETTER #36, January 1978.
Differentiation of a Synthetic Lubricacing Oil from Several
Petroleum Derived Automotive Lubricating Oils
*n response to recent requests for distinguishing between synthetic
and petroleum-derived automotive lubricating oils, our laboratory
analyzed a number of oils by several techniques. One synthetic
?il and several petroleum-derived oils were tested. The synthet-
ic oil proved to be distinguishable from the other oils through
its high content of carbonyl compounds, mainly esters. The pet-
roleum oils yielded broad, unresolved GC profiles, one moderately
broad HPLC peak, substantial absorption in the 1 ppm region ,
(relative to TMS) of the NMR, and absorbance in the 2930 cm ,
1450 cm" , and 1360 cm spectral regions of the IR. The synthet-
ic oil produced large, well resolved peaks by GC, a second sharp
Peak by HPLC,and additional adsorptions at approximately 3.8 ppm
and 1740 cm by NMR and IR, respectively. The GC profile of the
synthetic oil was clearly atypical of petroleum oils, while its
additional absorptions at approximately 3.8 ppm by NMR and
1740 cm by IR were indicative of carbonyl compounds. These
Unique spectral and chromatographic features of the synthetic
°il were also used to determine our ability to detect adulter-
ations of the petroleum oils with the synthetic oil. Detection
limits were established at approximately 10%. (R. Frederick,
pTS 340-6627, Coml. 201-321-6627/N. Pangaro.)
See page 2.
69
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REPRINTED FROM:
Proceedings of 1973 Conference on Prevention
and Control of Oil Spills, Washington, D.C.
March 13-15, 1973. pp. 179-193. Available
from API, Washington, D.C.
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 A tomic 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
chromatography, molecular emission and absorption spec-
trophotometry, atomic absorption spectrophotometry, gas
chromatography, computerized mass spectrometry, et. 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, A PI 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 eva''
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 o»s
(Table 2) by mass spectrometry and gas chromatography t°
determine high molecular weight paraffins, naphthenes
(cyclic paraffins), and polynuclear aromatics. Emissioij
spectroscopy is used to determine bulk vanadium-nick6
content; X-ray spectroscopy and Kjeldahl analysis are use"
to determine bulk sulfur-nitrogen content. Adsorpti0"
chromatography with several different columns is used tjj
isolate paraffins, naphthenes, and aromatics from the °'
matrix and to separate the aromatics from the aliphatic8'
A schematic presentation of these analysis steps is pr°"
vided (Figure 1).
Portions of the authentic and weathered2 samples &
first separated for determination of bulk nickel-vanadiu1*
and sulfur-nitrogen content. Separate portions are distil'e
'The Baird Atomic study was funded through the EPA Analyti*^
Quality Control Laboratory, NERC, Cincinnati.
'Weathering of oils is simulated by using a continuously recircul"',
ing salt water system with controlled temperature, agitation, 1'S
and wind conditions.
70
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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 Method*
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 Chro matography
Gravimetric/Adsorption Chro matography
ASTM Method8
ASTM Method*
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
ASTM Method cited in text.
° remove components boiling below 400° F; these volatiles
. * Discarded. Accurately weighed portions of the higher
ar In8 residues are dispersed in 50 cc n-pentane, insolubles
tin s.eParated by centrifugation and discarded. Each solu-
n_n 's then fed to a clay column and successively eluted with
eiu ntane' benzene-acetone, and acetone. The n-pentane
ate preferentially contains the paraffins, naphthenes, and
Co°matics, and the acetone-benzene and acetone eluates
ter i the more P°!ar samPle fractions. These polar ma-
>als are thought to be more water soluble and therefore
reuable indices; they are not used in the analysis scheme.
2
2
2
1
1
1
4
1
2
2
2
2
2
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.
1
2
3
4
5
6
7
8
Sample Type
Crude oil
Crude oil
Crude oil
Crude oil
Crude oil
No. 2 Heating oil
No. 4 Fuel oil
No. 5 Fuel oil
Refined and
formulated from
Venezuelan stock
Oil Field
Tia Juana
Lago
Grande Isle
Nigeria
Zuitina
Location
Venezuela
Venezuela
Indonesia
Nigeria
Libya
71
-------
Sulfur Analysis
X-Ray
Spectroscopy
Pentane 1
Benzene + Acetone 2
Acetone 3
Pentane 1
POLLUTANTS
./• \
u
1
to
400°F
tion
F
1
bJickeJ./ Vanadium
Analysis
Emission
Spectroscopy
\
r
Nitrogen
Analysis
Kjeldahl
Residue
Centrifugation
Insolubles,
1
Clay Separation
Acetone + Polars
Benzene
Pentane
Paraffins -t- Naphthenes
Aromatics
Silica Gel
Separation
Aromatics,
Acetone
n-Paraffin Aromatic Analysis - Low Voltage
Analysis Naphthene Analysis (P + N fraction) - High Voltage
Figure I: Analysis Schematic of the Esso System.
72
-------
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 % of Sample
Pentane
Polars Insolubles
19.3
24.7
12.3
10.6
15.2
0.0
8.6
12.1
18.4
20.4
21.5
9.0
15.5
1.2
1.0
3.1
0.2
1.0
5.8
10.9
9.4
10.9
Fractions
95.9
101.8
102.6
93.5
96.5
96.5
99.0
102.5
97.0
97.9
98.4
"Data based on unweathered samples.
r
?r various polynuclear aromatic types. Twenty-six promising
"^erprint indices derived by these techniques (Table 4) are
L jected to Discriminant Function Analysis to identify the
tr st discriminators for the test oils (Table 5). This statistical
eatrnent is needed to isolate five indices having the lowest
P °bability of mismatching two oils (Table 5). Estimation
jj c°nfidence levels for oil classification could also have
en Performed with the Bonferroni T Statistics.
and -l*"s study> Esso uses only four fingerprint indices
an ' *'*^ k'gh statistical confidence, distinguishes among
y Possible pairs of the oils, even after the oils have ex-
a"enced extensive laboratory weathering. These indices
an ,?enerally unaffected by simulated weathering, but their
iiuf ab'lity to other oils nas not been establ'sned- Otner
1Ces "lay be required for individual situations.
A«other Esso Approach
c, In a supplemental study Esso evaluates a combined gas
f0^0niatographic-ultraviolet spectrophotometric procedure
ar Passive tagging oils (Figure 2). Individual polynuclear
I °matics (PNA's) separated by gas chromatography are col-
, ed and measured by ultraviolet absorption spectro-
w otpmetry. After 1-gram portions of the weathered and un-
cy | ered oils are extracted with caustic and dissolved in
>'ohexane, triphenyl benzene is added as internal
col fd' Eacn solut'on 's fed to a deactivated alumina
cycll'k11 *2^H O) that is successively eluted with cyclohexane,
Cy :°"e*ane/benzene, benzene, and benzene/methanol. The
n- lo"exane/ benzene fraction, which is enriched in three-
dis8 a°d heavier PNA's, is stripped of solvent and the residue
tj, Persed in toluene. A gas chromatogram of the PNA's is
ga n obtained (Figure 3). Selected components appearing as
abs ° • mat°8raPhic peaks are trapped and their ultraviolet
aj>D°rpt'-0n sPectra obtained. These spectra together with
mlese Quantitative results and visual examination of the
d.^ornatograms for passive tagging oils. Preliminary data in-
te that this approach is promising.
The Phillips Study
iisef , Hips surveys a multitude of parameters that may be
u* 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 CM
and Cao 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
73
-------
POLLUTANTS
Ni
Table 4: Some Promising Esso Finger
2—3 Ring Naphthenes
2 (P+N)
N
-20
ZnParaffin
c=20
c=40
2—4 Ring Naphthenes
2 (P + N)
2— 5 Ring Naphthenes
2 (P+N)
'21
2nParaffin
c=20
c = 40
CnH2n-6
2 CnH2n_
18
-24
2nParaffin
c=20
c = 40
CnH2n -14
-18
C25
2nParaffin
c = 40
2-1 Ring + 2 Ring Naphthenes
2—5 Ring + 6 Ring Naphthenes
2nParaffin
2nParaffin
C30
2nParaffin
131
2nParaffin
c=20
c = 40
c=20
c = 40
c=20
c = 40
c=20
c = 40
2C20+C
21
'22
+ €31 +
2i
€30 + C31 4- C32
2 C24 + C25
C27 + C28
(Benzenes)
2 Aromatics
2CnH2n_6 c=20
v~I r~~ c = 36
2/ Aromatics
(Indenes)
2 Aromatics
SCnH2n_10 c=20
2 Aromatics
Cn H2 n _ 14 ( Acenopthenes)
2 Aromatics
Cn H2 n _ 16 ( Acenopthalenes)
2 Aromatics
2nParaffins
2 (P + N)
Cn H2 n _ is (Phenanthrenes)
2 Aromatics
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 an
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 di*'
74
-------
IDENTIFICATION OF OIL
Table 5: Most Promising Esso Fingerprint Indices
Ni
SnParaffins
2(P + N)
Z-5 Ring Naphthenes
2 (P + N)
C20
£nParaffinc=4o
£-1 Ring+2 Ring Naphthenes
2-5 Ring+6 Ring Naphthenes
*°lved
and
ltl
>n carbon disulfide (ca 5-10% W/V) and injected;
Coi}centrations need not be known. The gas chroma-
injector is equipped with a glass liner that is re-
f°r clear"nS' Column specifications are 50 feet
'nch Packed w'th nonpolar liquid silicone OV-101
a rated at 25,000 effective plates. Oil chromatograms
and C°mpared visually, and certain features are abstracted
list tabulated wnen analyzing many samples and for sta-
'cal 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 oil, 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-Ci5 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 Ci5 to any signal parameter at Cn or Ci8. 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.
Extract uich Caustic
O g)
Add Cyclohexane and
Internal Standard
tu Santple
S« pa rite on A1203 (+ 2Z K?.0)
Clut* by Cyclohexane,
Cyclohexane/Benzene, Benzene
Benzene/Methanol
Front Cue Point
at Appearance
of Color
Figure 2: Analysis Schematic of Another Esso Approach.
75
-------
POLLUTANTS
Figure 3: Gas Chromatogram by Esso of Polynuclear Aromatics in a
(TPB) Is Used as Internal Standard.
Crude Oil. Marked Peaks Are Identified in Table 6. Triphenyl Ben/'-
Table 6: GC Components Trapped by Esso for UV Analysis
•I
Peak Aa fluorene -
Peak B methyl fluorene
9 10
Peak C phenanthrene
Peak D 2-methyl phenanthrene
Peak E 1-methyl phenanthrene
Peak F dimethyl phenanthrene
aGC peaks are identified in Figure 3
76
-------
IDENTIFICATION OF OIL
CRUDE OIL
Isothermal
Distillation
0.15 ma
RESIDUE, WT. %
(600 F + Fraction)
Liquid-Solid
Chromatography
Silica
Measure
API Gravity
Pour Point
Sulfur Content
Nitrogen Content
Nickel + Vanadium Content
Carbon Isotopic Composition
Sulfur Isotopic Composition
GLC Profile
DISTILLATE
SATURATES
MM
Measure
%(w/w) In 600°F+ Fraction
Carbon Isotopic Composition
Adduction
AROMATICS
Measure
ASPHALTICS
Measure
In 600F-t-
Fraction
Carbon Isotopic
Composition
In 600F+
Fraction
Carbon Isotopic
Composition
Measure
GLC Carbon Number Distribution-
Calculate OEP Ratio
Figure 4: Analysis Schematic of the Phillips Study.
77
-------
POLLUTANTS
Type U-2 Profila
C20
1 ,
Figure 5: Coding of Gas Chromatogram Profile by Phillips — Type U-2 System.
indices of individual oils remain quite stable from column
to column.
WHO1 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) a'
low temperatures as a method for identifying oils. Their ef-
78
-------
IDENTIFICATION OF OIL
Type B Profile
Figure 6: Coding of Gas Chromatogram Profile by Phillips - Type B System.
diScr'S a Probe to evaluate the utility of the technique for
vantaminating among oils and to determine whether an ad-
study86. derives frorn "sing cryogenic temperatures. This
pc0c *}cals mostly with unweathered heavy crude and
tested^01 oils (Table l2): only two weathered oils were
W^h thand n° su''stant'a' effort was made to correlate them
sir,g ti'e aPPr°Priate unweathered oils. Emphasis is on asses-
distj . utility of low temperature molecular emission for
the lftU,lsll'n8 among different oils and not to demonstrate
\vea(, lly of this technique for correlating appropriate
•p, re" and unweathered oils; i.e., passive tagging.
77°K Li°''S are c'issolved '" methylcyclohexane and cooled to
glass' Methylcyclohexane is a good solvent that forms a clear
and (ffftlle cry°8en'c temperature. The use of other solvents
metavi nt °'l concentrations is described; 10 ppm oil in
29Q ^cyclonexane yields the most satisfactory results.
'engthsM and 340 m/i are the diagnostic excitation wave-
spectr 'Sec' to compare the oils. In every case oil emission
'han tf resu'ting from excitation at 340 m/x are more intense
at this °\SC resultin8 from excitation at 290 m/z, but excitation
"ter wavelength yields broader emission spectra; all
the oils yield characteristic emissions in the 380-400 m^t
region when excited at 340 m^i. 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)
79
-------
POLLUTANTS
DEFINITION OF TERMS
CHROM'ATOGRAM SERIES NO:
COLUMN
NO.
INJECTION
NO.
n-C134- CHART SPEED 4—
I/2"=IMIN = 6°C
-PROGRAM START!
CS2 SOLVENT PEAK
LINE ir-BASELINE
INCREASING TEMPERATURE
INJECTION!
Figure 7: Gas Chromatograrn by Woods Hole Oceanographic Institution with Terms Defined.
Table 9: Woods Hole Oceanographic Institute
Fingerprint Indices3
Pristane/Phytane
C17/ Pristane
C18/Phytane
Cp/Back ground
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 WHO1 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 1,3, 30 Days
4 Re-refined Freshwater, Saltwater,
Beach Sand
With or Without 1 of 4
Spill Control Chemicals
Added.
aError attributable to presence of oil-based spill control chemical.
''The source of these samples was not supplied to Woods Hole by EPA.
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
80
-------
IDENTIFICATION OF OIL
Table 11: Ability by Woods Hole Oceanographic Institute
to Distinguish Among 30 Oils and OiJ 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.
is 'r'n£ a weathered oil to one of only a few unweathered oils
atj.e(lu'red, then nickel-vanadium, sulfur-nitrogen, and GC
lar S may suff'ce; but- where comparison with many simi-
""Weathered oils is to be made, the entire Esso Method
5|j
lias be USed- This assumes> of course, that a computerized
"leth Spec.trometer 's available. Therefore, the extent of
ahl • ut'''zat'°n depends on problem complexity and avail-
ie'nstrumentation.
Ad • ^sso Method is somewhat inconvenient because
ne 0rPtion chromatography is used repeatedly and because
m Actions among the 26 fingerprint indices (Table 4)
com ^ to be made for different analyses. Additionally,
com
Put
erized mass spectrometry is not available in most
therebv limiting the usability of the Esso
ext °?- The method should permit unique correlations with
fro SlVe libraries of oil indices, however. It also benefits
its j ^e extensive weathering studies that were performed in
evelopment by Esso.
pr0c *°'her Esso approach evaluates a combined GC-UV
for D .c tnat may provide additional fingerprint indices
of (L3ssive tagging oils. Weathered and unweathered portions
it, t,e sanie oils are used in order to establish method integrity
sr,o H Presence of weathering. If successful, this technique
Ultra ^ w'('e acceptance because gas chromatographs and
anaj V'.°'et spectrophotometers are generally available in
Vant-'°a' 'aboratories. It could also be combined to ad-
with those portions of the Esso Method that require
PolynCOtnmon'y available instruments. The chromatograms of
"clear 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
ual 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 WHOI System is a ready-to-use passive tagging
method requiring minimum sample pretreatment and using
only one instrumental technique. WHOI provides explicit
81
-------
POLLUTANTS
CONCENTRATION In
:• 11
1IME CONSTANT ".")
GAIN 3U/" " I).01/MAX
TEMPERATURE
EXCITATION vVAVUEf-GlM •' /|;
EXCITATION WAVELENGTH
GAIN 30/0
FLUORESCENCE WAVELENGTH
SLITS ll/.'?
GAIN >/" l( ij.ill/MAX
TEMPERATURE "u"fl
PL. 3'iOMU
1IIU
HUO 500
WAVCLfNCTII (iKinoii.oli.Ts)
PL. 3ltOMU
CONCENTRATION 101'1-M
SLITS 22/11
TIME CONSTANT ".3
GAIN 30/6 R 0.1 MAX
TEMPERATURE "°K
EXCITATION vVAVELENGTH 29(i
EXCITATION WAVELENGTH 3'i()
GAIN 30/0
FLUORESCENCE vVAVELENGTH
SLITS 11/22
GAIN 3U/6 R U.01 MAX
TEMPERATURE 77°K
"30JT
400 -~
WAVELENGTH (nanomclers)
"SiJCT
200
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.
82
-------
IDENTIFICATION OF OIL
CONCENTRATION lo PPM
SLITS 22/12 R o.oi MAX
TIME CONSTANT 0.3
GAIN 30/iu
TEMPERATURE 77°K
EXCITATION WAVELENGTH 290MU
P1IOSPHOROSCOPB 16.5 VOLTS
OIL (A)
200
300
400 500
WAVELENGTH (nonomuluis)
600
700
"'
Difle''ent Number 6 Fuel Olls al 77U|C h>; Baird Atomic. Concentrations Are ID ppm in
tiSSUeC s for seParat'ng oils from water, sand, and animal
GC j niatr'ces. The oils are then injected directly into the
suspenstrument m carbon disulfide solution. Unweathered
and j" °'ls are also merely dissolve.! in carbon disulfide
adsornjeCteti' No Preweatnel"ing, high speed centrifugation,
Sues chrornato8raPhic, or multi-instrumental tech-
\VjjQJ SUcn as those used by Esso and Phillips, are used by
iristril ^e meth°d requires some modification of most GC
instru'Tlents' nowever. These are now generally available
ThU'IUS '" mosl analvtical laboratories.
a"d M C ^HOI Method was evaluated by using weathered
Lab0rnvveatnered portions of oils supplied by EPA's Edison
Un'4u T°ry' Most- but not a" of the weathered oils were
able» -ry. corre'atc<^ several correlations were only "prob-
ParjSo . WHO] Method, therefore, seems usable for com-
c0rre|,ns to small numbers of standard oils and possibly for
achieal °ns ^ith sma" libraries of oil indices. But, it may not
tne "definite" correct correlations with extensive
°f Oil indiccs that should be achievable by the Hsso
,'and h>' a method that may derive from the Phillips
- tt ol additional fingerprint indiccs, such as nickel,
' Sl1"1"' and ni'rogcn content, in conjunction with
I Method, should yield further improvement.
requirement for support-coated open tubular (SCOT)
s> 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
83
-------
POLLUTANTS
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/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
Countyj 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 C
Nueces Co.
Bra/oria Co.
Lake Maracaibo
Lake Maracaibo
An/oatequi
84
-------
REPRINTED FROM:
Environmental Science & Technology,
Vol. 7, No. 1, July 1973.
fraction of Dispersed Oils from Water for Quantitative
Analysis by Infrared Spectrophotometry
W|CHA
EL GRUENFELD
Protection Agency, Edison Water Quality Research Laboratory, National Environmental
S(Jarch Center (Cincinnati), Edison, N.J. 08817
Per °^ne parameters tnat optimize the extraction of dis-
frar H °''S from water for quantitative analysis by in-
Prn sPectrophotometry (ir) are examined, and an im-
ttjn 6c* extraction procedure is recommended. Trichloro-
p-J^oethane, an essentially nonpoisonous solvent (1000
is j). ?k V), 's compared with carbon tetrachloride, which
pp 'Shly poisonous to operating laboratory personnel (10
pet ,} V)- Both solvents are usable for extracting dis-
by : ,oils fr°m water, and for their quantitative analysis
**tt' • trichlorotrifluoroethane is preferred. Changes in
ftciHaCt'°n efficiency following small additions of sulfuric
ftientanc* sodium chloride are examined. Great improve-
pt results in extraction efficiency, but. no further im-
^e einent derives from addition of more salt. Absorbance
ce|| Surei«ents are at 2930 cm"1 in 10-mm path length
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 Nassarius obsolctus 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.
85
-------
Table I. Fraction of Oil Removed by Individual Extracts from Synthetic Dispersions Containing 5 Ml of 50% H2SO4
and 5 Grams of NaCt
Percent recovered"
No. of
25-ml
extrac-
tions
1
2
3
4
South Lou
Crude
Freon 1 13
92.6
99.3
99.8
100
isiana
Oil
ecu
94.4
99.7
100
No. 2
Fuel Oil
Freon 113 CCl,,
97.2 97.8
99.5 100
100
Bachaquero
Crude Oil
Freon 113
90.0
98.7
99.7
ecu
95.4
99.5
99.9
No. 6
Fuel Oil
Freon 113
91.1
98.7
100
ecu
92.2
98.6
100
100 100
a Determined as A/A, x 100 + B: A. infrared absorbance at 2930 cm ~' due to the extract of interest; At, sum of absorbances at 2930 cm~' of all the ex-
tracts (2x1); 8, total percent oil recovered by the previous extracts.
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 (CCiJ
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 saJt. 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 CHa 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.
.,30
0.70
0.60
0.10
1.0
3200 300O 28OO
WAVENUMBEB 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"
points A and fl
is determined as the difference between
86
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 Corn-
Volume 7, Number 7, July 1973
-------
A FREON 111
« C CI4
NUMBED OF 25ml EXTRACTIONS
Figure 2. Number 2 Fuel Oi! extracted from 1-liter duplicate
synthetic oil-water samples containing no added acid or salt
LEGEND
O NO ACID OR SALT
0 Set 507. SUIFURIC ACID
• 05 GRAMS SODIUM CHLORIDE
X 5 GRAMS SODIUM CHLORIDE
A 25 GRAMS SODIUM CHLORIDE
A 75 GRAMS SODIUM CHLORIDE
Q ISO GRAMS SODIUM CHLORIDE
• 250 GRAMS SODIUM CHLORIDE
0 1 2 3 4 5 t 7 15 10 II 12 13 U IS
NUMBER OF 2> -I EXTRACTIONS
Figure 3. Number 2 Fuel Oil extracted from similar 1-liter syn-
thetic oil-water samples containing acid or salt
Acidified sample is extracted with Freon 113; untreated sample and sam-
ples containing the indicated amounts of salt are extracted in CCU
Environmental Science & Technology
pany, Inc.), and carbon tetrachloride, spectroanalyzed
(Fisher Scientific Co., Catalog no. C-199).
Procedure. Synthetic 1-liter oil-water samples contain-
ing identical concentrations of emulsified oil were pre-
pared by shaking 5-ml portions of each test oil with 1%
liters of tap water for 1 min in 2-liter separatory funnels.
The mixtures with No. 2 Fuel Oil, No. 6 Fuel Oil, and
South Louisiana Crude Oil were allowed to separate un-
disturbed for 15 min, while the mixture with Bachaquero
Crude Oil was allowed to separate for 1 hr. The homoge-
neous oil-water emulsions obtained as bottom layers were
used to prepare the duplicate synthetic 1-liter samples.
This was accomplished by mixing two 500-mI portions of
each emulsion with an equal amount of tap water, in 2-
liter separatory funnels. Subsequent extraction of the oils
from the synthetic samples was also carried out in these
funnels.
The relative extraction efficiencies of Freon 113 and
carbon tetrachloride and the influence of acid and salt
were examined by extracting the 1-liter synthetic samples
with successive 25-ml portions of each solvent; acid and
salt were added to some of the samples (Figures 2-4). The
quantity of oil in each extract was monitored by measur-
ing the oil absorbance band intensity at 2930 cm-1 in
10-mm path length silica cells (Figure 1). Extraction ef-
ficiencies and the effects of sulfuric acid and sodium chlo-
ride were determined by plotting the absorbance of each
extract as a function of the number of 25-ml extractions
(Figures 2-4). Removal of oil from water was considered
complete only after attaining zero absorbance. That the
absence of an infrared absorption band indicates "com-
plete" extraction of oil was also shown by submitting re-
sidual water, emulsified with No. 2 Fuel Oil and extracted
with Freon 113, to analysis by the gravimetric procedure
of the American Public Health Association (1971). No oil
was found in this water. The fraction of total emulsified
oil separated by each extract was estimated by determin-
4.53
4.46
LEGEND
FREON 113 •
C CI4 '
01 2345
NUMBER OF 25ml EXTRACTIONS
Figure 4. South Louisiana Crude Oil extracted from 1-liter dupli-
cate synthetic oil-water samples containing 5 grams of sodium
chloride and 5 ml 50% sulfuric acid
Absorbance intensities greater than 1.0 are calculated values
87
-------
idg 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 a"'"
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 b*
performed. Initial dilution is to 100 ml. Seawater samples
are an exception because they already contain adequa'6
salt and can therefore probably be analyzed without addi-
tion of sodium chloride. Such samples were not examine''
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 e*'
traneous solvent extractable organics.
Acknowledgment
Special thanks are given to Joseph Lafornara for provid-
ing valuable background information, and to Henry
iewski, Michael Killeen, Susan Rattner, and Peter
for their assistance.
Literature Cited
American Petroleum Institute, "Manual on Disposal of Refine')1
Wastes," Vol. IV, Method 733-58, 1958.
American Public Health Association, "Standard Methods for tne
Examination of Water and Wastewater," 13th ed., APh^'
AWWA, and WPCF, New York, N.Y., 254-6, 1971.
Beckman Instruments, Inc., Mountainside, N.J., Infrared ApP11'
cation Note 68-2, 1968.
Harva, O., Somersalo, A., Suomen Kern., 3l(b), 384-7 (1958). .
Jacobson, S., Woods Hole Oceanographic Institution, Woo""
Hole, Mass., personal communication, June 7, 1972. i
Sax, I. N., "Dangerous Properties of Industrial Materials," ™
ed., pp535, 1192, Reinhold, New York, N.Y., 1968.
Received for review August 10, 1972. Accepted March 26, 1973.
88
-------
Resented at the National Bureau of Standards Conference on "Standard
Reference Materials for Offshore Drilling-Petroleum," Santa Barbara,
-a-ifornia, October 1975.
REVIEW OF METHODS & STANDARDS USED FOR OIL IN BRINE ANALYSIS BY CONTRACTING
LABORATORIES ON THE LOUISIANA COAST
by
Uwe Frank
Industrial Environmental Research Laboratory-Ci
U.S. Environmental Protection Agency
Edison, New Jersey 08817
INTRODUCTION
The Increased demand for energy is expanding the exploration and
development of offshore petroleum production. Wastes generated by off-
shore drilling operations and discharged into the marine environment are
therefore an increasing menace and are of vital concern to the Environ-
mental Protection Agency (EPA). Production platforms on the Outer
Continental Shelf offshore Louisiana alone produce ca. 410,000 barrels
of waste water (brine) per day^ , by 1983 this figure will have risen to
(2\
and estimated 1.54 million barrels per dayv '. These brines contain oils,
toxic metals, a variety of salts, solids and organic chemicals. To curb
the indiscriminant discharge of these pollutants into our environment,
the Federal Water/Pollution Control Act Amendments of 1972 require that
anyone discharging wastes into the nation's waters from a point source
must acquire a permit. Once a discharger has applied for a permit, EPA
and State pollution control officials decide on effluent limits and con-
ditions after soliciting and considering public comments. In addition
to meeting effluent limitations, dischargers are also required to monitor
and analyze their discharge continuously and report the results to the
State Agency and EPA. The explicit numerical limits for petroleum de-
rived oils are listed in Table P ''
89
-------
Petroleum values reported by the offshore oil industry are usually
determined by contracting laboratories. To establish the merit of these
values the EPA recently conducted a "Field Verification" study. I
participated in this study and reviewed analytical methods used by
contracting laboratories on the Louisiana coast. In this presentation
I will discuss and evaluate these methods and examine the necessary
standards.
DISCUSSION
The methods used by the Louisiana laboratories for offshore brine
analyses basically entail extraction of the oil and determination by
one of four techniques, i.e., infrared, gravimetric, ultraviolet
fluorescence and colorimetric techniques. However, the accuracy of
these techniques varies widely. Factors such as brine characteristics,
physical and chemical properties of oil and standards used, all in-
fluence their accuracy. The extent to which each technique is affected
ts primarily governed by the oil constituents which are measured. Ideally*
the determinative technique measures all, and only, the oil constituents.
However, due to the complex chemical composition inherent to petroleum
oil, each technique measures only some specific oil constituents. In
the case of the infrared, fluorescence and colorimetric techniques the
constituents measured are then used as a "handle" for determining the
total otl.
Table II lists the determinative techniques, the respective con-
stituents measured and the primary factors influencing the accuracy of
each technique.
90
-------
Standards play a critical role in all but the gravimetric technique.
It appears, therefore, that this technique is more universally applicable
than the others. Its accuracy however is not on par with the fluorescence and
infrared techniques. A recent collaborative study conducted by the
^erican Society for Testing and Materials (ASTM) shows that the gravi-
metric results were consistently much lower. This is illustrated in
Table in, which shows the results obtained by our laboratory using both
tne infrared and gravimetric techniques.
As a consequence of measuring only a fraction of the total oil
c°nstituents and using it as a "handle", the infrared, fluorescence and
c°lorimetric techniques need standard oils of comparable composition to
the sample oil for accurate results. Any imbalance in the ratios of com-
being measured to the total composition in the standard and sample
results in either erroneously low or high results.
The standard of choice is an oil identical to the oil being measured.
However, the availability of such source oil standards is not always
assured, necessitating the use of a substitute. In the case of the Louisiana
ldboratories, an oil of different identity than the sample oil, was often
Used. This recourse critically affects the accuracy of results obtained
wUh the fluorescence and colorimetric techniques. Both techniques use only
^nor oji constituents as a "handle" for total oil determinations. Slight
dr1ations in the composition of these constituents in the oil will there-
f°re significantly affect the accuracy.
Conversely, the infrared technique uses the major constituents of
' as a "handle". Its accuracy in comparison is therefore only slightly im-
by compositional variations of oil. The infrared tech-
also allows the use of a synthetic or arbitrary standard consisting
91
-------
of a mixture of isooctane, hexadecane (cetane) and benzene. The merit of
this standard is based on the assumption that the infrared absorptivities
of oils from different sources are very close and are similar to the
synthetic standard absorptivity. Gruenfeld' ' and others'5'6' have shown
that the absorptivity of a large variety of oils are in fact very similar
and close to the synthetic standard.
An alternate approach suitable for the infrared technique and routinely
used in our laboratory entails the derivation of standard oil from the
sample itself. The standard oil is prepared by stripping the solvent from
the sample extract. Although some volatility losses are incurred during
solvent stripping they do not significantly alter the ratio of components
measured by infrared to the total oil composition. The availability of
an adequate oil standard is thereby always assured.
CONCLUSIONS
This study has shown that the concentration of oil in brine is
defined by the determinative technique used. Only minor and specific
component classes of oils are measured by the fluorescence and colorimetric
techniques. Accurate quantisation of oil in brine samples, to assure
compliance with Federal effluent limitations, is therefore only attained
by using standards of Identical compositions as the sample oils. In
situations where such standards are unavailable, the Infrared technique
was found to provide more accurate results because a major class of oil
components (paraffinic hydrocarbons) is measured. The gravimetric
technique does not require standards and is applicable in situations
\
where the loss of volatile oil components does not af/ect the accuracy
92
-------
of results. The technique is not applicable to measurement of oils that
contain significant amounts of petroleum compounds that volatilize at
temperatures below 70°C.
93
-------
REFERENCES:
(1) Proposed 1973 Outer Continental Shelf Oil and Gas General Lease Sale
Offshore Mississippi, Alabama, Florida, Draft Environmental Impact
Statement, U.S. Department of the Interior, Bureau of Land Management,
Washington, D.C.
(2) Determination of Best Practicable Control Technology Currently Avail-
able to Remove Oil from Water Produced with Oil and Gas, Offshore
Operators Committee, Sheen Technical Subcommittee, 1974, Prepared by
Brown and Root, Inc., Houston, Texas.
(3) Development Document for Interim Final Effluent Limitations Guidelines
and New Source Performance Standards for the Offshore Segment of the
Oil and Gas Extraction Point Source Category, U.S. Environmental
Protection Agency, Washington, D.C. 20460 (September 1975).
(4) Gruenfeld, M., 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.
(5) Atwood, M. R., Hannah, R. W. and Zeller, M. V., Determination of Oil
in Water by Infrared Spectroscopy, Infrared Bulletin No. 24, Perkin-
Elmer Corporation, Norwalk, Connecticut (1972).
(6) Kahn, L., Dudenbostel , B. F., Speis, D. N. and Karras, G. M., Deter-
mination of Mineral Oils and Vegetable/Animal Oils in the Presence
of Each Other, Abstracts of Pittsburgh Conference on Analytical
Chemistry and Applied Spectroscopy, March 3-7, 1975, Cleveland, Ohio,
Suite 215 Whitehall Center, Pittsburgh, Pennsylvania 15227 (1975).
94
-------
TABLE I. Effluent Limitations for Point Sources
Petroleum Derived Oil - mg/1
Near Offshore produced water
r*r Offshore produced water
Near Offshore produced water
Offshore produced water
Maximum for
any one day
Average of daily
values for thirty
consecutive days
shall not exceed
Compliance by July 1. 1977
72
72
48
48
Compliance by July 1, 1983
No discharge
5'2 30
95
-------
TABLE II. Determinative Techniques Used by the
Louisiana Laboratories
Determinative Technique
Factors
Main Constituents Measured Affecting Measurement
Infrared
Paraffinic Hydrocarbons
Standards Used
Gravimetric
Non-volatile, Residual
Oil Constituents
Volatility of Oil
Ultraviolet -
Fluorescence
Polynuclear Aromatic
Compounds
Standards Used
Colorimetric
Dark Colored Oil
Constituents
Color of Oil,
Standards Used
96
-------
TABLE III. IERL Results of ASTM Collaborative Study
Mg/1 of Petroleum Hydrocarbons
Avg. of 28 Labs
Sample No. Infrared Gravimetric Infrared
1 261 215 261
2 272 221 263
3 130 70.8 131
4 118 82.5 110
5 47.8 26.3 49
6 26.8 24.2 21
97
-------
Michael Gruenfeld*
Quantitative Analysis of Petroleum
Oil Pollutants by Infrared
Spectrophotometry
REFERENCE: Gruenfeld, Michael. "Quantitative Analytii of Petroleum Oil Pollutant*
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/cni are measured in 10 and 100-mm path length cells.
with and without ordinate scale expansion. Solution concentrations in the range O.S to
40 mg/100 ml oil in solvent yield linear plots that pass through the origin. 7he concen-
tration O.OS 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 xS. This is considered the practical detection limit of these oils by the
infrared (IK) 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 spectrophotomcters. 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, Jacobsoti |/JJ
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. N.J. 08817.
'The italic numbers in brackets refer to the list of references appended to this paper.
98
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS
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
99
-------
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
100
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS
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 docs not constitute endorsement by the
U.S. Government.
101
-------
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 20
0.70
0.80
0.90
1.0
3300 3000 2800
WAVENUMBEI CM*'
FIG. 1—Infrared absorption band of No. 2 Fuel Oil dissolved in Freon 113 (0.034% w/v).
Hung 10-inm path length silica cells: Freon 113 in in the reference beam. Absorbance at
2<)30/c>n is ilelcrmineil 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. E|0mml% 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
102
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS
100
2930cm'1
Absorbance = loglo
2930cm'
5B
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 OH dissolved in f'reon 113: (/) without
ordinal r scalp expansion, and (2) with ordinate scale expansion *5.
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 Eiomm'1"0 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
103
-------
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-Bouyuer 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
r - r • x
* ~ s ~A~
rls
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
Cs = 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 [
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS
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 2 FUEL Oil
NO 6 FUEL OIL
BACHAOUERO CRUDE OIL
SOUTH LOUISIANA CRUDE OIL
10 20
CONCENTRATION
40 50
l) Oil IN SOLVENT
f\(j, )--Oil solutions in carbon tetrachloride measured in 10-mni path length cells with-
out ordinale scale expansion.
105
-------
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
BACHAOUERO CRUDE OIL
SOUTH LOUISIANA CRUDE OIL
10 20 30 40 50
CONCENTRATION (mj/100 ml) Oil IN SOLVENT
,/'G' *~?i' S°'"tio"s in 98 Percent Fre°" "3/2 percent carbon tetrachloride measured in
lU-mm path length cells without ordinate scale expansion.
106
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS
KET
• NO 2 FUEL OIL
• SOUTH IOUISANA CRUDE Oil
10 30 30 40 50
CONCENTRATION |mg/IOO ml) OIL IN SOLVENT
FIG. 5—Oil solutions in Freon 113 measured in 10-mm path length cells without nrdinate
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
107
-------
WATER QUALITY PARAMETERS
FREON-113
» 987. FREON.1I3 -
1% CARBON IETRACHLORIDE
20 3.0 4.0 50 60
CONCENIRAIION (m0/IOO ml) Oil IN SOIVCNI
FIG. t>—Solatia of No. 2 Fuel Oil measured in WO-mm path length cells without
orainute scale rxjwnsiun.
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
108
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS
• NO. 2 FUEL OIL
A NO. 6 FUEL OIL
* BACHAQUERO CRUDE OIL
• SOUTH LOUISANA CRUDE OIL
1.0 2.0 30 40 50
CONCENTRATION (mg/100 ml) OIL IN SOLVENT
FIG. 7—Oil solutions in carbon li'lrachloriilt' measured in 100-mm paih length cells willi-
nul orilintilf .VCH/C 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 x5 (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-tnl portions of solvent. Improved
109
-------
WATER QUALITY PARAMETERS
NCk 2 FUEL OIL
NO. 6 FUEL Oil
BACHAOUERO CRUDE OIL
SOUIH LOUISIANA CRUDE Oil
1.0 2.0 30 40 5.0
CONCENTRATION (mg/100 ml| Oil IN SOLVENT
,™IG> S~°Ll S"lutions '" 98 PerceM Fre"n "3/2 P^cent carbon tetrachloride measured in
WU-mmpatH length cells without ordinate scale expansion. A higher than normal IR instru-
ment gain setting was used.
110
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS
0.6
z
0 0.5 -
04-
0.1
KEY
NO 1 FUEl Oil
SOUTH LOUISIANA CRUDE Oil
1.0 2.0 30 40 5 0
CONCENTRATION |m9/'00 ml) Oil IN SOI VENT
FIG. 9—Oil solutions in Freon 113 measured in 100-mm path length cells withnnt or-
tlinate scale expansion. A higher than normal IR instrument gain setting wtif iist'H.
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 IR technique. These measurements
111
-------
WATER QUALITY PARAMETERS
KEY
NO. 7 FUEL OIL
NO 6 FUEL OIL
BACHAOUERO CRUDE OIL
SOUTH LOUISIANA CRUDE OIL
10 2.0 3.0 4.0 5.0
CONCENTRATION (mo/100 ml) OIL IN SOLVENT
KIG. 10—Oil solutions iii carbon letrachloride measured in 10-mm path length cells with
tinlinutt' scah- i-xpansiuiixS.
112
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS
O.I 0.2 03 04 05 06
CONCENTRATION (mg/100 ml) Oil IN SOLVENT
FIG. II — Cnrhiin tetrachloride solutions of ' Bachaquero Crude Oil measured in 100- mm
iwth length cells with ordinal? scale expansion x5.
yield a recognizable absorption band at approximately 2930/cm (Fig. 1)
tor 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 plofs demonstrates that the
lines diverge and, therefore, that the oils have different absorptivitics.
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 Bccr-Bongner Law plots that
113
-------
WATER QUALITY PARAMETERS
• IN CARBON TETRACHIOSIDI
• IN FREON-II3
10 20 30 40 SO
CONCENTRATION (mg/100 ml) OIL IN SOLVENT
KIG. 12—Solutions of No. 2 Fuel Oil measured in 10-mm path length cells without
ontinate scute expansion.
114
-------
GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS
• IN CARBON TETRACHIORIDE
• IN FREON 113
'0 20 30 40 50
CONCENTRATION (mg/100 ml) OH IN SOIVENT
IIG. 1.1—Stilutinns nj South Louisiana Cnulc Oil measured in 10-mm path length <•<•//.*
without nnlimiti' sculp expansion.
are obtained from the same oil in different solvents (Figs. 12 and 13): No.
2 Fuel and South Louisiana Crude oils yield ahsorptivities in Freon 113
that differ from their absorptivities in carbon tctrachloride. 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 oi! absorptivilics.
115
-------
WATER QUALITY PAHAMLTEHS
TAUL.l: 1—Ll/cct o/ solvent distillation on oil absorptivity. (No. 2 Fuel and South Louisiana
Crude Oils are in Freon I I.I solution; No. ft Fuel and Bachaquero Crude Oils are in 98%
Freon 113/2% carbon tetrachloride solution).
Before Distillation After Distillation"
Oil in Solvent Absorptivity'' % Loss Absorptivity %
Oil (mg/I(X)ml) (lijo mm1"7"' (by weight) (E)o niml%> Change
No. 2 Fuel
South Louisiana
Crude
No. 6 Fuel
Hachaquero
Crude
104
14.5
101
10.5
10.6
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 (stripped) from 100-ml solutions having known oil content, using the
procedure of the Al'HA [2|. The weighed residues are rediluted to 100-ml and the solution
absorbances measured at 2930/cm.
''HIQ mm'"'" values are absorbanccs at 2930/cm, measured in 10-nim path length cells.
normali/.cd lo 1% (weight/volume) dissolved oil.
following prolonged solution storage was also examined. Oil solutions in
carbon letrachloride, 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
|/| Jaeobson. S., personal communication, Woods Hole Oceanographic Institution, Woods
Hole, Muss., 7 June 1972.
\2\ Standard Methods for the Examination of Water and Wastewater. 13lh ed., American
Public Health Association, New York, 1471, pp. 254-256.
|.?| Harva, O. and Somersalo, A.. Suoim-n Kemistilehti. Vol. 3l(b). 1958, pp. 384-387.
\-t\ Manual on Disposal ofRefinery Wastes. Vol. IV, Method 733-58, American Petroleum
Institute, 1958.
|5| Infrared Application Note 68 2, Heckman Instruments, Inc., Mountainside, N.J., 1%8,
|6| Gruenfeld, M., Environmental Science and Tft'hnolo^y, Vol. 7, 1973, pp. <>36-63Q.
|7| Sax, I. N., DutiKt'ruus Properties n/'Industrial Materials, 3rd ed., Keinliold. New York,
1968, pp. 535, 1192.
|*| Atwood, M. R., Hannah. K W., and Zeller, M. V.. Infrared Bulletin No. 24. The
Perkin-Hlmer Corp., Nnrwalk. (dun. 1972.
116
-------
REPRINTED .FROM: Journal of Water Pollution Control
Federation, February, 1977, PP. 216-226
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.6-8
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 estimated " 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.18
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 l« 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,
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. 117
-------
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. Mya 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 (IH) 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 Na2So4> and re-
frigerated for 24 h. The tissue extract mix was
Soxhlet extracted in n-hexane for 6 h. These
extracts were divided into subtractions 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
spectro.scopy and cc-mass spectroscopy.
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
118
-------
Tanacredi
, F
NCRt ASING TIME ANO rEMPEHATUHE
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 C(i-CM 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 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; Cn-
pristane/C1s-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 C,. through C;jfi 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 CC-MS system.
uv-fluorescence spectroscopic method. Re-
cent investigators 1U 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 fhioresce 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 200 mM while scanning the emission
.spectrum from 240 to 540 nry. Thurston and
Knight " had used 340 m^ as the excitation
wave length for the characterization of petro-
*Perkin-Elmer Corp.
Journal WPCF
119
-------
Petroleum Hydrocarbons
leum entities; however, it has been recently
demonstrated "3 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^ to
440 mm (at 20-nijn 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-
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.
280 300 320 340 360
EXCITATION FREQUENCY
380 400 420
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 miu.
Helium carrier gas at 90 ml/s was measured
at the column outlet. Only organism sub-
February 1977
120
-------
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-paraffin
mix) cc 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
C17-pristane, Cjg-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
/Ag/1. 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
—
—
9/1 S
16.4
20.0
10.7
—
9/17
7.1
34.9
12.0f
4.9
9/24
2.0
28.9t
7.2
1.3
9/29
3.S
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.Of
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.
Journal WPCF
121
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Petroleum Hydrocarbons
TABLE II. Effluent Sample Correlation Data.
Fluorescence Maxima
Profile Fit
Maxima Region Fit
332 TOM Peak Fit
Date
1973
Sample
Correlates
With WCCO
Slight
Correlation
Correlates
With WCCO
Slight
Correlation
Correlates
With WCCO
Slight
Correlation
7 Sept.
10 Sept.
IS 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 Wrard
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
4-
-f
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.2S-29 Chromatograms of the Mya II-
saturated portion revealed the presence of iso-
meric compounds or other members of the
homologous series. The Mya HI aromatic
portion (Figure 7B) was considerably less
complex than the Mya II aromatic fraction;
this suggested that there was less exposure of
the organism's to petroleum hydrocarbons out-
side the Bay than within. Brown and Huff-
man sn showed this when they observed lower
concentrations of aromatic hydrocarbons in
February 1977
122
-------
Tanacredi
100 -
• • wcco
(2>« No. 2 Fuel Oil
x * 9/7/73 26th Ward
"• 9/7/73 Jamaica
* - 9/7/73 Rockai/vav
-- 9/7/73 Conev Is.
•» " 9/24/73 Rockawav
i ' 9/24/73 Jamaica
P^S*fa^=tJ$j
300 320 340 . 360 380 400 420 440 460
240 260
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^, an
additional peak of greater intensity occurred at
370 mfj.. This peak could be attributable to
contamination from other petroleum pollutants
such as residual fuel oil, which peaks between
350 and 400 m/*.3' 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
NYNI6
NYN09A
NYJ01
NY 102
NYJ03
NY JOS
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
1 All values in mg/l.
Journal WPCF
123
-------
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 C20, a characteristic of lube
oils.29 They did exhibit unresolved envelope
portions with some samples revealing the C17-
pristane/C,s-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 subtractions 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 m^ Peak F
Slight Slight Slight
Correlates Corre- Correlates Corre- Correlates
Date Source With WCCO lation With WCCO lation With WC
7 November NYN16
1973 NYN09A
NYJ01
NYJ02
NYJ03 +
NYJOS +
NYJ07
9 January NYN16
1974 ' NYN09A
NYJ01 +
NY J02 +
NY 103 +
NYJOS +
NY }07 +
February 1977
124
-------
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
A 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
200 nip
Slight
Corre- Corre-
lation lation
12/3/73
12/3/73
Mya Ilf
Mya III|
t Collected at Diamond Point, Jamaica Bay.
t Collected at Rockaway point, Atlantic Ocean.
Journal WPCF
125
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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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tional Academy of Sciences, Arlie, Va., 3
(1973).
17. Analytical Quality Control Laboratory News-
letter (EPA, Cincinnati, Ohio), 18, 8
(July 1973).
18. Personal Communications with Mr. Michael
Gruenfeld, Industrial Waste Treatment Re-
search Laboratory, EPA, Edison, N. J.
19. Keizer, R. D,, and Gordon, D. C., Jr., "Detec-
tion of Trace Amounts of Oil In Sea Water
by Fluorescence Spectroscop." Jour. Fish.
Res. Bd. of Canada, 30, 1039 (1973).
20. Goldberg, M. C., and Devonald, D. H. Ill,
"Fluorescent Spectroscopy—A Technique for
Characterizing Surface Films." Jour. Res.
U. S. Geol. Survey, 1, 714 (1973). '
21. Riccher, R. E., Amer. Assoc. Petrol. Geol.
Bull., 46, 60 (1962).
22. Thurston, A. D., and R. W. Knight, "Charac-
terization of Crude and Residual-Type Oils
by Fluorescence Spectroscopy." Env. Sci.
and Tech., 5, 64 (1971).
23. Frank, U., Analytical Quality Control Labora-
tory Newsletter (EPA, Cincinnati, Ohio),
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 ind personal communications with
Dr. B, E>udenbostel, Region II, Surveillance
and Analysis Lab., EPA, Edison, N. J., on
"Computerized Gas Chromatography/Mass
Spectroscopy" presented October 9, 1973.
February 1977
126
-------
Tanacredi
28. Meinschein, W, G,, "Origin of Petroleum." carbons In Open Ocean Waters." Science
Bull. Am. Assoc. Petrol. Geol, 43, 925 191,817(1976).
(1959), 31. Zitko, V., "Determination of Residual Fuel Oil
29. Meinschein, W. G., "Hydrocarbons: Saturate, Contamination of Aquatic Animals." Bull.
Unsaturated and Aromatic." In Elington, Env. Contam. and Tox,, 5, 560 (1971).
G., and Murphy, M, I. J. (Eds.) Organic 32. Wedgewod, P., and Cooper, R. L., "Detec-
Geochemistry (New York: Svinger-Verlag), tion and Determination of Traces of Poly-
346 (1969). nuclear Hydrocarbons in Industrial Effluents
30. Brown, R. A., and Huffman, H. L., "Hydro- and Sewage." Analyst, 80, 651 (1955).
Journal WPCF
127
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REPRINTED FROM:
Proceedings of 1977 Oil Spill Conference (Prevention,
Behavior, Control, Cleanup), New Orleans, Louisiana,
March 8-10, 1977, pp 487-491. Proceedings available
from API Washington B.C.
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 by 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 hydrocarbons and organic* that are of
recent biological origin. The techniques include chromatographic proce-
dures using alumina and silica gel for separating hydrocarbons from other
orgamcs, followed by instrumental methods such as gas chromatograpln-
fluorescence spectroscopy. ultraviolet absorption spectroscope, et al The
various oil parameters that are used to demonstrate the presence of petro-
leum oils are discussed, and the most effective ones are recommended. In
addition, a recent study is a/so described ,n 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 al.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, et a/.,™ Biert, et a/.,:< 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 a/.2" 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,1 this unresolved envelope is characteristic of the homologous and
isomeric hydrocarbons in fossil fuels. According to Clark and Finley,"-" 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. rial.2"
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
Figure 1. Gas chromatogram of a Bachaquero crude oil exhibiting the
presence of an unresolved complex mixture
12 8
-------
1977 OIL SPILL CONFERENCE
DEFINITION OF ERMS
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
over (Costly C-i.-,-C:is). and a strong predominance of odd-numbered
n-a||(. en~m|mbered compounds. The exceptionally broad distribution of
phasj?ie's 'Ci-Ciin) in crude oils and most refined products is also em-
0filc *'m"nance of one or two n-alkanes over all others. Use of GC
:iKo s environ mental samples to demonstrate petroleum incorporation is
'ndjc . "eslt'd by Clark and Finley." According to these authors, the lirst
"Hreso|°n °' Petroleum uptake is often seen by the presence of a large
"'"Ika V^ enve'°Pe below the n-alkane peaks, and by the presence of
"'tilde T'5eii'2 which have the same order of mag
lot ap e n-alkanes are quite susceptible to microbial catabolistn, and may
"*&& h* '" ^ Pro'i'cs <>f substantially weathered oils. Such chromato
^ey. °U't' reta'n their characteristic inverted cup and saucer appearance.
'so
aPpe
'4-
chr(
Urma,
"latograms of petroleum oils often contain major peaks that
tetr- addit'°n to tllose of n-alkanes. Two isoprenoids. pristane I 2, 6, 10,
"Cca^^'hylpentadecane) and phytane (2. 6. 10, 14-tetramethylhexa-
sarnp|e are ()t particular interest in evaluating the presence of petroleums in
^Wezn', en relatively non-polar Maliimary phases are used (e.g
"lat i,d:" - Ov-1. OV-101. orSt-.W). pristane and phylane yield C,( peaks
" a"ian UIUJ arc usua"y on'y partially separated from the CIT and Ci»
C(St|lnio Peaks (f;i8ure ->• This characteristic tour peak configuration is
"lilrj,^ ^to many oils and indicates the likely incorporation of petroleums by
llrgi)nis'"nvir()nmental samples, Pristane is commonly produced by marine
'"'on c>1S' 'll'Wl-'ver. and consequently does not confirm petroleum incorpn-
^'ano k"' 'le<-ort'ing t() Anderson, el at,' and Ehrhardt and Heineniann.1"
as not been found as a natural component of marine organisms. It.s
^
ln samples therefore suggests oil incorporation.
Sc'ing components
lict at'C '1.vJrOL'arbons are abundant in most crude and refined petroleum
1 and their presence in marine samples is often thought to indicate
petroleum incorporation. For example. Margrave and Phillips'" use the
presence of triaromatic and greater substituted aromatic compounds as a
useful indication of petroleum pollution. The U.S. Department of Com
in i :e" report emphasizes that aromatic hydrocarbons in marine waters are
currently believed to he from petroleum iources. Similarly. Brown and
Huffman' cite evidence suggesting that marine organisms do not produce
aromatic hydrocarbon mixtures. Conversely, Blumer and Youngblood"
describe forest lues and prairie tires as a likely source of many poly nuclear
aromatic hydrocarbons in marine sediments. Similarly. Anderson. i'i <;/.'
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 polymiclcar aromatic hydrocarbons in
marine samples are widely thought to indicate the presence of petroleums.
Several publications describe methods for measuring aromatic livdrocar-
hon* in marine samples. Fluorescence spectroscopy is the most highly
recommended technique. According to Gordon, i-t ul." fluorescence
methods are rapid, sensitive, and simple. While these authors believe
fluorescing materials to be aromalics, they describe the uncertainty of actual
compound identity as LI major drawback. Gordon and Kcizer13 describe
several fluorescence methods for measuring aromatic* in water, while
/itko.JI and Margrave and Phillips1' provide some useful fluorescence data
describing petroleum incorporation by animals and sediments. Several
fluorescence spectra of oils are illustrated in figure .1.
FOSTERTON CRUDE
VENEZUELAN BUNKER C (xO.33)
REDWATER CRUDE
KUWAIT CRUDE
lUANlPA CRUDE
PEMBINA AND
LEDUC CRUDE
380 420
EMISSSION WAVELENGTH (nm)
Figure 3. Fluorescence spectra of several petroleum oils13
129
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OIL SPILL BEHAVIOR AND EFFECTS
Naphthalenes and substituted naphthalenes
The toxicity of higher boiling anomalies 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, et al., l6
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:
1. gas chromatograms exhibiting the presence of an inverted "cup and
saucer effect"-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» alkane peak
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 197.1. 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. et a/..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 50 ft OV-101 support-coated
open tubular (SCOT) column, using a flame ionization detector (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.'2 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
-------
1977 OIL SPILL CONFERENCE
i
I
40
260
300
340
380
420
460
500
Pi EXCITATION WAVELENGTHS (nm)
We 5 c,
^dirng . J~luorescence spectra of sediment sample extracts: (upper)
n0rHrnn m an oil spi" impacted area; (lower) sediment from a
^acted area
isopreno.d phytanc peaks are not evident. This is probahly a consequence of
extensive microbial degradation that lias occurred during the preceding three
years. Substantial fluorescence profiles resembling those of known oils were
obtained from the contaminated sediments, hut only weak profiles differing
substantially from those of known oils were obtained from the unconrimi-
nated sediments (Figure 5). NMR was used in addition to fluorescence
spectroseopy. to confirm the presence of aromatics in the contaminated
sediments. Absorption peaks in the range 6.5-8.1) ppm resulted from the
contaminated sediments, but not from the uncontaminaied sediments (Fi-
gure 6). This absorption region is typical of aromatic compounds and
confirms the presence of petroleum oil in the sediment samples that'were
presumed to be contaminated. This conclusion is also supported hy the ens
chromatogiaphic and fluorescence data.
REFERENCES
I. Anderson. J. W., R. C. Clark, and J. J. Stegeman, 1974. Petroleum
hydrocarbons. Proceedings ,,f Marine Rinussavx W,,rksl,,,,> Spon-
sored by API. EPA and Marine Technology Society. Marine
Technology Society, Washington, D.C.
. *STM. 1976. Annual Book of ASTM Standards, Part .11 Water
American Society for Testing and Materials
I. Bieri. R. H.. A. L. Walker, B. W. Lewis, G. Lasser, and R. J. Hugget.
1974. Marine Pollution Monitorins f Petroleum). NBS Special Pub-
lication 409. Proceedings of a Symposium and Workshop held at
National Bureau of Standards, Gaithersburg, Maryland May H-17
Blumer, M. and J. Sass. 1972. Indigenous and petroleum-derived
hydrocarbons in a polluted sediment. Marine Pollution Bulletin v ?
pp92-94
5. Blumer M G Souza. and J. Sass, 1970. Hydrocarbon pollution of
edible shellfish by an oil spill. Marine Biologv v5 pp195-20''
6. Blumer, M. and W. W. Youngblood, 197S. Polycyclicaromatic hydro-
carbons m soils and recent sediments. Science. v!88 pp53-55
Brc,wn, R. A. and H. L. Huffman, 1976. Hydrocarbons in open ocean
waters. Science. v!9l, pp847~849
8. Clark R.C andj. S. Finley, 1973. Techniques for analysis ot paraffin
hydrocarbons and for interpretation of data to assess oil spill effects
10
'ttr^J^Y^^^
8
6 4
p.p.m.(g)
nn NMR spectra of sediment sample extracts: (upper) sediment from an oil spill impacted area- (lower) sediment from
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
131
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OIL SPILL BEHAVIOR AND EFFECTS
in aquatic organisms. Proceedings of Joint Conference on Preven-
tion and Control of Oil Spills. American Petroleum Institute,
Washington, D.C.
9. Clark, R.C. andJ, S..Finley, 1974. 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, 1974. 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
11. Farrington, J. W. and G. C. Medeiros, 1975. Evaluation of some
methods of analysis for petroleum hydrocarbons in marine or-
ganisms. Proceedings of Joint Conference on Prevention and 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 Seawater by 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-
19
20.
21
carbon contamination in the water column of the northwest Allan"
Ocean. Marine Chemistry. v2, pp251-261
15. Hargrave, B. T. and G. A. Phillips, 1975. Estimates of oil in aqua"
sediments by fluorescence spectroscopy. Environmental Poll"11
v8, pp193-211
16. Rossi, S.S..J. W.Anderson, and G. S. Ward, 1976. Toxicityof*8'6
soluble fractions of four test oils for the polychaetous anne'1 '
Meanthes arenaceodentata and Capilella capitata. Environment
Pollution. vlO, pp9-18 ...
17. Nadeau, R. J. and E. T. Bergquist, 1977. Effects of a Major Oil Sp"
Near Cabo Rojo, Puerto Rico on Tropical Marine Community
Proceedings, J977 Oil Spill Conference. American Petroleum I"5"'
tute, Washington, D.C. ..
18. Neff, J. M. and J. W. Anderson, 1975. An ultraviolet spectrophoto^"
ric method for the determination of naphthalene and alKj
naphthalenes in the tissues of oil-contaminated marine ani"1
Bull, of Environ. Contain. Toxicol. v!4, pp 122-128 . .
U.S. Department of Commerce, 1976. Measurement and Interpreta"
of Hydrocarbons in the Pacific Ocean. Dept. of Commerce Rer,
AID.6BA. '
April 1976
AID.6BA. 76/EPR.3EX.76. Final report on Contract No. 4
• ' r,f
,.35266.
Zafiriou, O., M. Blumer, and J. Myers, 1972. Correlation of °ilsi?|C
Oil Products by Gas Chromatography. Woods Hole Oceanograp
Institution. WHOI-72-55 . flf
Zitko, V., 1971. Determination of residual fuel oil contamination
aquatic animals. Bull, of Environ. Contain. Toxicol. v5, pp 5'*~
132
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REPRINTED FROM: Rapp. P.-v. Reun. Cons. int. Explor. Mer,
171: 33-38, 1977.
THE ULTRASONIC DISPERSION, SOURCE IDENTIFICATION, AND
QUANTITATIVE ANALYSIS OF PETROLEUM OILS IN WATER
MICHAEL GRUENFELD and RAY FREDERICK
Industrial Environmental Research Laboratory-Ci
U.S. Environmental Protection Agency, Edison, New Jersey 08817 U.S.A.
We describe three current projects involving the development of methods for the quantification and
source identification of water dispersed oils, and for the preparation of stable oil-in-water dispersions.
Adaptations of existing solvent extraction, IR quantification, and GC identification methods are discussed,
and some newly developed techniques are presented. The techniques include: 1) a method for inducing
comparable volatility losses in milligram and sub-milligram amounts of oils, and thereby enhancing their
source identification; 2) a rapid adsorption method for separating hydrocarbons from other organics,
and thereby achieving more selective petroleum oil quantification; 3) a method for using an ultrasonic
device to prepare stable oil-in-water dispersions, having a known oil content.
lc*cntifyi
PRODUCTION
"etroleum oils enter the water column of the aqueous
eiU'ironment through many pathways. Offshore drilling
Derations, ships' cleaning operations, damaged tankers
arid storage tanks, industrial and municipal outfalls,
and natural seeps all contribute their share. Discharged
Petrolcum oils thereby constitute a major pollution
Problem, that is under intensive evaluation by the U.S.
Environmental Protection Agency ( EPA), other govern-
^ent agencies, various industrial organizations, and by
Segments of the academic community.
standardized laboratory methods that are cur-
used to monitor oil pollution are procedures for
ying the point of discharge of visible oil slicks
shoreline residues, and methods for quantifying the
extractable organics in water. Few corresponding
ds exist for identifying the source of sub-milli-
quantities of water dispersed oils, and for estima-
the petroleum oil content of waters that also con-
substantial amounts of other extractable organics.
ur paper addresses these needs. We describe two
t.Urrcnt projects to develop methods for the quantifica-
and source identification of water dispersed pe-
oils, and a procedure for preparing stable oil-
dispersions, having known oil content. These
Projccts arc st||j undcnvay; anci our conclusions are
lerefore tentative. We hope to publish final results in
ne "car future.
°IL 'DENTIFICATION
°" identification methods were recently publish-
f'lc American Society for Testing and Materials
They include a gas chromatographic (GC)
procedure (ASTM D3328-74T), and a method for
using nickel, vanadium, sulfur, and nitrogen compo-
nents of oil for the identification of oil (ASTM D3327-
74T). Other recently published, and soon to be publish-
ed ASTM methods are also relevant. They include a
sample preservation technique (ASTM D3325-74T), a
sample preparation method (ASTM D3326-74T), an
infrared (IR) procedure (provisional ASTM designa-
tion D3414-75T), and a data handling technique (pro-
visional ASTM designation D3415-75T). When pro-
perly combined, these methods can be used to identify
the source of discharge of oil pollutants, i.e., to "finger-
print" oils.
While these ASTM methods emphasize the identi-
fication (fingerprinting) of oil pollutants that occur as
visible surface slicks and shoreline residues, our present
interest as described in this paper, is the development
of a method for fingerprinting much smaller quantities
of oils, i.e., oils that are extracted from the water
column. We decided to utilise and integrate existing
procedures wherever possible. Consequently, ASTM
D3328 was selected for matching oils by GC. Similarly,
existing procedures were used for solvent extraction
(Gruenfeld, 1973) and IR quantification (Gruenfeld,
1975). The need for quantification prior to identifica-
tion is discussed below.
Optimum intercomparison of oils is enhanced by
stripping volatile components from weathered and un-
weathered oils, to yield residues with similar volatility
losses. ASTM D3326 achieves this by distillation: 50
ml portions of slicks, shoreline residues, and unweather-
ed oils are heated to yield 280°C + distillation residues
that are then matched by GC, IR, and the elemental
133
-------
Michael Gruenfeld - Ray Frederick
analysis methods. But, the requirement for 50 ml quan-
tities of oil greatly exceeds the amount of oil that is
usually available from the water column; i.e., water
samples normally yield only microlitre (mg) quantities
of dispersed oil. Therefore, we developed a procedure
for treating 0-5 and 30 mg quantities of oil to yield
residues with GC profiles that closely resemble the
residue profiles of ASTM D3326.
It should be noted that other methods are available
for GC identification of water dispersed oils (CON-
CAWE, 1972; Dell'Acqua et al., 1975). But these pro-
cedures do not attempt to optimize intercomparison of
oils by yielding residues with similar volatility losses,
nor do they benefit from the incorporation of a stan-
dardized ASTM method.
OIL QUANTIFICATION
Many methods are available for estimating the
amount of oil in water. Some measure extractable
aromatics by UV absorption or fluorescence techniques
(Zsolnay, 1973; Levy, 1971; Gordon and Keizer, 1974).
Others measure extractable hydrocarbons by IR, follow-
ing Florisil or silica gel adsorption chromatography
(CONCAWE, 1972; Brown et al., 1974; Yu and Cole-
man, 1975), while still others measure total extract-
able organics by IR and gravimetric procedures (EPA,
1974; Gruenfeld, 1975). All of these methods can
estimate S-ig/ml levels of oil in water, but only the UV
absorption and fluorescence methods, and one IR proce-
dure (Brown et al., 1974) estimate 1-10 ng/litre levels.
This IR method is somewhat cumbersome,, however,
because exceedingly large water samples are need-
ed (usually 20 litre), while the UV absorption and
fluorescence methods suffer from substantial variability
in response from oil to oil, thereby handicapping se-
lection of meaningful reference standards.
Our current interest as described in this paper, is the
development of a method for estimating the dispersed
petroleum oil content in water columns near surface
slicks, ships' ballast water discharges, offshore waste
disposal sites, and offshore platforms inter alia. The
method should be capable of distinguishing petroleum
oils from other extractable organics, and be appropriate
for estimating oil in water concentrations below 10 tig/
litre. We are evaluating, as the method of choice, a
rapid silica gel adsorption technique (Longbottom,
1974). followed by IR measurement of hydrocarbons.
The work is still incomplete, however, and consequently
discussion is limited to evaluation of the silica gel ad-
sorption step.
OIL DISPERSION
Adequate methods are needed for preparing stable
oil-in-water dispersions that can be further diluted with
30 20
RETENTION TIME (min.)
Figure 21. South Louisiana Crude oil. Untreated neat oil (upper
chromatogram). After ASTM D3326 distillation (lower chro-
matogram). Numbers refer to n-alkanes. Pr and Ph refer to
pristane and phytane, respectively.
water. Use of stable dispersions having known oil con-
tent can enhance bioassay tests of oil toxicity, and per-
formance evaluations of oil quantification methodology.
Most dispersions are now prepared by vigorously shaking
or mechanically mixing oils with water, but separation
of oils can quickly ensue. Consequently, we evaluated
the use of a commercially available ultrasonification
device for preparing stable dispersions. We then exa-
mined the resulting dispersions for stability and miscibi-
lity with water. We also tested the ultrasonically dispers-
ed oils for spectral and chromatographic alterations
that may interfere with their quantification and iden-
tification.
EXPERIMENTAL
APPARATUS
Gas chromatographic determinations were performed
with a Perkin-Elmer Model 900 GC instrument1,
equipped with a flame ionization detector. A 3 m by
3mm O.D. (0-6 mm wall) stainless steel column was
used, packed with 10 % OV-101 on 60/80 chromosorb
W (AW-DMCS treated). Helium at 40 ml/min was
the carrier gas. Temperature programme: initial-50°C
for 2 min., final-325°C, programme rate-8°C/min., in-
jector-300°C.
Infrared spectroscopic determinations were perform-
ed with a Perkin-Elmer Model 45 7A IR instrument.
Absorbances were measured in 10 mm rectangular silica
1 Mention of trade names or commercial products does not
constitute endorsement by the U.S. Government.
134
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The ultrasonic dispersion, source identification, and quantitative analysis of petroleum oils in water
30 20
REIENHON IIV1E (mm.)
>-,.
"Sure 22. South Lousiana Crude oil. After ASTM D3326
Distillation (upper chromatogram). After the 70 nig neat oil
treatment procedure (lower chromatogram). See legend of
21 for key to numbers and letters.
(Beckman Instruments Incorporated, catalogue
Dumber 580015), using Perkin-Elmer cell holders (ca-
U°gue number 186-0091).
Ultrasonic dispersions were prepared with a Branson
Power Company (Danbury, Connecticut, USA)
W185 Sonifier Cell Disrupter, using a Va inch
horn with plain conical tip.
opectroanalyxed carbon tetrachloride (Fisher Scien-
If'c Company, Catalogue Number C-199), and Freon
3 solvent (E. I. Du Pont De Nemours and Company,
He.) vvere used. Freon 113 is a Du Pont designation
°r 1,1,2-tricliloro- 1,2,2-triflnoroethane, and this rea-
«ent is also available from other manufacturers under
Xarious trade names.
^°paration of hydrocarbons from other organics was
with Davidson Type 923 silica gel ( 100—
ll lfl<'ntification
O»
. ' "Hulatc'd oil in water samples vvere prepared by
( .
. rasonically dispersing a South Louisiana Crude oil
cr. Sample extraction was achieved with four, 25
Portions of Freon 113, following addition of acid
' ^ (Grwnfehl. 1973). Quantification of oils was
by IR spectroscopy (Gruenfeld, 1975),
by further dilution to yield final 100 ml Freon
A ^Utl°ns containing 0-5 or 30 mg oil. Simulation of the
* M D3326 distillation was accomplished by strip-
ping the solutions to final 1—2 rnl volumes, in 150ml
beakers, with the aid of a steam table and a filtered
air stream and the concentrates were transferred to 10
by 30 mm glass vials. The vials containing 30 mg por-
tions of oil were suspended in a 40°C water bath, and
a filtered air stream was used to remove final solvent
traces. This condition was maintained for 10 additional
minutes. The vials containing 0-5 mg portions of oil
were maintained at room temperature, the air flow
was turned off just before total solvent removal and
final solvent evaporation occurred spontaneously. This
condition was maintained for 10 additional minutes.
Distilled solvent was used for the lower concentration
determinations. Small amounts of CCli (10—20(xl)
were then added to each vial for GC injection.
In order to enhance intercomparisons of neat oils
with dispersed oils, the neat oils were treated as fol-
lows: vials containing 70 mg portions of neat (un-
dispersed) oils were suspended in a 40°C water bath
for 15 minutes, in the presence of a filtered air stream.
RETENTION TIVE (min.l
Figure 23. South Lousiana Crude oil. After the 70 mg neat oil
treatment procedure (upper chromatogram). After the 30 mg
dispersed oil treatment procedure (lower chromatogram). See
legend of Figure 21 for key to numbers and letters.
135
-------
Michael Gruenfeld - Rav Frederick
RETENTION TIME (min.)
Figure 24. South Louisiana Crude oil. After the 70 mg rient nil
treatment procedure (upper chromatogram). After the 0-5 nig
dispersed oil treatment procedure (lower chromatogram):
(A) - shows slight column degeneration, (B) - new column.
See legend of Figure 21 for key to numbers and letters.
Portions of the residues were then injected onto the GC
column.
Oil quantification
While modifications of existing procedures for oil ex-
traction (Gruenfeld, 1973) and quantification (Gruen-
feld. 1975) to improve sensitivity are planned, our
present discussion deals only with a silica gel adsorption
technique for separating petroleum oils from animal
and vegetable oils, directly in CCU solution. The latter
oils contain many components that typify the water
dispersed non-hydrocarbon organics that separate to-
gether with petroleum oils during solvent extraction.
Carbon tetrachloride solutions of South Louisiana
Crude and Number 2 fuel oils, and vegetable, olive,
and cod liver oils were prepared in 100 ml volumetric
flasks, at the following concentrations: petroleum oils -
20 ing/100 ml, non-petroleum oils - 100 nig/100 ml.
These solutions were vigorously stirred for 5 and 10
minute intervals with a magnetic stirrer, after additions
of 3 g activated and partly deactivated silica gel. Acti-
vation was achieved by maintaining 100 g portions of
silica gel at 150'" C for two hours. The degree of ad-
sorption of the non-petroleum oils by silica gel. and the
impact of silica gel adsorption on the petroleum oil5
were monitored by IR (Gruenfeld, 1975). The degree
of oil removal from solution was compared graphically
with the degree of silical gel deactivation and solution
stirring time.
Oil dispersion
Ultrasonic dispersion was achieved by inserting the
instrument probe into 50 ml graduate cylinders con-
taining 50ml water and accurately weighed amounts
of oil. Maximum dispersing energy was applied i
two minutes, while suspending the cyclinders in an &
bath. Dispersions of a South Louisiana Crude oil (•*'*'
cSt at 38°C) and a Bachaquero Crude oil (1070 cSl
at 38 °C) were tested for stability and miscibility Wi"1
water. Portions of the South Louisana oil were also re-
covered after dispersion, and examined for ultrasonical"
ly induced chromatographic and spectral changes. Gas
chromatography, and IR. UV, and fluorescence spec-
troscopy were used.
RESULTS AND DISCUSSION
OIL IDENTIFICATION
As previously explained, our immediate goal was t*
develop a procedure for enhancing comparisons of i'n
dispersed oils, with 0-5 and 30 ing quantities of water
dispersed oils. We hoped to induce losses of volatiles »
minute oil residues, that approximated losses induce*
by ASTM D3326.
The ASTM method strips off almost all component
below the Cn normal alkane (Fig. 21). Our procedure
for treating neat oils yields equivalent losses (Fig- 22 J-
Similarly, our procedure for treating 0'5 and 30 m=
portions of dispersed oils yields chromatograms th*
nearly match the chromatogram of the neat oil (l*1^ '
23 and 24). Therefore, within the constraints of °i»
limited tests (one oil was used) it appears that c
method can successfully correlate water dispersed 01"
with neat oils, for the purpose of source identilicatK
(fingerprinting). However, evaluation of the nietnt
with more oils is needed, especially following short ten
weathering in the water column. This work is
underway and will be reported in the near
Meanwhile, our method offers a convenient te^
whereby reproducible and intercomparable chrornat
grams of milligram and sub-milligram amounts of '
can be obtained.
OIL QUANTIFICATION
As part of our work to develop an appropriate °
quantification method, we evaluated a rather uruq1'
136
-------
Tin- ultrasonic dispersion, source- idcniilicalion. and quantitative analysis of petroleum oils in water
110 r-
90
•
a
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
D Olive Oil 10 min.
p.
5
PERCENT DEACTIVATION
"). Adsorption of nils by silica gel in CC1, solution. IR
ni(1asurcniriits arc at 2!I30 cm-1, using 10 mm path length cells.
'' ;'
',' ;'"|"ion technique for separating- petroleum oils
^"1 animal and vegetable oils. Separation was achiev-
j:, J'V merely adding small amounts of silica gel to
'' ' solutions of oils, and then stirring brielly. The
.^-petroleum oils were removed by silica gel, while
0 Petroleum oils remained in solution. Consequently,
'e expect that this procedure will successfully separate
Ho.st water dispersed petroleum oils from the non-hy-
°carhon organic* that are also extracted from the
,Vatcr column. It should be noted that Flori.sil can also
tj' "S(;d in a similar manner (CONCAWE, 1972), but
lat silica! gd is more effective {Longbottom, 1975).
'n order to optiim'/e the adsorption procedure, we
Ciliated the interaction of silica trcl dcactivation and
v'mion stJn-in<: time, \vith oil separation efficiency
2")'i. Maximum separation of the petroleum oils
°'T1 the non-petroleum oils was achieved with fully
(|.'V;lti'd silica u'el, and a 10 minute stirring period
"'"'ring vate was not monitored). These conditions did
.'!* f.'iu.se a visible loss of the petroleum oils. Increased
lr;i U'i'1 deactivation. and reduced stirring time ad-
versely affected the method. The method appears quite
practical for separating petroleum oils from non-hydro-
carbon organics, prior to IR measurement. But, fully
activated silica gel, and ample magnetic stirring (10—
15 min.) are recoinmended.
OIL DISPERSION
Preparation of stable oil-in-water dispersions by ultra-
sonification offers some attractive possibilities. The con-
centrates are stable, infinitely miscible with water, and
have a known oil content. They are useful for critical
method evaluations, and can be added to water co-
lumns for various biological and toxicity tests. Our
method was used recently to advantage in a study of
the mode of accumulation of Number 2 fuel oil by the
soft shell clam Mya arcnaria (Stainken, 1975). Disper-
sion stability appears to diminish with increasing oil
viscosity. The South Louisiana oil yielded 1 % (10 000
Hg/ml) dispersions that remained stable beyond four
days. But, the Bachaquero oil yielded less concentrated
dispersions, that were also less stable.
Since ultrasonification causes considerable heatin^
resulting in oil vapourization losses, we incorporated an
ice bath in the method to prevent these losses. Follow-
ing ultrasonification of 6 and 30 mg portions of the two
oils, recoveries in the range, 96—100 %, were obtained.
In addition, ultrasonically treated and untreated por-
tions of the South Louisiana oil were compared by gas
chromatography, and IR, UV absorption and fluorescen-
ce spectroscopy. No significant chrornatographic or
spectral changes were observed.
CONCLUSION
^ Current work to develop methods for the quantifica-
tion and source identification of water dispersed petro-
leum oils, and for the preparation of stable oil-in-water
dispersions, is discussed. Tentative procedures are pre-
sented for: 1) separating hydrocarbons from non-hydro-
carbon organics for the purpose of oil quantification;
2) inducing comparable volatility losses in milligram
quantities of oils to enhance their source identification;
!i) preparing stable oil-in-water dispersions with known
oil content, that are water miscible. Preliminary con-
clusions are provided regarding the successful applica-
tion of the methods, but these conclusions are subject
to future tests with more oils, and to integration of our
procedures with already existing methods. Final results
will be published in the near future.
ACKNOWLEDGEMENTS
The authors are grateful to Messrs. Uwe Frank and
Fred Behm. and Misses Pamela S/eely and Janet Buz-
xaro for the data acquisition that has made this paper
possible.
137
-------
Michael Gruenfeld - Ray Frederick
REFERENCES
Brown, R. A., Elliott, J. J. & Searl. T. D. Measurement and
characterization of non-volatile hydrocarbons in ocean water.
In Marine pollution monitoring (petroleum). NBS (U.S.) Spec.
Publ. 409. 293 pp. National Bureau of Standards, Washington,
D.C. 20234, U.S.A.
CONCAWE 1972. Mineral oil in water by infrared spectropho-
tometry. In Methods for the analysis of oil in water and soil.
Report No. 9/72. Stichting CONCAVVE, The Hague.
Dell' Acqua, R., Egan, J. A. & Bush, B. 1975. Identification of
petroleum products in natural water by gas chromatography.
Environ. Sci. Technol. 9: 38-41.
EPA 1974. Oil and grease, total, recoverable. In Methods for
chemical analysis of water and wastes. EPA-625-/6-74-003.
298 pp. U.S. Environmental Protection Agency, Office of
Technology Transfer, Washington, D.C. 20460, U.S.A.
Gordon, D. C. & Keizer, P. D. 1974. Estimation of petroleum
hydrocarbons in sea water by fluorescence spectroscopy:
improved sampling and analytical methods. Technical Report
No. 481. Environment Canada. Fisheries and Marine Service,
Marine Ecology Laboratory, Bedford Institute of Oceano-
graphy, Dartmouth, Nova Scotia.
Gruenfeld, M. 1973. Extraction of dispersed oils from water for
quantitative analysis by infrared spectrophotometry. Environ.
Sci. Technol. 7: 636-9.
Gruenfeld, M. 1975. Quantitative analysis of petroleum oil pol-
lutants by infrared spectrophotometry. In Water quality para-
meters. ASTM STP 573. 580 pp. American Society for Testing
and Materials, 1916 Race Street, Philadelphia, Pennsylvania
19103, U.S.A.
Levy, E. M. 1971. The presence of petroleum residues off the
east coast of Nova Scotia, in the Gulf of St. Lawrence, and
the St. Lawrence River. Water Res. 5: 723-33.
Longbottom, J. E. 1974. Oil and grease analysis. Analyt. Quality
Contr. Newsl. 22:7. U.S. Environmental Protection Agency,
Cincinnati, Ohio 45268, U.S.A.
Longbottom, J. E. July 1975, personal communication. U.S-
Environmental Protection Agency, Cincinnati, Ohio 45268,
U.S.A.
Stainken, D. M. 1975. Preliminary observations on the mode of
accumulation of No. 2 fuel oil by the soft shell clam, Mya artnana-
In Proceedings of the 1975 Conference on Prevention and
Control of Oil Pollution. 612 pp. American Petroleum Institute,
1801 K Street N.W., Washington, D.C. 20006, U.S.A.
Yu, T. S. & Coleman, W. H. 1975. A quantitative method for
determining apparent oil concentration in water containing
detergents. NSRDC TM-28-75-10. Department of the Navy.
Naval Ship R & D Center, Bethesda, Maryland 20084, U.S-A-
Zsolnay, A. 1973. Determination of aromatic hydrocarbons_in
submicrogram quantities in aqueous systems by means of higj1
performance liquid chromatography. Chemosphere 6: 253-61"
138
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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 quantisation
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.
This is the oral presentation of the proceeding paper.
139
-------
Oil quantitatlon 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 spectra!
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
140
-------
room temperature for 10 additional minutes. Finally, small amounts of car-
bon tetrachloride (10 - 20 ul) were added to each vial for gas chromato-
9raphic 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
°ils were then injected onto the gas chromatographic instrument without
further dilution.
/ci-j >>\ n vaporization
(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
°f 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-
Natogram 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
°il; 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
ar>d phytane from the normal alkanes was achieved with a new column. I
should emphasize that although oils that actually weathered in the water
141
-------
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 guantitation 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 }% (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
142
-------
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 -
143
-------
Slide 1
1 4
-------
Slide 2
145
-------
Slide 3
-------
• ifffl if)
.6
".:
• ttHMIIOM TIME (ML)—20-
Slide 4
-------
Slide 5
148
-------
I
Slide 6
149
-------
Slide 7
1 50
-------
no p-
1004
A
A
0)
_Q
- 90
T3
O
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
D Olive Oil 10 min.
3 5
PERCENT DEACTIVATION
Slide 8
151
-------
Slide 9
152
-------
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.
153
-------
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 =4°
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 Mari«e
Pollution Monitoring Symposium, NBS Special Publication 409:127
Belcher, R. S. (1974): "Determination of Mineral Oil In Water", In: Examinati011
of Waters: Evaluation of Methods for Selected Characteristics; Australia0
Water Resources Council Technical Paper No. 8:79-83
Bieri, R. H. , et al. (1974): "Identification of Hydrocarbons in an Extract
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.. _3:309
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", Marine Pollution Bulletin, 5.: 101-105
Bogatie, C. F. (1974): "Rapid Identification of Oil and Grease Spills from P°lp
and Paper Mills by Infrared Spectroscopy", Tappi , 57:130-134
Brown, C. W. , Lynch, P. F., Ahmadjian, M. (1974): "Monitoring Narragansett
Oil Spills by Infrared Spectroscopy", Environmental Science and Technology- '
j8:669-670
Brown, C. W. , Lynch, P. F., and Ahmadjian, M. (1974): "Novel Method for
Oil Spills and for Measuring Infrared Spectra of Oil Samples", Anal.
46:183-184
154
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Brown, R. A., Klliot, J. J., and Searl, T. D. (1974): "Measurement and Charac-
terization of Nonvolatile Hydrocarbons in Ocean Water", NBS Special Publi-
cation 409: 131
"town, 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.
B*uce, 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
Bu<*ininkas, 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
PL
nernatskaya, A. N. (1974): "Determination Using Modern Methods of Impurities
Polluting the Waste Waters from Petroleum Refineries", Khimiya Tekhnolo-
£iya 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
Cla*k, 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
n
tetney, w. J., and Wong, C. S. (1974): "Fluorescence Monitoring Study of Ocean
Weather Station "P"", NBS Special Publication 409:175
, N. G. (1974): "Determination of the Content of Petroleum Products
in Water by an Optical Acoustical Method", Izmeritel 'naya Tekhnika, Ji:66
, M., and Heineman, J. (1974): "Hydrocarbons in Blue Mussels from the
Kiel Bight", NBS Special Publication 409:221
^
*rrington, 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
p.
r*lngton, J. W., et al. (1974): "Analysis of Hydrocarbons in Marine Organisms:
Results of IDOE Intercalibration Exercises", NBS Special Publication 409:163
S
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Seawater Along the Halifax-Bermuda Section: Lessons Learned Regarding
Sampling and Some Results", In the Proceedings of the Marine Pollution
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Fluorescence Spectroscopy of the Present State of Petroleum Hydrocarbon
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Petrol Origin by Way of Fluorescence Spectroscopy", Zeits Anal. Chem. (Ger/>
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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
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Combustibili. 28^367-371
Iliffe, T. M., and Calder, J. A. (1974): ''Dissolved Hydrocarbons in the
Gulf of Mexico Loop Current and the Caribbean Sea", Deep Sea Res., 2Jj481
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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
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156
-------
Jeltes, R. (1974): "Prompt Detection and Tracing of Oils and Other Detrimental
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Kawahara, F. K. (1974) : "Recent Developments in the Identification of Asphalts
and Other Petroleum Products", NBS Special Publication 409:145
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Heavy Residual Fuel Oils and Asphalts by Infrared Spectrophotometry Using
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McClynn, J. A. (1974): "A Review of Techniques for the Characterization and
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Medeiros, G. C. , and Farrington, J. W. (1974): "IDOE-5 Intercalibration Sample:
Results of Analysis After Sixteen Months Storage", NBS Special Publication
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Mitra, G. D., et al. (1974): "Gas Chroma tographic 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, &'
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
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 Oil8'
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Rashid, M. A. (1974): "Degradation of Bunker C Oil Under Different Coastal Envi~ *
ronments of Chedabucto Bay, Nova Scotia", Estuarine & Coastal Mar. Sci., 2}
Ray, S. M., Oja, R. K. , Jeffrey, L. M. , and Presley, B. J. (1974): "A Quantitatijf
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
by Two-Dimensional Precision Gas Chromatography", Journal Chroniatography_»
9^:603
Sackett, W. M. , and Brooks, J. M. (1974): "Use of Low Molecular-Weight-Hydrocafb°
Concentrations as Indicators of Marine Pollution", NBS Special Publication
409:171
Sleeter, T. D. , et al . (1974): "Quantitative Sampling of Pelagic Tar in the
Atlantic, 1973", Deep Sea Res.. 2^1:773
Straughan, D. (1974): "Field Sampling Methods and Techniques for Marine Orga
and Sediments", NBS Special Publication 409:183
Sutton, C., and Calder, J. A. (1974): "Solubility pf Higher-Molecular-Weight
n-Paraffins in Distilled Water and Seawater", Environmental Science
nology. 8:654-657
158
-------
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, _8:657-663
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
v°nHellman, H,, and Holeczek, M. (1974): "Kohlenwasserstof fe in Quellwassern-
Olverschmutzung oder Naturstof fe?11, Tenside Detergents. H :197
Warner, J. S. (1974): "Quantitative Determination of Hydrocarbons in Marine
Organisms", NBS Special Publication 409:195
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Electrolytic Stripping Cell", Jour. Chromatog. Sci . . 21=845
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Sampling, Analysis, and Interpretation", by Institute of Petroleum, Oil
Pollution Analysis Committee, Applied Science Publishers Ltd., Essex, England
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Ecosystem", South African Journal of Science, 70:141
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Perkin-Elmer Infrared Bulletin 22
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Acidification", Perkin-Elmer Infrared Bulletin 41
8°lnay, A. (1974;: "Determination of Aromatic and Total Hydrocarbon Content in
Submicrogram and Microgram Quantities in Aqueous Systems by Means of High
Performance Liquid Chroma tography", In the Proceedings of the Marine Pollu-
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8°lnay, A. (1974): "Determination of Total Hydrocarbons in Sea Water at the
Microgram Level with a Flow Calorimeter", Journal Chromatography , 90:79
159
-------
AS 'I'M D3325-74T (1975): "Tentative Method for Preservation of Waterborne Oil
Samples", ASTM Standards. 31:565-567
ASTM D3325-74T (1975): "Tentative Method of Test for Preparation of Sample
for Identification of Waterborne Oils", ASTM Standards , 31; 56 1-564
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., 42:374-378
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 Butter field Rd., Oak Brook, 111. 60521
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element True Boiling Point Gas Chromatography for Monitoring Oil Pollution »
Joint Conference on Prevention and Control of Oil Pollution, San Francisco •
93-97
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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_gS£
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Composition", Anal. Chem.. 4^:1573-1583
Farrington, J. W. , 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
oy"
Frank, U. (1975): "Identification of Petroleum Oils by Fluorescence Spectroscope
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.: 331-34°
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ance of Aromatic and Aliphatic Components from Small Sea Surface Slicks »
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160
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En y i ro nine n l a 1 S c I e u c e and Tech no logy, 9:469-472
s
-------
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193-215 "
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Passive Tagging Procedures for the Identification of Crude Oil Spilled
on Water", Joint Conference on Prevention and Control of Oil Pollution,
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Chromatography, Gravimetry/Densitometry Comparison", Environmental Scien££
and Technology, 9_:241-246
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of Australian Crudes and Condensates by Gas Chromatographic Analysis",
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Journal Water Pollution Control Federation, 47:796-809
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Infrared Spectrometry to the Identification of Petroleum", Anal. Chern^,
47:1696-1699
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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
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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
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Method for the Determination of Naphthalene and Alkylnaphthalenes in
the Tissues of Oil-Contaminated Marine Animals", Bulletin of Environmen
Contamination and Toxicology, 14:122-128
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Aromatic Hydrocarbons in Crude Oils, Heating Oils, and Marine Tissues', ^
Joint Conference on Prevention and Control of Oil Pollution, San Francis
103-115
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Triterpane Fingerprinting of Crude Oils", Anal. Chem. , 4_7_: 1617-1622
162
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II, PETROLEUM OILS
C, ENVIRONMENTAL STUDIES
163
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(*) 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)
* See page 2.'
-------
REPRINTED FROM: Proceedings of 1975 Conference on Prevention and Control
of Oil Pollution, San Francisco, CA. March 25-27, 1975,
pp 463-468. Available from API Washington B.C.
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
<,Cc "emical analysis has shown that various components of oils can
Wich within marine invertebrates. Several mechanisms by
e*pe may occur n°ve been conjectured. This paper offers
frjip,. rtffll' verification of a mechanism by which a commercially
snej n°nt bivalve, Mya arenaria, can accumulate oil within its tis-
are' e Piper also documents the behavioral response of Mya
have shown that various components of oils can accumulate with
marine bivalves [11,6,7,33,10).
deleterious ecological side effects resulting from oil
WubT"8 My° (25~35 mm) were ex?°sed t° #2 f»el oil and an oil
*vate.e ~?e (OH Reci 0) which were ultrasonically emulsified in
ppm ' P>e concentrations tested were 50 ppm, 100 ppm and 150
4°Ca *po®*res were done in both natural and artificial seawater at
"Q 2 C. Exposure periods ranged from 3 hours to 4 days.
fffec?Cr°SCOp'c oljservat'ons were performed to determine the
Wick °l t>le dyed oil contactinS the gill surfaces and the means by
'espo °'l was either ingested or ejected. Definite patterns of
tfegi Se to the dyed oil were established. Essentially, the clams
°'l micelles and globules as food or detritus particles. The
oil micelles are passed by ciliary currents directly to the
Cfeiid"'' I:arger globules are bound by mucus secreted by the gill
bincj/f.1"' f'as chromatography and mass spectrometry confirmed the
°f tf,eg ?f oH-mucus. The oil-mucus is ingested or rejected by means
listn m c'l'ary Pathways. Implications of the oil-mucus mecha-
^ed tfle eie°tton of this mucus into the environment are dis-
ODUCTION
cHnarj sP'llage continues to be a serious problem in coastal and
'% nc waters. Many spills reported from barges, tankers, and
'Vbe nstallalions involved fuel oils (8,2,32,22,20]. Spilled oil
0)1ida( a red bV many factors including evaporation and photo-
l^,2i ,'°n- The water soluble portions of the oil, mostly aromatics
'''oca r'" d'sso!ve and be dispersed. Many of the petroleum
f°rir>at'r -S may be d'sPersecl throughout the water column by
ghetnjcl()n °f ernulslons. either by turbulent wave action [13,12] or
XlSjQ dlsPersion. Moms [23] documented the occurrence of oil
a
-------
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. Harrington
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-
praded 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 soiuble dye. Oil Red
0, Adapting a procedure developed by Gruenfeld and Behm [14],
#2 fuel oil and Oit 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
dams were placed in specimen dishes with natural filtered seawater
(26%) or artificial Rik 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-oU-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 crac'!en'
small red oil globules would appear on the water surface. Even wn
some of the dyed oil emulsion accumulated on the surface, t"c ^
was still a visible red tint in the water column. This may have be
due to the solubilized (colloidal micelles) and soluble hydrocai''
components in the dyed fuel oil emulsion-seawater mixture [
Because some of the dyed emulsion did crack with time,
tu
IIV, «• ,
exposure concentrations are relative only to time 0 when the crn
sion was added. The Oil Red 0 (C.I. 26125) is a neutral dis-azo or
which does not contain water solubilizing groups and hence is "J .
uble in aqueous media but is soluble in oils, fats, waxes, etc. [1 'j
The Oil Red 0 dye was totally insoluble in aerated and nonaera
control mixtures of dye and artificial seawater and dye * ^
natural seawater. Earlier toxicity and ancillary experiments
confirmed that the patterns of mucus secretion and be . „<>
response were elicited by the emulsified fuel oil. The dye had
effect on the clam. -\
In preliminary experiment I at 22°C, seven clams (25-35 rn
were used. Tests were run for 3 hours. All clams behaved simlja aj
The dyed oil emulsion present in the water while the clarn ^
filtering resulted in periodic "coughing" and periodic reject!" ^
dye-oil in a mucus binding. This periodic coughing is a rapid .
traction of the adductor muscles which forces water out the i ^
ant siphon. It is a mechanism commonly used by bivalves to
themselves of accumulated detritus and pseudo feces. . ^t
Within 2 to 3 hours, two clams had dyed oil-mucus visible i«^ ^
stomach. One of these plus two others now had dyed oil-mucw^(
the digestive diverticula and around the style. Most commonly^ ^
dyed oil was found bound in mucus near the palps. The behavi ,
the dyed oil on the gills and palps was closely followed. It was n ^
that the oil droplets and globules were passed to the edge 01 .
gills, bound in mucus as a food or detritus particle, and Pa'
towards the palps. Smaller oil droplets passed directly to the P ^
The oil usually followed the ciliary pathways outlined by »
[181- ,..edin
The results from preliminary experiments II and III are B -yC,
tables 1 and 2 respectively. In preliminary experiment IV at ^
clams were exposed to 50 ppm and 100 ppm of dyed #2 f" ef
emulsion for 24 hours. Large clams were used (40-50 mm), "."^joil
concentration. At 50 ppm exposure, one clam accumulated ay j,
and oil-dye-mucus in the stomach and diverticula. All hao jpfl
oil-dye-mucus on the mantle, gills, and some on the palps. A ^j
ppm exposure, three died. One accumulated oil in the storna*
diverticula, with oil-dye-mucus on gills and mantle.
The first accumulation experiment at 4"C was a 24-hour ^
sure of clams (20-25mm) to the dyed-oil emulsion at concentra
of 50 ppm and 100 ppm. Fourteen clams were used in the test ^
ppm exposure. Four clams accumulated dyed oil in the stornac .,
diverticula. Two clams had no oil visible and ten had dye"
mucus on the gills, palps, mantle, and foot. Some dyed
exited through the pedal aperture. Seven clams were exposed
100 ppm concentration. Five clams accumulated dyed °"^,}
stomach and digestive diverticula. All clams had oil-dye-rnu
gills, mantle, and palps. , . >
The results from accumulation experiment II at 4°C are W ^
table 3. In these experiments, clams were exposed to the ay
emulsion for 1,2, 3, and 4 days. Figure 2 illustrates the res ^
accumulation experiment II at 4°C. Experimental ptoblcw
eluded the measurement of the incorporation of the 150 pP
centration of the dyed oil emulsion on day one.
Chemical confirmation of the oil being bound or
mucus is shov/n in figure 1. The data was obtained from a "j^"^
series of experiments in which Mya were exposed to s"^^!^'!
centrations df #2 fuel oil. Complete results from these eXP6^!^
will be published at a later date. The mucus extracted was s .^
by clams exposed to an initial concentration of 100-ppm elT1 {ed- ,
#2 fuel oil. Four weeks later some of the mucus was colle up
had a gray-green flocculcnt appearance and formed ^"/.^^
clumps on the surface and in the water column. A sample ° fitf
and mucus was taken from the water column (there was n°
t, d'"
166
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EFFECTS
Table 1. Preliminary experiment 11-22°C, natural filtered
seawater, clams (20-25 mm) were placed in specimen dishes
and dyed oil added
rouP 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.
rouP 2-83 ppm, exposure 5 hours (5 clams)
All clams had packaged the. oU-dye within mucus. This
oil-dye-mucus was always found on and around the palps.
„ A small amount was sometimes found on the gills.
rouP 3-83 ppm, exposure 5 hours (kept anaerobic l'/z 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
oit-dye-mucus was being shunted out the pedal aperture
Q and siphon in several clams.
roup 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.
°uP5-83 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.
°UP 6-Kept anaerobic I'/z 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.
°UPS 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.
Gr,
Gr
Gr,
of ••' and filtered through no. 1 Whatman paper. Infrared analysis
apn Water at tne time of sampling indicated a concentration of
j^'oxirnately 0.466 ppm oil in the water column. Solvents were
\va * distilled and checked for impurities. All glassware was pre-
^as r With solvents- Approximately 40 ml of mucus-water sample
,, tillered and then washed with artificial seawater. The filter was
C|,j" extracted in an erlenmeycr flask with 50 ml of carbon tetra-
'Wr6 With constant shaking for ten minutes. The sample was
» Y *''tered through prewashed sodium sulfate and concentrated to
"H of °-15 ml under nitr°gen- During this procedure, all
*0n0n tetracnl°ride evaporated. Gas chromatographic analysis was
With* US'ng a Pcrkin El"101 Model 900 gas chromatograph equipped
lWa ^mc ionization detector and a six-foot stainless steel column
°P«t • With 8% Dexsi' on 80/100 mesh of Chromosorb W. Standard
4e(ea'ln8 conditions were as follows: carrier gas N2, 2 cc/min;
de, '°f H2, 20 cc/min, air 40 cc/min; injector temperature 200°C,
Hi! temperature 300°C, manifold temperature 300°C; column
Of 80plture 70°C with two-minute hold, programmed at an increase
""Men Per minute to 300"C; injection sample 0.1 micro liter. The
Uteki'^mple gas chromatographic tracing was quantitated gravi-
by comparison to the peak area of 16 micrograms of
dissolved in hcxane. The calculated quantity of oil
m the mucus was 833 micrograms. The hydrocarbon con-
-- water was 4.66 ppm or 0.466 micrograms per milliliter.
f°ie, the most the hydrocarbon background of the water
Table 2. Preliminary experiment ///-22°C, clams (25-34 mm)
placed in 1,600 ml Rila seawater mix (26%), 12-hour 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): 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.
50 ppm (#2 Fuel): All clams had oil-dye and or oil-dye-
raucus 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.
100 ppm (#2 Fuel): 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.
50 ppm (So. Lo. Crude): 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 Dntributed by adhering to the filter was 18.64 micro-
grams, and iiie 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 Jersey. 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 [ 1 ] 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 in 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 will 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
167
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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 clams)-Four clams accumulated dyed oil in stomach
and digestive diverticula. 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)-AU 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
oil-dye-mucus commonly occurring in large masses on, near, and
around the palps of Mya. 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
the 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
Q- S0«
Oly 1
Day
Figure 2. Accumulation experiment II. Clams exposed to dyed
#2 fuel oil emulsion at 4°C
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 narc zation 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 con tracts 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
168
-------
EFFECTS
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
"arcotization of the clam.
If the clam's gut is full of detritus and silt there is little visual
Cumulation. The most rapid accumulation of oil seems to be cor-
, 1ed with a partially empty gut. When the dyed oil is in the
fornach, it may be in mucus fragments or as minute droplets. Occa-
^Onally, the style tip may be tinted red. Next, the digestive divertic-
la gradually become tinted from the stomach outward toward the
*nit of the digestive diverticula where the tissue becomes undiffer-
e«tiated gonad.
The ability of the clam to accumulate oil, concentrate it in
ty}|cus. and release it, may have deleterious ecological side effects.
ni]e tjje c|am js accurnuiating oil within its own tissues, it may also
, m accumulating oil in their tissues and rejecting some in the form
concentrated oil-mucus. This situation is greatly aggravated in the
^* of coastal spills when dispersants and sinking agents are used.
. ne effect of sinking oil or dispersing it is primarily cosmetic and
peases the potential for oil accumulation by filter and detritus
lee and
«nder. Trevallion et al. {34] reported that the siphons of clam
f maJ°r source of food for plaice. Many crabs, including those
numan consumption and those utilized by marine predators
n as ^riped bass, also feed on clams [30]. By these means, low
Wrations of oil may rapidly be disseminated through the
"te sediments and food chains.
OWLEDGMENT
Cjj "e analyses for this investigation were performed at the analyti-
'fldu 'ty of the U-S- Environmental Protection Agency (EPA),
Jej strial Waste Treatment Research Laboratory in Edison, New
Sup y- Use of these facilities was provided by an EPA program that
Ports graduate level research of the environment.
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u{"er, M.; Sass, ].; Souza, G.; Sanders, H.; Grassle, F.; and
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carbons in seawater extracts of crude oil and crude oil frac-
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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-
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Marine Biology 17:243-250.
10. Ehrhardt, M. 1972. Petroleum hydrocarbons in oysters from
Galveston Bay. Environmental Pollution 3:257-271.
11. Farrington, J.W., and Quinn, J.G. 1973. Petroleum hydrocar-
bons in Narragansett Bay. I. Survey of hydrocarbons in sedi-
ments and clams (Mercenaria mercenaria). Estuar. & Coastal
Mar.ScL 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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13. Gordon, D.C., Jr.; Keizer, P.O.; and Prouse, N.J. 1973. Labora-
tory studies of the accomodation of some crude and residual
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15. Gurr, E. 1960. Encyclopedia of microscopic stains. Baltimore:
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16. Hanks, R.W. 1963. The soft shell clam. U.S. Fish & Wildlife
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17. Jtfrgensen, 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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19. Kennedy, V.S., and Mihursky, J.A. 1972. Effects of tempera-
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22. Michalik, P.A., and Gordon, D.C., Jr. 1971. Concentration and
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170
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REPRINTED FROM: Fate & Effects of Petroleum Hydrocarbons in Marine
Organisms and Ecosystems, Proceedings of a Symposium,
Seattle, Wa. November 10-12, 1976, pp 313-322,
THE ACCUMULATION AND DEPURATION OF NO. 2 FUEL OIL BY THE
SOFT SHELL CLAM, Mya arenaria L.
by
Dennis Stainken
Oil & Hazardous Materials Spills Branch
Industrial Environmental Research Laboratorv-Ci
U.S. Enivronmental Protection Agency
Edison, New Jersey 08817
Abstract
Young soft shell clams, Mya arenaria, were exposed to subacute concentrations of No. 2 fuel
oil-in-water emulsions under simulated winter (4 C) spill conditions. A pattern of accumu-
lation and discharge of petroleum constituents, an experimental depuration time (biologi-
cal half-life, TB,-,-.), and a potential transport mechanism of aromatic compounds from the
fuel oil to the clams were experimentally determined. The clams were exposed to single
dose concentrations of 10, 50, and 100 ppm of No. 2 fuel oil-in-water emulsion for 28 days.
Clams accumulated the greatest amount of hydrocarbons within one week after the initial
exposure. The accumulated hydrocarbons decreased each week as the hydrocarbon content of
the water decreased. Mass spectrometric analysis determined that the principle compounds
accumulated and retained after three weeks of oil exposure were monomethyl, dimethyl, and
trimethylnaphthalene isomers. Depletion of oil from the water column and accumulation and
discharge of fuel oil constituents appeared to involve a mucus-oil complex formation by the
clam.
The depuration period was determined when the clams were transferred to an uncontaminated
system for 14 days subsequent to the 28 day oil exposure. Accumulated hydrocarbons were
rapidly, although incompletely discharged. At the end of the depuration period, mass spec-
trometric analysis revealed that many of the hydrocarbons present in the clams were
dimethyl and trimethylnaphthalene isomers. The biological half-lifes (TB,,.) calculated
were: lOppm (50 days); SOppm (11 days); lOOppm (13.5 days). i0
Key Word Index: Mya arenaria, oil accumulation, depuration, retention, No. 2 fuel oil,
aromatic molecules, oil emulsion.
Introduction
Chemical analyses have shown that various components of petroleum oils are accumulated
within marine invertebrates. Blumer et_ al_ (1970), Ehrhardt (1972), Clark and Finley (1973)
and Farrington and Quinn (1973) have reported the occurrence of petroleum oils in bivalve
molluscs sampled from the environment. These molluscs were sampled from areas which had
either been exposed to oil spills or from areas considered industrially contaminated. Lab-
oratory studies of the accumulation of petroleum oils and oil fractions by mussels or
oysters were investigated by Lee et al (1972), Blaylock et_ al (1973) and Stegeman and Teal
(1973). Their work indicated that various fractions of petroleum oils can be accumulated
and retained by mussels and oysters.
Oil spillage continues to be a serious problem in coastal and estuarine waters. Field
studies of spills and their effects on Mya arenaria were documented by Thomas (1973) and
Dow and Hurst (1975), and the occurrence of oil in Mya arenaria subsequent to environmental
contamination was reported by Zitko (1971) and Scarratt and Zitk.0 (1972).
Soft shell clams, Mya arenaria, occur frequently in areas receiving acute and chronic oil
exposures and are often harvested for human consumption. This study was therefore per-
formed to determine experimentally, a pattern of accumulation and discharge of petroleum
constituents, a depuration time (biological half-life, TBrg), and a potential transport
mechanism of aromatic constituents of the oil in soft shell clams exposed to No. 2 fuel
oil-in-water emulsions. Few studies of oil accumulation and discharge have been performed
at winter temperature conditions and experimentation was therefore performed at 4 C to
simulate the winter state during which time spills and discharges were more likely to occur
resulting from winter storms.
A number two fuel oil was chosen for study because it is commonly shipped in coastal waters,
used in coastal industrial installations, and has already been involved in a well docu-
mented spill (Blumer, e_t_ al_, 1970). The oils were added in an emulsified form to simulate
a potential naturally occurring condition. Forrester (1971), Gordon et_ a_l^ (1973) and
Kanter (1974) have reported the formation of oil emulsions in sea water by various mechan-
isms. 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.
Emulsions tend to be relatively stable and a mechanism for the accumulation of emulsified
oil by soft shell clams has been reported by Stainken (1975).
171
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Materials and Methods
The No. 2 fuel oil was supplied by the U.S. Environmental Protection Agency, Industrial
Environmental Research Laboratory-Ci, Edison, N.J. The specific gravity of the oil was
2.40 centistokes. The oil was composed of 14% aromatics and 86% nonaromatics according to
ASTM method No. D2549-68. Oil-in-water emulsions were ultrasonically prepared according
to a procedure developed by Gruenfeld and Frederick (1977) .
Clams for the experiments were collected from Sequine Point, Staten Island, N.Y. Young
clams with a mean shell length of 25 mm (Newcombe, 1936; Pfitzenmeyer, 1965) were util-
ized because young bivalves tend to have greater respiration and filtration rates than
those of older bivalves (Prosser and Brown, 1961; Walne, 1972).
An exposure period of 28 days to No. 2 fuel oil emulsions having concentrations of 10, 50,
and lOOppm was utilized. Four 20 gallon covered aquaria containing 60 liters of filtered
sea water per aquaria (Salinity - 20%. ) were employed. The sea water was collected from
Sandy Hook Bay and filtered through a coarse plankton net to remove macro debris. One
aquaria served as a control. Each of the remaining aquaria received a sufficient volume of
a stock oil emulsion to attain a final concentration of either 10, 50 or lOOppm of oil
emulsion per aquaria. The time at which the stock emulsions were added to the aquaria was
termed Time 0. The water was continuously aerated and the temperature was maintained at
4°C. The clams were acclimated to the experimental conditions for a duration of 6 days be-
fore the stock emulsions were added. Sampling for hydrocarbon contents of water and clams
was performed every 7 days after Time 0. The controls were sampled at Time 0 and every
7 days subsequent to Time 0.
Chemical confirmation of the oil being bound or adsorbed to mucus was provided by extract-
ing mucus secreted by the clams exposed to 100 ppm emulsified No. 2 oil. The mucus was
collected after the four weeks oil exposure. It had a gray-green flocculent appearance
and formed flocculent clumps on the surface and in the water column. The methodology used
for hydrocarbon analysis has been described by Stainken (1975).
The depuration of the clams was determined for 14 days after the 28 day oil exposure. Each
experimental group of clams was removed from their aquaria and rapidly placed in clean 20
gallon aquaria containing 60 liters of fresh coarse filtered Sandy Hook Bay sea water main-
tained at the same temperature and salinity. As clams were transferred, the valves were
wiped clean to remove deposited pseudofaeces, etc. Samples for chemical analysis were ob-
tained on day 7 and 14.
The hydrocarbon contents of the Sandy Hook Bay sea water and the experimental aquaria water
were determined using a Freon extraction technique (Gruenfeld, 1973; Zeller, 1974). Each
week, 400 ml samples were removed from the center of each tank and at the same depth.
Prior to the experiments, the hydrocarbon content of the sea water was analyzed to ensure
external contamination did not occur.
The extraction and column chromatograph method used for chemical analysis of tissue was a
modification of that employed by Blaylock, et^ al^ (1973). All solvents were of distilled
spectral grade (Burdlck & Jackson, Muskegon, Mich.). All glassware was prerinsed with
hexane. Once a week, 5 clams (5-8 gm tissue weight) from each experimental exposure were
placed in a round bottom flask (500 ml) having a standard taper ground glass neck, and
mixed with 95% ethanol (150 ml) and KOH (10 gui). Clams were shucked and the water from
the mantle cavities were drained before being placed in the flasks. The shells were not
extracted. The mixtures were then refluxed at approximately 65-70 C on a heating mantle
and under a Friedrich reflux condenser for m hours. After cooling to ambient temperatures,
the interior of each condenser was washed with 5 ml hexane. The washing was collected in
the receiver flask.
The digested material was transferred to a Teflon stoppered separatory funnel (1 liter)
using distilled water (80 ml) and two portions (50 ml each) of hexane. The mixture was
then shaken by hand for one minute. The two phases were then drained into separate flasks
and the aqueous phase was returned to the separatory funnel using a hexane (50 ml) wash.
The extraction and separation was repeated for a total of three times.
The combined extracts were washed (minimum 3 times) with aliquots (500 ml each) of distill-
ed water and transferred to an Erlenmeyer flask (300 ml)'using additional hexane. Water
was removed from the extract by adding 2-3 gm of anhydrous\Na-SO, . The extracts were then
concentrated to approximately 40 ml under a stream of filtered nitrogen in a warm water
bath (50°C) and transferred to a conical flask (50 ml) whe/e concentration was continued to
a final volume of 2 ml.
172
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The concentrated extract was placed on a glass column (25 cm length x 1.0 cm I.D.). The
column was packed with 6.5 cm of 5% water deactivated silica gel (100-200 mesh, (Jrade 923,
Davlson Chemical, Baltimore, Md.). Another 6.5 cm of 5% water deactivated alumina
(Neutral, 90-20 mesh, Brockman Activity 1: Fisher Chemical Co^) was layered over the
silica. Both the silica gel and alumina were activated at 110 C for 24 hours and then de-
activated prior to column packing. Sand was layered over the alumina (0.5 cm). When
packed, the column was rinsed with one column volume of hexane.
The residual concentrate was charged onto the column and then elutriated with 50 ml hexane.
Subsequent analysis demonstrated that all petroleum hydrocarbons in the sample had passed
through. The column was rinsed with 50 ml of benzene but few hydrocarbons were found in
this fraction and it was later discarded. The eluate was concentrated for gas chromato-
graphic analysis to a volume of 0.1 ml under nitrogen.
The identification of sample hydrocarbons was achieved by comparison with gas chromatograms
of hydrocarbon standards and of No. 2 fuel oil in hexane. A Perkin-Elmer (Model 900) gas
chromatograph was employed, equipped with a flame lonlzation detector and a 6 ft stainless
steel column packed with 8% Dexsil on 80/100 mesh of Chromosorb W. Standard operating con-
ditions were as follows: carrier gas: Nj, 2 cc/min; detector: OH2> 20 cc/min; air: 40
cc/min; injector temperature: 200 C; detector temperature: 300 C; manifold temperature:
300°C; column temperature: 70 C with a two minute hold, and programmed at an increase of
8°C per minute to 300°C; injection sample: 0.1 microliter. Further identifications of
compounds were accomplished by mass spectrometric analysis provided by the U.S. EPA (IERL-
Ci), Edison, N.J.
Hydrocarbons were quantified by cutting and weighing the chromatograms. The total areas
under the peaks were obtained by subtraction of the baseline area of the first week con-
trol measurement. It was assumed that the area under the first week control measurement
would be most representative of background levels and therefore any material above this
baseline were considered accumulated petroleum hydrocarbons. The sample peak areas were
compared to that of an internal standard (octacosane, Cjg) having a known concentration
(2 ug/gm clam tissue).
Hydrocarbon quantification results were analyzed for regression. The regression analysis
used the No. 1987A/ST3 package from the 700 series Standard Statistical Program, Wang
Laboratories, on a taped program. The significance of the regressions were further
analyzed by a t-test (Zar, 1974).
Results
Water quality appeared excellent throughout the 28 day period. The dissolved oxygen and
ph remained optimum in all tanks and bacterial contamination, was not apparent. Control
animals were reactive to tactile stimuli and appeared to be feeding. Very little mortal-
ity occurred at all oil concentrations. The actual mortalities were: Controls - 0.0%,
lOppm - 0.5%, SOppm - 2.0%, lOOppra - 3.0%.
During the 14 day depuration period, the lOppm clams seemed to recover their tactile
irritability, in comparison to the controls. The 50 and lOOppm clams were sluggish and
after shucking many smelled foul. Their visceral mass was less firm than controls and
their color had paled from the normal gold-brown. There was an Increase in mortality in
the clams previously exposed to the concentration of 50 and lOOppm. The actual mortality
at the end of the depuration priod was: Control 0.0%, lOppra - 0.0%, SOppm - 18.3% and
lOOppm - 13.6%.
The sea water before all tests and the controls during the tests did not have detectable
hydrocarbon contents. Within the oiled tanks, some of the oil emulsion had broken within
two hours after addition. Much of the oil emulsion and water soluble fraction remained in
in the water column as indicated in Table 1. One week later, the detectable hydrocarbon
concentrations in the water column of the oiled tanks were similar. Much of the oil was
bound in mucus which adhered to the glass cooling colls and tank walls or formed floating
organic flocculent conglomerates. The IR spectra also showed the emergence of an apparent
aromatic band at 3030 cm"1. The hydrocarbon peaks at 2930 cm"1 often masks the aromatics.
Apparently, the water soluble aromatics of the fuel oil were becoming unmasked as more of
of the non-aromatic hydrocarbons became dissipated from the water column. The Week 2 data
were similar to those of Week 1 and a peak emerged at ca. 3000 cm"1.
173
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Time 0*
0
4.5
43.7
60.7
Week 1
0
1.31
1.04
1.52
Week 2
0
0.56
0.71
0.78
Table 1. Hydrocarbon concentration (ppm) in the water col mini
during the 28 day exposure period
Week 3 Week A
0 0
0.37 0
0.37 0.29
0.32 0.47
*The Time 0 sample measurement was made two hours after addition of the emulsified oil.
By Week 3, the measurable hydrocarbon content had dropped further and in the 10 and SOppm
tank samples the possible aromatic peak became less apparent. The lOOppm tank sample still
had a strong peak at 3030 cm"1. At the end of the fourth week, the hydrocarbon content
in the tanks had fallen to almost trace concentrations and peaks were becoming indistin-
quishable.
The tanks used in the oil exposure experiments were drained after the fourth week. There
was an extremely strong ddor of No. 2 fuel oil, especially in the lOOppm tank. As the
mucus was wiped off the sides and cooling oils, the odor became readily apparent in all the
oiled tanks. This indicates that much of the oil was bound by the mucus which had settled
on the cooling coils and sides of the tanks. The mucus-oil complex formation may have
aided in the dissipation of hydrocarbons from the water column. The mechanism of the
binding of oil in nmcua by the clams has been documented (Stalnken, 1975) . Mass spectre-
metric analysis demonstrated that the mucus contained predominantly dimethyl and trimethyl-
naphthalenes with paraffins, mainly in the C-14 and C-15 regions. Figure 1 illustrates the
gas chromatograras of No, 2 fuel oil and an extract of hydrocarbons from a mucus sample. A
close correlation of peak heights Is readily noticeable. The chromatogram pattern of the
mucus is similar to No. 2 fuel oil but some of the lower boiling compounds are missing.
During the depuration period, measurable amounts of hydrocarbons in the aquaria water were
not detected. There was an emergence of a peak at 3000cm during the second week in the
experimental tanks.
Gas chromatograms for the control clams are contained in Fig. 2. The basic pattern for
the normal control clams remained unchanged during the experiment. The chromatogram for
Week 1 was chosen as a baseline for the exposed animals to compare the amount of No. 2
fuel oil hydrocarbons accumulated. The chromatograms for the clams exposed to lOppm No. 2
fuel oil are Illustrated in Fig. 3. The chromatograms A-C show a characteristic well
defined pattern of peaks. Mass spectrometric analysis of the third weeks sample (chromato-
gram C) revealed that the bulk of the chromatogram consisted of monomethyl, dimethyl and
trimethylnaphthalenes. By the fourth week, the resolution of the peaks had diminished and
the chromatograms of the clams placed In fresh sea water would superficially Indicate
there were few hydrocarbons present. However, mass spectrometric analysis revealed that
the sample (Chromatogram F, Fig. 3) contained dimethyl and trimethylnaphthalenes at the
end of the depuration period.
Figure 4 depicts the chromatograms from the clams exposed to SOppm of No. 2 fuel oil
emulsion. The same pattern of peaks as those of the lOppm is present. The peaks have
much higher resolution and are still apparent even after the depuration period. A higher
attenuation (10x64) had to be used because the samples contained a much higher hydrocar-
bon concentration. Mass spectrometric analysis of the Week 3 sample revealed that most
of the sample was comprised of monomethyl, dimethyl and trimethylnaphthalenes. Mass
spectrometric analysis of the clam sample at the end of depuration (Chromatogram F, Fig. 4)
showed t.iat the sample was composed of alkyl benzenes, monomethyl, dimethyl and trimethyl-
naphthalenes. The chromatogram of Week 1 control was drawn -under chromatograms E and F,
Fig. 4, to illustrate the quantity of petroleum hydrocarbons retained by the clams.
Chromatograms of clams exposed to a concentration of lOOppm No. 2 fuel oil are illustrated
in Fig. 5. The same recurrent pattern Is readily visible and the peaks have sharper res-
olution. Mass spectrometric analysis of the week 3 sample showed it to consist of naph-
thalene, monomethylnaphthalene, a mixture of dimethyl and trlmethylnaphthalene isomers,
phenantherene, and Cj2 a"d Cj^ paraffins. Mass spectrometrkc analysis of the clam sample
after the two week depuration (Chromatogram F, Fig. 5) confirmed that the sample consisted
of naphthalene, a mixture of dimethyl and trimethylnaphthalene Isomers and no paraffins.
174
-------
Fifc. 1. Gas chromatograms.
(A) Sppm #1 f(JH OIL IN HEXANE, ATTENUATION 1x128
IB) CARBON TETRACHLORIDE EXTRACT OF ClAM MUCUS, ATTENUATION 10x1024
175
-------
Fig. 2. Control animal gas chromatograms.
(A) TIME 0, WITH COLUMN BLEED DRAWN BELOW; (B) WEEK 1; (C) WEEK 2, (0) WEEK 3; (E) WEEK 4,
-------
Fig. 3.
Gas chromatograms clams exposed to 10 ppm 112 Fuel
(A| WEEK .; (B) WEEK 2; (C) WEEK 3; (p) WEEK 4; (E) CLAMS PLACED IN FRESH SEA WATER WFFI
(F| CLAMS PLACED IN FRESH SEA WATER, WEEK 2, W,TH CONTROL WEEK 1 DRAWN AS BASE LINE
ALL CHROMATOGRAMS AT 1x128 ATTENUATION
177
-------
Fig. A.
Gas chromatograms clams exposed to 50ppm fl2 Fuel Oil.
(A) WEEK 1: Ik) WEEK 3; K) WHK 3. (01 WIEK 4; (El CIAM5 PIACID IN fUESH SEA WAH», WEEK I. WITH
CONTKOl WEfK 1 AS BASEL.NE; |F| CLAMS IN -«E$H SIA WATER. WE« 3. WIIH CONIROl WEEK . AS BASELINE
ALL CHUOMATOGBAMS AT I0»64 AI1ENUAIION
178
-------
Fig. 5.
Gas chromatograms clams exposed to 100 ppm #2 Fue] 011
Ml WEEK ... ,., WEEK 8i |C| WEEK 3, ,„, WEEK 4; (II CLAMS F-LACED ,N „„„ SEA WATER, W|« , w.IH
CONTROL WEEK . AS BASIUNf (F| CLAMS ,N FRESH SiA WATH. WIEK 2, CONTROL WEEK , AS
ALl CHROMATOGKAMS AT !0x4« ATTtNUATION
179
-------
150 _,
100 .
r- 50
10
lOppm
Time ( Weeks )
Fig. 6.
Accumulation and depuration of petroleum derived hydrocarbons.
Weeks 1-4 were the oil exposure period.
Weeks 5 & 6 were the depuration period.
(Dotted line is calculated regression.)
180
-------
The results of quantification of the gas chromatoprams are listed in Table 2 and illus-
trated in Fig. 6. Results are expressed as micrograms of petroleum hydrocarbons/gram
clam sample. After one weeks exposure to oil, the values diminish with time. The sudden
upsurges in Week 3 at lOppm and in Week 4, of the 50 and lOOppm are unexplained. The cal-
culated regressions are the broken lines in Fig. 6. A gradual loss of petroleum hydro-
carbons with time is demonstrated. Analysis of the regressions indicated that the 50 and
lOOppm regressions were not significantly different (.05P) although they are both signif-
icantly different from the lOppm regression.
Experimental concentration factors were calculated for the first week of exposure from the
data in Table 1 and 2. The hydrocarbon concentration of the water column, Week 1, was sub-
tracted from the Time 0 value. This net hydrocarbon concentration in the water column was
then divided into the hydrocarbon value accumulated by the clam after the first week of
oil exposure. The computed concentration factors were: lOppm - 8.5, 50ppm - 3.3, lOOppm -
1.8.
Table 2. Quantification of petroleum hydrocarbons (microerams
Week 1
, Week 2
Week 3
Week 4
Week 5
Week 6
per gram of clam tissue) .
10 ppm
27.28
22.92
31.65
17.27
(depuration) 12.94
(depuration) 14.95
50 ppm
143.15
120.50
97.03
146.80
66.80
55.34
100
109.27
99.23
90.73
117.52
83.56
57.05
The biological half lifes (TEgg) or residence times were calculated using a regression
analysis of the depuration period and plotting on semilog paper. The fourth weeks data
were calculated as Time 0 for the depuration period. The calculated TB5Q were: lOppm -
50 days; SOppm - 11 days; lOOppm - 13.5 days. A theoretical time period for the clams to
completely depurify accumulated petroleum hydrocarbons can be estimated if the regressions
in Fig. 6 are extrapolated. The extrapolation results in the following depurification
periods: lOppm - 61 days; 50ppm - 93 days; lOOppm - 109 days.
Discussion
The concentrations of oil in the water column, began to decrease several hours after the
addition of the oil emulsion. Several factors were probably responsible for this. 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 ejected from the clams and
were accumulated on the cooling coils. Subsequent chemical analysis revealed a large con-
tent of oil in the mucus. It is presumed that as the clams filtered water, they were ex-
posed primarily to naphthalene derived compounds. Earlier investigators reported that fuel
oil can form droplets and micelles in sea water, and that the major water soluble compon-
ents were the aromatic compounds, particularly the naphthalenes (Boylan & Trtpp, 1971;
Boehm & Quinn, 1974). The accumulation of large amounts of oil and aromatic molecules in
the mucus may be a potential mechanism of concentrating and disseminating petroleum hydro-
carbons in the environment. The mechanism for the formation of an oil-mucus complex was
discussed by Stainken (1975).
After Time zero, the oil rapidly disappeared from the water column. The amount of dis-
solved compounds attained a peak measured in the first week, and then decreased to an
equilibrium value for several weeks ranging from 0.5-0 .29ppm. Analysis of mucus and clam
tissue indicated that naphthalenes and methyl substituted naphthalenes were the apparent
predominant components in the water. This is further substantiated by other reports
(Frankenfeld, 1973; Anderson, ejL Si' » 1974) which indicate that the water soluble frac-
tions of oils often contain aromatic compounds, particularly naphthalenes.
181
-------
The peak accumulation of petroleum derived hydrocarbons by the clams was reached out- week
after exposure, followed by a gradual loss in accumulated hydrocarbons. The bull' of tin
material accumulated were naphthalenes and methyl substituted naphthalene i seniors, lit'icr
aromatics were detected and accumulated but the naphthalenes were the most persistent. The
results of this study are comparable to those of Vaughan (1973). He reported that oysters
exposed to No. 2 fuel oil often accumulated methyl and dimethyl naphthal enew. Other studie.s
also indicate that aromatic hydrocarbons may be accumulated by bivalves (Ehrhardt, 1972;
DISalvo, et^ al^, 1973).
Paraffins or aliphatics were not detected in clam tissue in this study, except in the higher
oil exposures. The aromatic compounds constituted a much greater percentage of the total
oil extracted from the clams. The extracted concentrations of aromatics from the clams were
also much greater than the original diluted concentrations when added at Time 0 as part of
the fuel oil emulsion. Similar results of Stegeman and Teal (1973) demonstrate that a high
aromatic content in the organism need not be the result of a high aromatic content in the
contaminating oil. This phenomena is probably due to the greater solubility of the aromatic
compounds (Anderson, ejt^ al^., 1974; Boehm and Quinn, 1974).
The greater uptake of hydrocarbons by clams exposed to SOppm may be due to a dose dependent
narcosis. Galtsoff, e£ al_., (1935) reported that water soluble substances of crude oil
could produce anaesthetic effects on the ciliated epithelium of the gills. The lOOppm oil
concentration may have reduced the filtration activity of the clams below that of the clams
exposed to 50ppm. The reduced filtration rate may account for the animals exposed to the
higher concentration (lOOppm) accumulating less oil than the animals epxosed to the lower
concentration (50ppm).
Depuration of accumulated petroleum derived hydrocarbons actually began as the hydrocarbons
decreased in the water column. When the clams were removed to fresh sea water, depuration
proceeded rapidly for the first week. The calculated half-time for depuration ranged from
11-50 days. If the calculated regression of the accumulation of petroleum hydrocarbons is
extrapolated (Fig. 6), 61-109 days are necessary for the clams to completely depurify the
accumulated petroleum hydrocarbons. Although extrapolation is not necessarily accurate, the
retention of hydrocarbons at the end of the depuration period indicated that after oil ex-
posure, depuration will proceed rapidly to a low concentration after which depuration pro-
ceeds slowly. Other investigators have reported similar findings in other bivalve species
(Lee, et aU, 1972; Stegeman & Teal, 1973; Vaughan, 1973). However, the actual tissue con-
centrations are related to the concentration of the initial oil exposure. The retention
of oil by Mya arenaria in this study is further substantiated by the observations of Thomas
(1973) in which clams were observed to eject oil for at least one month after an oil spill.
Oil retention can have several deleterious effects. Long term low level petroleum hydro-
carbon contamination may interfere or weaken the ability of the clam to withstand further
environmental stresses such as those of temperature, salinity, spawning, disease and insult
from other contaminants. Futhermore, oil retained in the sediment and clams may also be
passed on to predators, including man.
1. Clams accumulate large amounts of petroleum derived hydrocarbons after initial exposure
to petroleum (No. 2 fuel oil). The amount and the type of petroleum hydrocarbon accumu-
lated are related to the initial dose, time following initial exposure and the relative
"solubilities" of the various hydrocarbon components. Naphthalene and methyl substituted
naphthalene isomers (mono, di, tri) are the most common compounds concentrated by the clams.
Occasionally, some alkyl benzenes, phenanthrenes and paraffins in the C12 and C14 region
are accumulated.
Depuration by soft-shell clams of accumulated petroleum hydrocarbons may actually begin as
soon as hydrocarbon levels drop in the water column. When clams are placed in fresh hydro-
carbon free sea water, depuration proceeds rapidly within the first week after which the
rate slows. Dimethyl and trimethylnapthalenes were still present in the clams after two
weeks in fresh sea water.
2. Chemical analysis of clam mucus extract demonstrated that clams can concentrate and in-
gest or release to the environment extremely large amounts of petroleum hydrocarbons by
means of their mucus - oil binding mechanism. Analysis of petroleum compounds present in
the mucus revealed them to be methyl substituted naphthalenes and paraffins, all compounds
similar to those which the clams accumulate. Types of chemical compounds accumulated or
bound In the mucus are probably related to differential solubilities of petroleum con-
stituents .
182
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References
Anderson, J.W., Neff, J.M., Cox, B.A., Tatem, U.K., and G. M. Miditower., Character i si i t-s
of dispersions and water-soluble extracts of crude and refined oils and their toxic its'
to estuarine crustaceans and fish. Mar. Biol. 27,75 (1974).
Blaylock, J.W., O'Keefe, P.W., Roehm, J.N., and R.E. Wlldung., Determination of n-alkam>
and methylnaphthalene compounds in shellfish. Proc. of Joint Conf. on Prevent. & Contr.
of Oil Spills, Wash., D.C., A.P.I., 173 (1973).
Blumer, M., Sass, J., Souza, G., Sanders, H., Grassle, F. and (!. llampson. (1970) The West
Falmouth Oil Spill. Tech. Rept. No. 70-44, Woods Hole Oceanographic Institution.
Boehra, P.O. and J.G. Quinn., The solubility behavior of No. 2 fuel oil in sea water.
Mar. Pollut. Bull. 5(7). 101 (1974).
Boylan, D.B. and B.W. Tripp., Determinations of hydrocarbons in seawater extracts of
crude oil and crude oil fractions. Nature 230, 44 (1971).
Clark, R.C., Jr., and J.S. Finley., Paraffin hydrocarbon patterns in petroleum polluted
mussels. Mar. Pollut. Bull. 4(11), 1972 (1973).
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pollution origin in aquatic organisms of San Francisco Bay: Methods and preliminary
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LSU-SG-73-01, 205 (1973).
Dow, R.L., Hurst, J.W., Jr., The ecological, chemical and histopathological evaluation
of an oil spill site. Part 1. Ecological Studies. Mar. Pollut. Bull. 6(11) (1975).
Ehrhardt, M., Petroleum hydrocarbons in oysters from Galveston Bay. Environ. Pollut. 3,
257 (1972).
Forrester, W.D., Distribution of suspended oil particles following the grounding of the
tanker Arrow. J. Mar. Res. 29(2), 151 (1971).
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Transportation, U.S.C.G., Off. of R & D, Wash., D.C. (1973).
Galtsoff, P.S., Prytherch, H.F., Smith, R.O., and J. Koehring., Effects of crude oil
pollution on oysters in Louisiana waters. Bull. Bur. Fish. Wash., D.C. 18, 143 (1935).
Gordon, D.C., Jr., Keizer, P.O. and N.J. Prouse., Laboratory studies of the accommodation
of some crude and residual fuel oils in sea water. J. Fish. Res. Bd. Can. 30 1611
(1973). '
Gruenfeld, M., Extraction of dispersed oils from water for quantitative analysis by
Infrared Spectrophotometry. Environ. Sci. & Tech. 7(7). 636 (1973).
Gruenfeld, M. and R. Frederick., The ultrasonic dispersion, source identification, and
quantitative analysis of petroleum oils in water. Rapp. P.-v. Reun. Cons. Int Explor
Her. 171, 33 (1977). ' ~
Ranter, R., Susceptability to crude oil with respect to size, season and geographic loca-
tion in Mytilus californianus (Bivalvla). U. So. Cal., Sea Grant Prog. Publ. No.
USC-SG-4-74, (1974).
Lee, R.F., Saurheber, R. and A.A. Benson., Petroleum hydrocarbons: uptake and discharge
by the marine mussel Mytilus edulis. Science 177, 344 (1972).
Newcombe, C.L., Validity of concentric rings of Mya arenaria for determining age. Nature
137(3457), 191 (1936).
Pfitzenmeyer, H.T., Annual cycle of gametogenisis of the soft shell c]am, Mya arcnarta,
at Solomon, Maryland. Chesap. Sci. 6(1), 52 (1965).
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W.B. Saunders, Phila.
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183
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Ste£
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REPRINTED FROM: Bulletin of Environmental Contamination
& Toxicology, 1976, Vol.16 No.6, pp 724-729.
Springer-Verlag New York Inc.
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
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. 08817
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_ al_. (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
e^ a^. (1971), GORDON, et^ al^. (1973) and KANTER (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.
185
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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%.)- 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.
186
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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 LC5Q 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 LC50.' Mortality was not found in
the controls. Death was defined as a total lack of muscle re-
sponse.
At 14°C, two 96 hour LC50 values for No. 2 fuel were obtained
These were 475 ppm (Test IIA) and 535 ppm (Test IIIA). Compari-
son by a t test revealed no significant difference and a mean
LC50 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
LC50 (7 day) was found for No. 2 fuel oil to be less than 100
ppm, compared to test IIIA LC50 (96 hour) of 535 ppm. The LCcn
(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 LC50 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.
187
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TABLE I. Calculated toxicity (LC ) during a 96 hour exposure
period.
14°C
(IA) So. Louisiana Crude
cone: 50,100,200,400,800 ppm
LC5Q = none
(IIA) //2 Fuel Oil
cone: 50,100,200,400,800 ppm
L,C50 = 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
1X50 = none
(HIP) Phenol
50,100,200,400,800 ppm
LC50
4°C
(IVA) So. Louisiana Crude
cone: 100,200,400,800,1600 ppm
LC-0 = 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
LC50 = 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 LC50 was not found in any tests
(except VIA, 7 days), while at 14°C a mean LCjQ 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.
188
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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, et_ 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 LC50 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 LC5(, 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,
11 *!• » 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 LCso 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.
189
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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. KEISER, and N.J. PROUSE: J. Fish. Res. Bd.
Can. 30., 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. 7_, 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).
190
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REPRINTED FROM: Bulletin of Environmental Contamination &
Toxicology, December 1976, Vol.16, No.6,
pp 730-738. Springer-Verlag New York Inc.
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, jrt 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_ al. (1974) and GARDNER,
ejt a_l. (1975). Histological aberrations in bivalves have been
reported by BARRY, e£ 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 (4°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.
191
-------
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 1% 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
192
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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*
Control 1 0
10 ppm 2 4.5
50 ppm 3 43.72
100 ppm 4 60.71
Week 1 Week 2 Week 3 Week 4
0000
1.31 0.56 0.37 0
1.04 0.71 0.37 0.29
1.52 0.78 0.32 0.46
* 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
193
-------
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 Heraatoxylin 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 PA.ULEY and CHENG (1968) occurring in the pallial
blood sinuses. The leukocytes often formed a band underlying the
mantle epithelium similar to that illustrated by DBS 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.
194
-------
Cross sections of the clam visceral mass and pallium were stai
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 vacuoli2ation 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
3 of all clams appeared to contain many vacuoles and the eel
membranes were often indistinct. However, this effect was exacer-
in the oil exposed clams compared to controls (Figure 1-3)
Figure 1. Section of the digestive diverticula
Azure A/eoson B. Time 0.
195
-------
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.
196
-------
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
197
-------
tumors were also present in the gills. BARRY, £Jt a^. (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 histologlcal
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.
198
-------
References
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(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. 8t 1009 (1974).
DBS VOIGNE, D.M., and A.K. SPARKS: J. Invert. Path. 12, 53 (1968).
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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).
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16, 6 (1973).
HEIDELBERGER, C. : Eur. J. Cancer £, 161 (1970).
JEFFRIES, H.P.: J. Invert. Path. 2£, 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 _177_, 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. 11. 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. 23, 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. 22_, 37 (1973).
SHAW, B.L., and H.I. BATTLE: Can. J. Zool. J35, 325 (1957).
VAUGHAN, B.E.: A.P.I. Pub. No. 4191 (1973).
ZITKO, V.: Bull. Environ. Contain. & Toxicol. _5_, 559 (1971).
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199
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REPRINTED FROM: Proceedings of the Symposium on "Recovery Potential
of Oiled Marine Northern Environments", Halifax,
Nova Scotia, October 10-14, 1977, pp 637-642.
Effects of Uptake and Discharge of Petroleum Hydrocarbons on the
Respiration of the Soft-Shell Clam, Mya arenaria1
DENNIS M. STAINKEN
U.S. Environmental Protection Agency, Industrial Environmental Research Laboratory-Ci,
Oil and Hazardous Materials Spills Branch, Edison, NJ. 08817, USA
STAINKEN, D. M. 1978. Effects of uptake and discharge of petroleum hydrocarbons on
the respiration of the soft-shell clam, Mya arenaria. J. Fish. Res. Board Can.
35: 637-642.
A winter (4°C) spill condition was simulated in which young soft-shell clams, Mya
arenaria, were exposed to subacute concentrations of No. 2 fuel oil-in-water emulsions
for 28 d. Clams we're exposed at the beginning of the experiment to single dose con-
centrations of 10, 50, and 100 ppm. Hydrocarbons were rapidly accumulated by clams
within 1 wk after exposure. The accumulated hydrocarbons then decreased each week as
the hydrocarbon content of the water decreased. Methyl substituted naphthalene isomers
were the principal compounds accumulated and retained by the clams after 3 wk of oil
exposure. A dose-response relationship was observed in the respiratory rates as measured
by oxygen consumption (QO,.). Significant differences (P = .05) in respiratory rates were
found imclams exposed to low concentrations of oil. The lowest concentrations of oil
caused a doubling of the respiratory rates and greater oil concentrations caused a depres-
sion in rate. The respiratory rates of the clams exposed to low oil concentrations decreased
as the hydrocarbon content of the water and clam tissues decreased, but remained sig-
nificantly altered from the controls. Clams were transferred to an uncontaminated system
for 14 d subsequent to the 28-d oil exposure to determine effects of depuration on the
respiratory rate. During the depuration period, many of the hydrocarbons present in clam
tissue were again found to be methyl substituted naphthalene isomers. During this period,
the respiratory rates of the clams initially exposed to 10 ppm fuel oil emulsion remained
significantly altered above the controls. The respiratory rates of all groups of oil-exposed
clams remained altered from (he controls, but the magnitude of difference tended to
decline toward the controls. A dose-response narcosis may have been evident during this
period.
Key words: Mya arenaria, respiration, No. 2 fuel oil, petroleum emulsion, petroleum
accumulation, depuration
STAINKEN, D. M. 1978. Effects of uptake and discharge of petroleum hydrocarbons on
the respiration of the soft-shell clam, Mya arenaria. J. Fish. Res. Board Can.
35: 637-642.
Nous avons simule une condition hivernale (4°C) de deversement dans laquelle de
jeunes myes communes (Mya arenaria) furent exposees a des concentrations subaigues
d'emulsions de fuel-oil n° 2 dans de 1'eau durant 28 jours. Au debut de Fexperience, les
myes ont ete exposees a des concentrations, par doses uniques, de 10, 50 et 100 ppm. Les
hydrocarbures s'accumulent rapidement dans les myes en dedans d'une sem apres exposi-
tion. Les hydrocarbures accumules diminuent ensuite a chaque sem a mesure que la
teneur de 1'eau en hydrocarbures diminue. Les isomeres naphtalene a methyl substitue sont
les principaux composes qui s'accumulent dans les coques et qui sont retenus apres 3 sem
d'exposition au petrole. Nous avons observe une relation dosage-reponse dans les taux
respiratoires tels que mesures par la consommation d'oxygene (QOS). Nous avons observe
des differences significatives (P = .05) dans les taux respiratoires des myes exposees a de
faibles concentrations de petrole. Les plus basses concentrations entrainent une augmenta-
tion du double des taux respiratoires et les hautes concentrations produisent un abaissement
des taux, Les taux respiratoires des myes exposees a de faibles concentrations de petrole
diminuent a mesure que la teneur en hydrocarbures dans 1'eau et dans le tissu des myes
diminue, mais demeurent nettement difterents de ceux des temoins. Les myes furent en-
suite transferees dans un systeme non contamine durant 14 jours a;pres 1'exposition au petrole
200
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J. FISH. RES. BOARD CAN., VOL. 35, [978
de 28 jours dans le but de determiner les effets de 1'epuration sur le taux respiratoire.
Durant la periode d'cpuration, on constate que plusieurs des hydrocarbures presents dans
le tissu des myes sont de nouveau des isomeres naphtalene a methyl substitue. Durant
cette periode, les taux respiratoires des myes initialement exposees a 10 ppm d'emulsion de
fuel-oil demeurerent nettement superieurs a ceux des temoins, Les taux respiratoires de
tous les groupes de myes exposees au petrole se maintienneni a un taux different de ceux
des temoins, mais la difference a tendance a diminuer et les taux a se rapprocher de ceux
des temoins. II se peut qu'il y ait eu une reponse de narcose liee a la dose durant cette
periode.
Accepted February 28, 1978
BIVALVE molluscs in the environment are frequently
exposed to single spills or chronic discharges of petro-
leum oils. Chronic subdetectable oil pollution may occur
from seeps, undetected spills, leaks, marine vessels, and
urban and industrial sewage. Pollution incidents in
temperate and subarctic estuarine and coastal marine
Waters will probably increase as further exploration, de-
velopment, and transportation of oil increases. Lee et al.
(1972), Stegeman and Teal (1973), and Neff and
Anderson (1975) have reported the occurrence and
uptake of petroleum hydrocarbons by bivalve molluscs.
Various effects of petroleum oils and derivatives on
bivalves have been described. Some of the effects re-
ported were alterations of behavior (Galtsoff et al.
1935), and tissue structure (Barry and Yevich 1975;
Stainken 1976a).
Few studies have characterized the effects of oils on
the respiration of bivalves or related physio:Ogical re-
sponses to concentrations of contaminants in bivalve
tissues. Gilfillian (1973, 1975) and Gilfillan et al.
(1976) reported alterations in the carbon budgets of
mussels and soft-shell clams exposed to crude and fuel
oils and noted that O2 consumption was altered. Avolizi
and Nuwayhid (1974) found that exposure to crude
oils decreased the O? consumption of mussels. In con-
trast, Fong (1976) described an increase in respiration
rates of soft-shell clams exposed to crude oil. It is dif-
ficult to compare directly some studies because of the
varying test parameters (i.e. salinities, temperatures,
methods of oil addition, period of exposure, animal size,
species, etc.). Therefore, this study was performed to
determine the effect(s) of uptake and discharge of
petroleum hydrocarbons on the O2 consumption of the
soft-shell clam. Mya arenaria. Effects on O2 consump-
tion were determined during a 28-d exposure to sub-
acute concentrations of No. 2 fuel oil emulsions and a
subsequent depuration period of 12d. The effect on
the clams was then related to the body burden of petro-
leum hydrocarbons and specific fractions.
Mya arenaria was chosen for study because these
clams occur frequently in areas receiving acute and
chronic oil exposures. Oil spills and their effects on M.
Qrenaria have been documented by Thomas (1973), and
the occurrence of oil in M. arenaria has been reported
by Scarratt and Zitko (1972). Mechanisms by which
M. arenaria may accumulate oil have been described
by Stainken (1975) and Fong (1976). A No. 2 fuel
oil was chosen for study because it is commonly used
Accepte le 28 fevrier 1978
in coastal industrial installations and shipped in coastal
waters. Experimentation was performed at 4°C because
spills are more likely to occur during inclement winter
weather.
The oils were added in an emulsified form to simulate
a potential naturally occurring condition. Forrester
(1971) and Gordon et al. (1973) reported the forma-
tion of oil emulsions in seawater. 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.
Materials and Methods
The No. 2 fuel oil was supplied by the U.S. Environ-
mental Protection Agency, Industrial Environmental Re-
search Laboratory, Edison, N.J., (specific gravity, 2.40
centistokes; composition, 14% aromatics and 86% nonaro-
matics according to ASTM method No, D2549-68). Oil-in-
water emulsions were prepared according to Gruenfeld
and Frederick (1977).
Clams with a mean shell length of 25 mm (Newcombe
1936; Pfitzenmeyer 1965) were collected from Sequine
Point, Staten Island, N.Y. Young clams were used because
they tend to have greater respiration and filtration rates
than those of older bivalves (Read 1962; Walne 1972). The
clams were acclimat to the experimental conditions for 6 d
before the emulsions were added.
An exposure period of 28 d to No. 2 fuel oil emulsions
having concentrations of 10, 50, and 100 ppm was utilized.
Four, 20-gal covered aquaria containing 60 L of filtered
seawater per aquarium (salinity = 20&>) were used. The
seawater was collected from Sandy Hook Bay and filtered
through a coarse plankton net to remove macrodebris. One
aquarium served as a control. Each of the remaining aquaria
received a sufficient volume of a stock oil emulsion to attain
an initial concentration of either 10, 50, or 100 ppm of
emulsified oil. The time of addition was termed Time 0.
The water was continuously aerated and the temperature
maintained at 4°C. The photoperiod was approximately
16L:8D. Previous studies (D. M. Stainken unpublished
data) have shown that M. arenaria establishes normal O;
consumption patterns within these experimental parameters.
The hydrocarbon contents of the Sandy Hook Bay sea-
water and the experimental aquaria water were determined
using a Freon extraction technique (Gruenfeld 1973). Be-
fore the experiments, the hydrocarbon content of the
seawater was analyzed to ensure no external contamination.
Each week, a 400-mL sample was removed from the same
depth at the center of each tank. Concomittant studies of
hydrocarbon accumulation and depuration of the clams
(Stainken 1976b), and the effects (Stainken 1976a), were
2 01
-------
STAINKEN: EFFECT OF PETROLEUM ON CLAM RESPIRATION
performed. The method used for hydrocarbon analyses was
described by Stainken (1976b).
Beginning at Time 0, the O.- consumption of 18 clams
from each of the groups, control and oil-exposed, was
measured every 4 d for 28 d. At the end of this period,
each experimental group of clams was rapidly removed to
clean 20-gal aquarium containing seawater as described
above. As clams were transferred, the valves were wiped
clean to remove deposited pseudofeces, etc. The (X con-
sumption of 18 clams from each of the groups, control and
oil-exposed, was then measured every 4d for 12 d. Samples
for hydrocarbon analyses were obtained on days 7 and 14.
The procedure used for O2 consumption measurements
was modified from that of Kennedy and Mihursky (1972).
A Scholander respirometer (The Mark Co., Brockton,
Mass.) was used. Each respirometer vessel received 8 mL
of natural seawater collected with the clams. This seawater
had been stored at 4°C in the dark. The water in the
respirometer vessels was prefiltered through a 1.4- and 0.8-
filter (Millipore Corp.. Bedford. Mass.) and aerated 1 h at
4°C prior to use. To absorb CO.., 0.2 mL of 10% KOH
was placed in a plexiglass boat with a piece of No. 4
Whatman paper. The valves of the clams were gently wiped
dry before the animals were placed in the respirometer
vessels. Animals were equilibrated 11 h prior to measuring
Oj uptake. O2 consumption was than measured for 1 h.
Measurements were made in the morning or afternoon at
the same time for each experimental group. Procedural
blanks were run with each experiment. Shell length was
measured at the end of each experiment and dry tissue
weight (minus shell) was determined by drying the tissue
to constant weight at 75-80°C for 48 h. The Oj consump-
tion data was then calculated as ^L CX/mg dry weight per
hour or QO-. This method has been used by Kennedy and
Mihursky (1972) and Widdows (1973); it decreases much
of the variability inherent in molluscan oxygen consumption
rates.
Respirometric results were analyzed by linear regression.
For regression analysis the procedures of Ostle (1963) and
Draper and Smith (1966) were used. An analysis of variance
and "in level" comparisons of the mean QCX measure-
ments of oil-exposed and depurated clams were performed
according to Campbell (1974). Results were considered
significant at the P = 0.05 level.
Results
The dissolved O-, and pH remained optimal in all
aquaria throughout the 28-d exposure period; bacterial
contamination was not apparent. Control animals were
reactive to tactile stimuli and appeared to be feeding.
Very little mortality occurred at any of the oil con-
centrations employed. The actual mortalities in the
oiled phase of the experiment were controls 0.0%, 10
ppm 0.5%. 50 ppm 2.0%. 100 ppm 3.0%.
In the 10-ppm aquaria, the oil odor disappeared by
the 3rd wk and the clams appeared to react more
slowly than the controls to tactile stimuli. The 50-ppm
aquaria still had a faint odor of oil by the 3rd wk and
the clams reacted much more slowly to tactile stimuli.
In the 100-pm aquaria, there was still a faint odor of
oil detectable after 3 wk. and the clams appeared nar-
cotized and were relatively unreactive.
During the 14-d depuration period, the 10-ppm clams
TABLE 1. Hydrocarbon concentration (ppm) in the water
column during the 28-d exposure period. The Time 0 sample
measurement was made 2 h after addition of the emulsified oil.
Aquaria Time 0 Wk 1
Wk 2
Wk 3
Wk 4
Control
10 ppm
50 ppm
100 ppm
0
6.0
.31.5
55.3
0
0.8
1.2
1.5
0
0.4
0.7
1.5
0
0.3
0.6
0.4
0
0.3
0
0.3
appeared to recover their tactile sensitivity to a level
similar to that of the controls, but the 50- and 100-ppm
clams continued to react sluggishly. Mortality increased
during the depuration period among the 50- and 100-
ppm clams. The actual mortality in the depuration was
controls 0, 10 ppm 0.1%. 50 ppm 18.26%, 100 ppm
13.63%. The seawater before all tests and in the con-
trol tank during the tests did not have detectable hydro-
carbon contents (0.2 ppm). Within the oiled tanks,
some of the oil emulsion had broken within 2 h after
addition. A indicated in Table 1, much of the oil
emulsion and water-soluble fraction remained in the
water column after 2 h.
One week later, the detectable hydrocarbon con-
centrations in the water column of the oiled aquaria
were similar. Much of the oil appeared bound to mucus
which adhered to the glass cooling coils and tank walls
or formed floating organic flocculent conglomerates-
The IR spectra also showed the emergenc: of an ap-
parent aromatic band at 3030cm-1. The week 2 data
were similar to those of week 1 and a peak emerged at
ca 3000 cm-1.
By week 3. the measurable hydrocarbon content had
dropped further, and in the 10- and 50-ppm tank
samples the possible aromatic peak became less ap-
parent. The 100-ppm tank sample still had a strong peak
at 3030 cm-1. At the end of the 4th wk, the hydrocar-
bon content in the tanks had fallen to almost trace
concentrations and peaks were becoming indistingui*0'
able. .
The mucus-oil complex formation may have aideo
in the dissipation of hydrocarbons from the water
column. The mechanism of the binding of oil in mucus
by M. arenaria has been documented earlier (Stainken
1975). Mass spectrometric analysis demonstrated that
the mucus contained predominantly dimethylnaphtna-
lenes and trimethylnaphthalenes with paraffins, mainly
in the C,4 and C,5 regions.
During the depuration period, measurable amount
of hydrocarbons in the aquaria water were not detected.
There was an emergence of a peak at 3000 cm-1 during
the 2nd week in the experimental tanks.
Figure 1 illustrates the calculated regression of QQ°
of clams exposed to oil over the 28-d period, and Table
2 contains the correlation coefficient, slope, y-intercep •
upper and lower confidence intervals for the regressio
lines of Fig. 1. Table 3 contains the mean QOj an
standard errors for the depuration period.
202
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J. FISH. RES. BOARD CAN., VOL. 35, 1978
TABLE 3. Mean QO2 and standard error (SE) or clams during
the depuration period.
.01 -
8
24
28
12 16 20
TIME (DAYS)
FIG 1. Calculated regression of QOj of clams exposed
to No. 2 fuel oil for 28 d.
The normal QO3 rates of M. arenaria acclimated to
4CC ranged between 0.06 and 0.04 QO2 units. This
agrees with the results of Kennedy and Mihursky
(1972) who found that clams acclimated and measured
at 1 °C had a QO2 range from 0.07 to 0.40. Although
these rates are not directly comparable due to tem-
perature, latitude, and other unknown variables, it does
indicate that O2 consumption by M. arenaria is rela-
tively low.
4d
8d
12d
Control
Q02
SE
lOppm
Q02
SE
50 ppm
Q02
SE
100 ppm
Q02
SE
.041
.008
.065
,010
.023
.007
.043
.007
.099
.016
.154
.011
.040
.011
.120
.021
.046
.008
.087
.010
.038
.008
.058
.010
The 10-ppm regression of QO2 in Fig. 1 appears to
have translated to greater rates than those of the con-
trols. After the initial exposure to oil and elevation of
QOo, there is a continual decline toward the control
values. After 28 d, the 10-ppm QOL, remained almost
double the control values. During the depuration period,
O.> consumption by the 10-ppm-exposed clams remained
elevated compared to the controls.
The 50-ppm regression of QO2 in Fig. 1 was also
elevated above that of the controls. Following an initial
elevation, the 50-ppm rates gradually declined toward
the levels of the controls. After 28 d of oil exposure,
however, the 50-ppm rates remained elevated. During
the depuration period, the rates were depressed slightly
below those of the controls. This could be due to
several factors including the more rapid loss of hydro-
carbons possibly affecting cellular respiration, or gen-
erally depressed body condition subsequent to oil
exposure.
The calculated regression of QO2 of the 100-ppm
TABLE 2 Correlation coefficient (r), slope (a,), y intercept (a0), upper confidence intervals (UCI), and lower confidence intervals
(LCI) for the regression lines of the control, 10 ppm, 50 ppm, and 100 ppm QO2 rates of Fig. 1. 'For each day measurement,
Control 10 ppm 50 PP™ 10°nPP»w
ao= 0.072 a, = 0.132 aa = 0.103 a, = 0.039
a. = -0 001 01 = -0.001 a, = -0.002 a, = 0.001
r= -0.573 r= -0.712 r = -0.683 r = 0.482
959r confidence intervals*
Control
10 ppm
50 ppm
100 ppm
Day
UCI
LCI
UCI
LCI
UCI
LCI
UCI
LCi
4
g
12
16
20
24
28
.092
.082
.074
.069
.066
.065
.066
.044
.044
.044
.041
.036
.027
.018
.147
.138
.130
.124
.119
.117
.116
.107
.106
.104
.100
.093
.085
.076
.125
.112
.100
.092
.087
.085
.084
.067
.066
.064
.059
.051
.039
.026
.067
.065
.065
.067
.072
.079
.087
.016
.025
.033
.037
.040
.039
.036
203
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STAINKEN: EFFECT OF PETROLEUM ON CLAM RESPIRATION
•Jams rotated around that of the controls (Fig. 1).
Rates were depressed until the 16thd when the rates
approximated those of the controls, followed bya con-
tinual elevation in rate until the end of the experiment.
During the depuration period, oil consumption was
slightly greater than the controls.
A single factor analysis of variance calculated for the
mean QO2 of the 28-d exposure period and the 12-d
depuration period showed significance (P = 0.001) for
the exposure period. Further "in level" analysis showed
'.hat differences existed between the control and 10-ppm
rates (P = 0.01) and between those of the control and
50 ppm (P = 0.05). There was no significant difference
between controls and 100 ppm. Comparisons of the
depuration results were not significant (P = 0.05) for
the rates of the controls and of those clams previously
exposed to 50- and 100-ppm concentrations. However,
the difference between the 10 ppm and controls was
still significant (P = 0.05).
The total mean QQ2S for oil exposure and depuration
periods were calculated. Generally the oil-exposed clams
had greater O., consumption during exposure and
depuration periods than the controls, The incidence of
respiration was also increased by oil. The mean percent
of clams respiring during the oil exposure period was
controls 65%. 10 ppm 99.2%, 50 ppm 88%, and 100
ppm 75.4%. During depuration, the mean percent of
clams respiring was controls 77%, 10 ppm 90.7%, 50
ppm 46.3 %. and 100 ppm 72.2%.
Discussion
The concentrations of oil in the water column began
to decrease several hours after the addition of the oil
emulsion. Several factors were probably responsible for
this. Much of the oil was apparently removed from the
water column by the mucociliary feeding and ejection
mechanisms of the clams. The mechanisms for the for-
mation of an oil-mucus complex and its chemical
analysis were described earlier (Stainken 1975). A
large content of oil. especially di- and trimethylnaphtha-
lenes. was found in the mucus. Boehm and Quinn
(1974) found that fuel oil could form droplets and
micelles in seawater and that the major water soluble
components were aromatic compounds, particularly
naphthalenes.
After Time 0. the emulsified oil rapidly disappeared
from the water column. The amount of dissolved com-
pounds attained a peak measured in the 1st wk. and then
decreased to equilibrium values (ranging from 1.5 to
0.3 ppm) for several weeks. An analysis of mucus and
tissue from clams concomittantly exposed and sampled
indicated that naphthalenes, and methyl-substituted
naphthalenes were probably predominant components
in the water (Stainken 1976b). Anderson et al. (1974)
have also reported that the water soluble fractions of
oils often contains naphthalenes.
After 1-wk exposure, the peak accumulation of petro-
leum derived hydrocarbons was reached, followed by
a gradual loss in accumulated hydrocarbons. Most of
the materials accumulated were naphthalene and methyl
substituted naphthalene isomers. Details of these chem-
ical analyses were reported earlier (Stainken 1976b).
Although other compounds were detected and ac-
cumulated, naphthalenes were the most persistent.
Depuration of accumulated hydrocarbons began as
the hydrocarbon concentration in the water column de-
creased. When clams were placed in fresh seawater,
depuration proceeded rapidly within the Istwk, after
which the rate slowed. Di- and trimethylnaphthalenes
were still present in the clams after 2 wk in fresh sea-
water.
Gilfillan (1973) found that small amounts of oil
extract could decrease the amount of energy available
for maintenance, growth, and reproduction by decreas-
ing the net carbon balance of the animal. Fong (1976)
also found that the respiration rates of small M. arenaria
were significantly increased after 3-wk exposure to sea-
water containing 220-370 g oil/L. In contrast. Dunning
and Major (1974) reported that exposure to cold sea-
water extracts of No. 2 fuel oil tended to decrease O;
consumption by mussels. Avolizi and Nuwayhid (1974)
investigated the effects of a crude oil on two bivalve
species and they also found a depression of respiratory
rates at sublethal concentrations. Higher temperatures
and physiological variation may possibly have in-
fluenced their results.
The results with M. arenaria in this study confirm
that low levels of fuel oil cause a marked elevation tn
the respiratory rates. Higher concentrations cause a
depression in the rates. As long as the oil- or water-
soluble fraction is present, the rates remain affected.
The actual content of hydrocarbons in the water column
during the last 3 wk of the test ranged from 0.0 to !•*
ppm. Even at the highest concentrations tested (100
ppm), as the concentrations of the water-soluble frac-
tion decreased, the respiration rates increased. This ap-
parently reflects a dose-response relationship. Very low
sublethal concentrations of oil cause a doubling of the
respiratory rates. Further support of a dose-response
effect is evidenced in the accumulation of hydrocarbons.
The greater uptake of hydrocarbons by clams exposed
to 50 ppm may be due to a dose-dependent narcosis.
Galtsoff et al. (1935) reported that water-soluble sub-
stances of crude oil could produce anesthetic effects on
the ciliated epithelium of gills. The 100-ppm oil con-
centration may have reduced the filtration activity °r
the clams below that of the clams exposed to 50 pp1"1
The reduced filtration rate may account for the animals
exposed to the higher concentration (100 ppm) *f-
cumulating less oil than the animals exposed to tn
lower concentration (50 ppm).
The mechamsm(s) that cause the alterations ^
respiratory rates is unknown. Concomittant studies o
the effects of oil Exposure on tissue structure (Stainke^
1976a) revealed a depletion of glycogen and generalize $
leukocytosis particularly evident in the blood sin
of the pallium and mantle membrane. There was a
204
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J. FISH. RES. BOARD CAN., VOL. 33, 1978
an increase in vacuolization of the diverticulum,
stomach, and intestine. Effects appeared to be exacer-
bated at the higher exposure levels. Although the in-
crease in respiratory rates is similar to an uncoupling
ellect of oxidative phosphorylation, further work on
the cellular level is necessary. Altered O> consumption
may be due to the disruption of normal cell membranal
processes (Goldacre 1968) or cellular metabolism.
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marine mussel, Mytilits edulis. Science 177: 344-346.
NEFF, J. M., AND J. W. ANDERSON. 1975. Accumulation,
release and distribution of Benzo-a-pyrene-C" in the
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PFITZENMEYER, H. T. 1965. Annual cycle of gumetogenesis
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SCARRA- T, D. J., AND V. ZiTKo. 1972. Bunker C oil in sedi-
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STAINKEN, D. M. 1975. Preliminary observations on the
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THOMAS, M. L. 1973. Effects of Bunker C oil on intertidal
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205
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ADDITIONS
206
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REPRINTED FROM:
Proceedings of 1979 Oil Spill
Conference (Prevention, Behavior,
Control, Cleanup), Los Angeles,CA,
March 19-22, 1979. pp. 323-331.
Available from API, Washington, D.C
METHODS FOR THE SOURCE IDENTIFICATION AND
QUANTIFICATION OF OIL POLLUTION
U. Frank, D. Stainken, and M, Gruenfeld
Environmental Protection Agency
Industrial Environmental Research Laboratory
Oil and Hazardous Materials Spills Branch
Edison, New Jersey 08817
ABSTRACT: Petroleum oil analyses frequently are performed by
Government and industrial organizations and by academic institu-
l°ns to identify the source of discharge of oil pollutants and to
Quantify such oils in water, sediments and tissues. This paper pre-
ie"ts a concise review of oil analysis methods used by several U.S.
"iencies, industrial organizations and standard setting societies. Oil
""alysis methods published by these organizations are evaluated with
f^ard to their convenience and safety for general laboratory use,
"vilify to yield correct quantification data, and correctness in identi-
?'n8 the presence and source of discharge of petroleum oil pollu-
*"i. Several procedures also are addressed for isolating individual
y'roleum fractions and hydrocarbons from environmental samples
°r the purpose of monitoring for the presence of oil pollution in
'diments and tissues.
Although oil spill prevention and control techniques have ad-
anced in the past decade, the discharge of oil to the environment
0|«inues to (,e a serious problem. These discharges may be of acute
r chronic duration, and occur from both point and nonpoint
j°ufces. Consequently, petroleum oil analyses frequently are per-
j°rnied by government and industrial organizations and by academic
p'itutions to identify the source of discharge of oil pollutants and
° Quantify such oils in water, sediment and biota, Bentz10 discussed
. toe of the legal and scientific aspects of analyses for oil in spill
"'Wification.
6* he complex composition and dissemination of petroleum in the
i?.Vlronment complicates analyses for oil pollution. Petroleum rap-
in Undergoes various weathering phenomena, or becomes contam-
»ted with other environmental contaminants. Farrington" reviewed
*Jy of these analytical problems.
. rnere are a multitude of methods for analyzing oil and its com-
,"«nt subfractions. Many are too time consuming, are imprecise,
Jjj* 'oxic solvents, and are impractical for routine use. Complicating
' analysis of petroleum is the relative lack of standardized methods
analysis. The present standard method is that used for analysis of
a"d grease in water and wastewater." This method has been re-
in' several "official" publications. '•'•"" Comparison and
ents °^ t*ie eff'cacy °f °'' an£i 8rease methods have been re-
Although these methods are accurate when analyzing
Contaminated by refined nonvolatile oils (such as lubricants,
es> heavy fuel oils), their applicability to water contaminated by
ar|d fuel oils is questionable. Further problems in oil spill iden-
and quantification were discussed by Bentz."
He he complexity of petroleum composition, its weathering, environ-
Dissemination and assimilation led to the application of a
of column, thin layer and high pressure chromatography
^'ques for sample separation-preparation, and use of ultraviolet
"tfrared absorption, UV- fluorescence, gas chromatographic and
8 spectral techniques for analysis. However, many of the meth-
are not standardized and interlaboratory data varies. Interlabo-
ratdry and intercalibration studies"-"'"'"4 have been reported cov-
ering several facets of the problem.
Several frequently used methodologies with various modifications
gradually evolved from numerous investigative reports. Multimethod
approaches have often been used and Bentz10 described many of
them. Most involve some type of sample solvent extraction, sample
cleanup procedure, concentration and analysis by several analytical
techniques. Many analysts now use similar techniques with modifica-
tions. These approaches are evolving into prestandardized proce-
dures as more intertaboratory and intercalibration exercises are
performed.
These methods must ultimately meet several criteria, however, to
be useful for spill source identification and quantification of petro-
leum oil. Dependability of the quantification data and the ability of
the method(s) to establish the source of oil discharge are critical for
monitoring and damage assessment. Many methods now used
employ volumes of toxic solvents (such as benzene, carbon disulfide,
and CC14) which present difficulties in analyst safety, and problems
in proper waste solvent disposal. Some methods are criticized for the
time consumed in sample preparation. Those methods ultimately
chosen should be practical for routine laboratory use.
This paper presents a preliminary synopsis of oil analysis methods
now used by U.S. agencies, industrial organizations and standard
setting societies. A more comprehensive review is in preparation.
Methods evaluated were examined according to their accuracy in
identifying and quantifying waterborne oil, the relative toxicity of
solvents used, and the practicality of the method for routine use.
Also addressed are several methods for isolating individual petro-
leum fractions and hydrocarbons from environmental samples to
monitor the presence of oil pollution.
Identification of waterborne oils
Establishing the identity and quantity of spilled petroleum oil in-
volves complex methodology. Several reviews have addressed earlier
oil identification techniques. Brunnock* reviewed mostly European
practices for analysis of beach pollutants and Adlard1 reviewed
British and U.S. methods of oil spill analysis. Additional methods
for oil identification were discussed by Gruenfeld" and Bentz.''10
Many methods now are used by "standard setting" organizations,
government, industry and academic institutions to identify and quan-
tify petroleum oil. Oil spill identification methods recommended by
the principal standard setting organizations in the U.S. are reviewed
in this section. Also discussed are the primary methods used for the
quantitative determination of petroleum oil dispersed in water.
Methodology for the identification of waterborne oils covers the
broad concepts of sampling and analyzing spilled petroleum for
identification and comparison with oils taken from suspect sources
of discharge. The general procedure includes sample collection, pres-
ervation, preparation, pretreatment, and analysis.
Sample collection. Procedures for sample collection must assure
that a representative portion of the oil be taken, from a slick, shore-
207
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1979 OIL SPILL CONFERENCE
line residue or suspect source, lor example. Sampling can be compli-
cated by emulsion formation, insufficient oil film thickness, or con-
tamination. The collection procedure must assure that no contamina-
tion of the sample occurs. No standard method of sample collection
has yet been developed. However, the U.S. Coast Guard (USCG)
presented a procedure'0' in which Teflon strips attached to a rake-
like device are drawn through an oil slick, The oil adheres to the
strips and 10 ml of oil can be collected in one pass through a thin
slick.
Some collection procedures that may be implemented routinely in
the field are reported by Kawahara" and Kreider70. More complex
procedures are described by the USCG.' Several other field tech-
niques for collection of surface oil using aluminum foil" and Teflon
discs" also have been reported.
Sample preservation. Protection of the collected sample against
chemical and physical changes during transit or storage is essential to
ensure sample integrity. Microbes can alter or deplete the n-alkanes
in an oil sample" and volatilization and auto-oxidation may affect
the sample's composition/6
The American Society for Testing and Materials' described appro-
priate sample containers and a sample preservation procedure
(ASTM Method D3325) which minimizes contamination and degra-
dation of the collected samples. Narrow mouth borosilicate glass
bottles with aluminum or Teflon-lined caps are recommended as
sample containers. Metal and plastic containers are unsuitable, be-
cause of the introduction of contaminants from the container which
may invalidate subsequent trace element analyses. Biodegradation
was minimized by storing at 10°C, and displacement of sample bottle
air with N; or CO; prevented auto-oxidation.
Sample preparation and prclreatmenl. Prior to analysis, it is nec-
essary to separate the sampled oil from extraneous matter (water,
sand, debris), and to bring the unweaihered suspect oil samples and
the weathered waterborne oil samples to physical conditions as com-
parable as possible. Spilled oils weather as soon as they are exposed
to the environment and the changes affect the comparisons of sus-
pect oil to waterborne oil.
ASTM Method D33264 incorporates a method combining four
procedures for separating oil from water, solids and debris, and for
simulating weathering. Procedure A applies to samples of more than
50 ml volume containing significant quantities of hydrocarbons boil-
ing above 280°C. it involves dissolution of the sample in an equal
volume of chloroform or dichloromethane and centrifuging to re-
move water and debris. Simulated weathering involves distilling the
sample to 280°C using a nitrogen purge to remove the solvent and
volatile hydrocarbons.
Procedure B applies to samples of less than 1 ml of "heavy" oil
and uses pentane or hexane as solvents. The extract is dried with
anhydrous MgSCX and volatiles and solvent are stripped in a beaker
under a nitrogen stream. Procedure C covers "light" oils containing
significant components boiling below 280°C. It only includes separa-
tion of oil from the water phase by centrifugation and does not
address weathering. Procedure D is for samples containing both
petroleum and vegetable-animal derived oils. This procedure includes
the separation of petroleum hydrocarbons from the animal-vegetable
oils by column chromatography using a silica gel-alumina column.
The USCG oil analysis scheme'"' includes an elaborate oil weath-
ering procedure that simulates natural weathering. Fiberglass troughs
with circulating sea water and exposure to the environment are used
to weather the suspect oils.
A simulated weathering procedure for minute amounts of oil has
been developed" by which treatment of 70 mg of an unweathered
reference oil yielded a chromatogram virtually the same as that
obtained by ASTM Procedure A. The procedure is applicable to a
minimum of 0.5 mg of oil and uses a 10 by 30 mm glass vial heated
at40°C for 15 minutes in the presence of an air stream.
Analysis. The analysis of oil samples often involves a multiparam-
eter approach using complementary methods that exploit the com-
plex composition of petroleum. The frequent use of these methods is
a reflection of the methods' convenience for general lab use, and cor-
rectness in identifying the presence and source of discharge of petro-
leum oil.
Gas chromatography. The most widely used methods for oil spill
identification are based on gas chromatography (GC). Many differ-
ent columns have been used in an effort to attain resolution of the
alkane fingerprint components of oil. Both packed columns using
nonpolar liquid phases and various types of capillary columns have
been used to effect separations. Flame ionization (FI) and flame
photometric (FP) detection are used primarily to measure the eluted
components. FP detection using a sulfur specific 394 nm filter pr°"
duces a profile of the sulfur components present in oil. The com-
bination of a conventional Fl detection and the FP detection pr°"
vides useful fingerprints for identifying oils.
ASTM includes a method (D3328-78) using both packed columns
and dual FI/FP detection. Method A (a packed column procedure)!
uses a 10-foot stainless steel column packed with OV-101 on 60/°y
mesh Chromosorb W. Method B (a capillary column procedure) IS
similar but employs higher resolution support coated open tubuia
(SCOT) columns. Column effluents from both methods may be
delected by FI or split (1:2) between FI and FP detection for sulfur
compounds. FP detection can, in certain circumstances, yield chr
matograms that distinguish two oils when the FI chromatogram
cannot. The matching of samples is based on the premise that i^en'^
cal oils yield identical chromatograms. A detailed discussion ° .
various features of FI detection oil chromatograms and their use i
oil spill identification is provided by Gruenfeld and Frank."
detection chromatograms have fewer features, but give sulfur Pr
files which may be used for oil characterization.
The USCG method10' avoids the use of packed columns and uses
both OV-101 and Dexsil-300 SCOT columns. The Dexsil column dp*
not resolve pristane and phytane but does allow operation to
high
temperatures (350°C) and, therefore, provides additional inform
tion on high boiling components from heavy oils. The USCG »P
proach also recommends FI and FP detection for two simultane"
GC profiles.
The use of another high resolution GC column has been rec0^
mended4' for increasing the separations of oil components. This
fingerprinting technique used a column of lithium chloride supP°
on diatomaceous silica. The advantages of inorganic packings
high resolution and thermal stability, and no column bleed, ther
permitting the separation of higher boiling oil components. .£
Fluorescence spectroscopy. Numerous fluorescence spectroscop^
techniques using several approaches for obtaining oil fingerprint v _
files have been reported. Frank" reviewed many of the fluore
methods employed for oil identification, and a means of cornp ^
fluorescence methods was described earlier."1" Using this mode.
i u/jtni'
various fluorescence methods are compared and contrasted ™ ,
*HP tnf
the context of a three-dimensional system. This system uses me
interdependent variables inherent to fluorescence spectrosc
excitation wavelength (x), emission wavelength (y) and fluoresC[iar-
intensity (z). Within the scope of this system, the fluorescence <•
acteristics of petroleum oils are presented as "total fluore^
spectra," and the spectral information obtained by each tech
described as an appropriate portion of such spectra. Figure 1 *•• ^ as
hypothetical three-dimensional total oil spectrum (illustrate ^
mountains by solid lines), and the spectral information obta"1
several methods.
The more common approach involves the derivation of <
spectra and use of the spectral profiles as fingerprints. B
spectra are obtained by fixing a fluorescence spectrometer s ^
tion monochromator at a selected wavelength and scanning the ^
sion monochromator. In the three-dimensional system in B6 cf
emission spectra (dotted line) represent slices of total f'u°re.tation
spectra that are parallel to the y axis. By varying the fixed exc ^jr
wavelength, different spectral slices are obtained that vary '
position along the x axis. ^e
Both ASTM Method D3650-78 and the USCG"" nuore^ci'8'
methods are based on the emission spectra approach using an use
tion wavelength of 254 nm. Thruston and Knight"" initiated . tj0n
of emission spectra for fingerprinting oils, and used an ex ^^g
wavelength of 340 nm. Coakley24 developed a similar methoo ^i
excitation wavelengths from 280-300 nm. Goldberg and DeV. (200-
compared the diagnostic value of various excitation wavelengt" $
250, 300 and 350 nm), and demonstrated that the 250 nm w^ «njtiv«
used in the ASTM and USCG methods produces the most &
spectral fingerprints for identifying oils. . oils
The use of synchronously excited spectra for fingerpr"1 ^eO
has been advocated."'" In this procedure, oil spectra are ° jnSyH'
by scanning both the excitation and emission monochromator ^(te-
chronization, and separated by a wavelength interval. 'n pfts'11'
dimensional system, synchronously excited spectra a's°/angle'5
slices of total fluorescence spectra but are oriented at a 45 e)(Cjte''
the x and y axes (dashed line in Figure 1). Synchronously
208
-------
SENSING, MONITORING, AND ENFORCEMENT
FLUORESCENCE'
INTENSITY
• SILHOUETTE PROFILE
— TOTAL SPECTRUM
--SYNCHRONOUS EXCITATION
SPECTRUM
EMISSION SPECTRUM
EXCITATION
WAVELENGTH
Y
EMISSION
WAVELENGTH
\
Figure 1. Comparison of emission spectrum, synchronously
excited spectrum, and silhouette profile within the context of a
total oil spectrum—The emission spectrum includes only one
maximum, and the synchronously excited spectrum includes
two maxima. The silhouette profile includes all three maxima
and represents the "shadow" of the total spectrum.
spectra have been demonstrated7' to usually contain more spectral
information than emission spectra and, in many instances, to contain
the same informational content as several emission spectra
combined.
The more complex approach of obtaining fluorescence "contour
maps" of oils has been described"'" as the ultimate in fingerprinting
by fluorescence. In this method, a large number of emission spectra
derived for each oil undergo computer analysis for conversion into
maps. Within the context of the three-dimensional system, contour
maps represent total oil spectra reduced to the two-dimensional (x, y)
plane (Figure 2). Relative fluorescence intensities within the maps are
indicated with concentric lines. Although a maximum amount of
spectral information is obtained by this approach, the possible insuf-
ficiency of contour maps for fingerprinting weathered oils has been
discussed."
A simpler approach for obtaining maximum spectral information
from an oil involves the derivation of silhouette profiles of total oil
spectra. "•"•" This procedure can be performed manually with a rou-
tine laboratory spectrometer in a relatively short time. Silhouette pro-
files of oils are obtained by plotting the maximum fluorescence inten-
sities of oil emission spectra versus excitation wavelengths and con-
necting the plotted points with straight lines. Figure 1 shows that
silhouette profiles represent the shadows of total oil spectra as proj-
ected into the x, y plane of the three-dimensional system. The utility of
silhouette plotting for discriminating between crude and refined petro-
leum oils has been shown" and weathered oils successfully correlated
with unweathered oils. Also demonstrated" was the method's utility
in identifying weathered lubricating oils in waste treatment effluents,
surface waters, and marine organisms.
In addition to these primary methods, several secondary approaches
have been employed to increase fingerprinting capabilities. Some ana-
lysts'" have advocated the use of excitation spectra, in addition to
emission spectra, as oil fingerprints. Within the three-dimensional sys-
tem, excitation spectra are the perpendicular counterpart to emission
spectra; that is, they are slices of total oil spectra parallel to the x axis
(emission spectral slices are parallel to the y axis).
Several analysts advocate a multi-parameter fingerprinting proce-
dure deriving emission and excitation spectra of simple compound
groups chromatographically isolated from oils. Drushel and Som-
mers" isolated simple multicomponent fractons by GC and identified
fluorescing oil components by comparing emission and excitation
spectra of standards and oil fractions. Correlations of oils was per-
formed by comparing the type of compounds identified. McKay and
Latham" used a similar procedure substituting a complex compound
isolation scheme of ion-exchange, gel permeation and thin layer
chromatography. Lloyd" used gradient elution chromatography and
a combination of emission, excitation and synchronously excited spec-
tra to identify individual oil components. High pressure liquid chro-
matography (HPLC) and emission and excitation spectra have been
used to identify polynuclear aromatic oil compounds for use as finger-
printing indices.
Studies on the use of cryogenic temperatures to enhance details in
the spectral fingerprints of oils have been conducted by several ana-
lysts. Procedures for obtaining "cryogenic temperature spectra" in-
volve the dissolution of oils in selected liquids and cooling the solu-
tions to 77 °K with liquid nitrogen. The liquids used are limited to
those that form a transparent solid (glass) at that temperature.
Drushel and Sommers" used mixtures of methylcyclopentane-methyl-
cyclohexane and ether-isopentane-ethyl alcohol (EPA) to prepare
glasses at 77°K. Freegarde et al." also used EPA for cryogenic fluo-
rescence analysis of oils. Hornig and Eastwood" evaluated several sol-
vents for the preparation of solid media and concluded that methyl-
cyclohexane yielded an optimum glass. They also compared fluo-
rescence spectra of crude and refined oils in both solid and liquid
methylcyciohexane, demonstrating dramatic improvements in the res-
olution of spectra derived from the solid medium.
Infrared spectroscopy. Methods measuring the 4000-250 cm-' re-
gion frequently have been used for fingerprinting oils. The most use-
ful IR region for fingerprinting oils is the 1500-500 cm~' range,
termed the "IR fingerprint region." IR spectra are compared by
visual comparison or by comparison of IR absorbance ratios. ASTM
Method D3414-75T, for IR analysis of waterborne oils, involves re-
cording spectra between 4000 and 667 cm~ '. The spectra of the sam-
ple and the reference oils are compared visually or by calculating the
ratios of the intensities of a number of bands, such as 8107720cm-',
and 160071375 cm-1.
The USCG uses a similar approach, primarily using an overlay pro-
cedure to visually compare spectra. The USCG procedure also in-
cludes a very detailed IR procedure for routine use by relatively un-
trained personnel. Oil weathering effects and possible interferences
incurred from sample collection and preparation procedures are
thoroughly discussed.
The use of IR spectroscopy for oil spill identification has been de-
veloped to a high degree of sophistication using computer and statisti-
cal data treatment. Statistical means of evaluating and comparing IR
data,67'" and several computerized procedures for identifying oils by
IR spectra have been reported.' '•"
Other methods. A number of other methods for fingerprinting oils
are used, and may provide useful fingerprints in many instances.
FLUORESCENCE
EMISSION SPECTRA
CONTOUR LINES
OOQ& DATA POINTS
EXCITATION
WAVELENGTH
V
EMISSION
WAVELENGTH
EXCITATION
WAVELENGTH
X
Q
EMISSION
WAVELENGTH
(A)
(B)
Figure 2. Three dimensional presentation of the derivation of a
contour map of an oil having an inverted-cone-shaped total
spectrum—The data points used for plotting the map B are
obtained from five emission spectra in A.
209
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1979 OIL SPILL CONFERENCE
ASTM Method D3327-74T includes the determination of nitrogen,
sulfur and nickel/vanadium contents of oils as fingerprint indices.
Nitrogen is determined by the Kjeldahl procedure and sulfur by X-ray
spectroscopy or, alternatively, by a high temperature combustion
method. The nickel/vanadium content is obtained by either X-ray or
atomic absorption spectroscopy.
Other methods"' include the use of isotope mass spectrometry for
determining carbon and sulfur isotope ratios for fingerprinting oils.
Weiss"1 and Bentz' reviewed many methods, including mass spectro-
metric, nuclear magnetic resonance (NMR) spectroscopic, and ultra-
violet spectroscopic methods, used for oil spill identification.
Quantification of water-dispersed petroleum
The accurate measurement of oil in water, sediment, or tissue nec-
essarily involves a definition of the term "oil," particularly because
different quantitative methods measure different sample constituents
(that is, alkanes vs aromatics). A broad definition is that oil consists
of crude petroleum or its liquid derivatives used as fuels and lubri-
cants. Each analytical technique, however, expands or restricts this
definition and establishes an "operational definition" of its own.
The primary methods now available for quantitating oil dispersed in
water are classified as gravimetric or spectroscopic techniques. All of
these techniques include solvent extraction procedures, followed by
the appropriate determinative measurement. In gravimetric tech-
niques, this measurement consists of weighing the extracted residues.
In spectroscopic techniques, measurements are performed using either
IR, UV or fluorescence spectroscopy. Some methods also incorporate
an interference removal procedure for separating petroleum com-
pounds from other non-petroleum organics, thereby increasing the
specificity for petroleum hydrocarbons.
The use of appropriate standards is mandatory in all but the gravi-
metric techniques. Sample measurements usually are related to a stan-
dard of a single known concentration or to standards spanning the
concentration range of the samples. The choice of standards critically
affects the accuracy of results and, in an ideal situation, the standard
is identical to the oil being monitored.
Spectroscopic methods. IR spectroscopy is the most commonly used
spectroscopic technique for quantitating water-dispersed oils. In an
American Petroleum Institute procedure,' dispersed oils are extracted
from water with carbon tetrachloride (CCI.). The absorption maxima
of oil at 2850 and 2930 cm~' is then 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 CCI. solution containing an accurately known concentra-
tion of the oil. In this method, a blend of hydrocarbons (37.5% isooc-
tane, 37,5% cetane, and 25% benzene) is used to approximate the IR
absorptivity of an average petroleum oil. This standard solution was
used when a portion of the dispersed oil was not available. This
hydrocarbon blend also has been used to represent "typical oils."'
I nan IR method by Beckman Instruments, Inc., 100 ml of dispersed
oil in water samples are extracted with two ml of CCU. The absorption
maxima at 2930 cm~' is then measured using 10 mm path length near
IR silica cells. The 2850cm-' band was not used. Reference solutions
are prepared and similarly measured as oil-in-water dispersions of
known oil content. A concentration vs absorbance plot is then derived
for quantitating dispersed oil in water samples.
The two preceding methods use the same extraction solvent, but dif-
fer in most other respects. The American Petroleum Institute method
uses a large oil extraction flask and salt and acid are added. Extraction
is performed with successive 100 ml portions of CCI. and two IR
absorption band maxima are then measured. Sample comparison is
made to only a single standard solution. The Beckman method uses a
small separatory funnel and one two ml portion of CCU to extract a
100 ml sample volume. Acid or salt are not added, and one IR absorp-
tion band maximum is measured. A concentration vs absorbance plot
derived from several oil in water reference solutions is used for sample
determination.
The influence of salt and acid has been discussed elsewhere by one
of the authors," who also compared the extraction efficiencies of
CCI. and Freon 113 (I,l,2-trichloro-l,2,2-trifluoroethane). Freon 113
can also be used for IR measurement of oil at 2930 cm-'. One liter of
dispersed oil in water samples was extracted with four consecutive 25
ml portions of solvent, using two-liter separatory funnels. Use of
Freon 113 is of special interest, because it is less toxic than CCI.. Ac-
cording to other analysts," CCI, is highly toxic (10 ppm TLV) when
inhaled or absorbed through the skin, while Freon 113 is much safer
(1,000 ppm TLV).
A study of the accuracy and sensitivity of the IR technique when
used for the quantitative determination of petroleum oils by single
point analysis has been reported.'0 Carbon tetrachloride, Freon 113-
and a mixture of these solvents were used. The detection limits of oils
by IR, the stability of oil absorptivities during prolonged solution
storage, and the utility of these absorptivities for oil identification
were also examined.
In an attempt to distinguish petroleum oils from other extractable
organics, several analysts have combined chromatographic adsorpti°n
techniques with IR measurements. Silica gel adsorption chromatogra-
phy has been used to separate petroleum hydrocarbons from n°n'
petroleum organics. "•'•'•"' Others developed a rapid silica gel adsorp-
tion technique which can be easily used for routine analysis."'" 'n
their procedure, three g of activated silica gel are added to the solvent
extract and the extracts are stirred for 10 minutes with a magnetic
stirrer.
The IR method has now been incorporated into the APHA' and
EPA106 procedures. These two methods are essentially the same.
Either soxhlet or solvent extractions with Freon are used. ASTM' a's°
includes the rapid silica gel adsorption procedure mentione
above."'"
Fluorescence spectroscopy is also frequently used for quantify"*
oil in water, sediment and tissue. Although more sensitive than II*
spectroscopy, fluorescence spectroscopy measures primarily the aro-
matic cons tituents of oil. H ornig" discussed some advantages of using
fluorescence-based methods, noting that these methods are especially
useful when the concentration of residual oils in water is less than I
ppm. Table 1 summarizes the numerous fluorescence approaches tn
have been used for measuring oil in extracts of water, sediment
tissue.
The oil concentration in the extract is determined by excitation »
one of several wavelengths, measurement of the fluorescence 'ntens'J[
at a certain wavelength, and relation of the sample's intensity to t )t
intensity of a reference oil. In a rapid procedure described elsewhere.
oil is measured in the water phase after the addition of 2-propa°
(which acts as an intermediary solvent between water and oil). By
of the synchronous excitation approach for quantifying oil in * ..y
Rayleigh-Tyndall and Raman radiation interferences may be tota 1
avoided. "•" This resulted in increased sensitivity and shorter anaiy
time. in
UV spectroscopy has been sparingly used for quantitating o u
water. Several UV methods have been reported, measuring U"
sorption in the 260 or 262 nm regions. "•"•" However, some an*^e()
noted that measurements of oil in the 260 nm region can be com"
with elemental sulfur." . „
Gravimetric methods. These methods include solvent extracti >
solvent evaporation and gravimetric measurement of the residues. ^
one technique, 100 ml of benzene is used to extract 2.5 liter Portionon.
water. The benzene then is evaporated and oil residues dried 1° (
slant weight. Another also uses benzene as the extraction solven''
sodium chloride is added to the sample to decrease emulsion P ^
lems. That technique was valid for petroleum with boiling P0"1 jja
I40°C or greater. TKe APHA and EPA standard methods cornpe^
also include gravimetric methods. These are essentially the s .{
using Freon as the extraction solvent instead of the more
benzene. _ 0,
Other methods. Methods based on chromatography (<•> "J ^
HPLC) have infrequently been used for quantifying oil in w* ^e
GLC procedure employing Fl detection has been reported in ^f^C
results were quantified in terms of individual oil components, -j jn
has been compared to IR spectroscopy for the quantification p ^,
water and proved more suitable because it yielded data about in ^
ual oil compounds." Freon has been used as an extraction solve ^g
samples analyzed by HPLC.'!0 Quantitative values nowevejej the
given as phenanthrene equivalents. Other analysts recommend ^ ^
use of total organic carbon (TOC) analysis for quantitating
water."
Analysis of petroleum fractions and hydrocarbons
The complex composition of petroleum and its environment*^ j(J
often preclude obtaining distinct oil chromatograms, spectra,
210
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SENSING, MONITORING, AND ENFORCEMENT
Table 1. Fluorescence speclroscopic methods
for the quantification of oil
Author
Zitko and Carson"'
Zilko1"
Levy"
Zilko and Tibbo'"
Cretney and Wong"
Keize'r and Gordon"
Frank"
Gordon and Keizer"
Cordon elal."
Margrave and
Phillips"
Tanacredi"
Cordons/ a/."
Frank"
Sample medium,
extraction solvent
Tissue, hcxane
Tissue, hexane
Water, CCU— cvap.
residue in hexane
Tissue, hexane
Water. CH,C1,—
evap. residue in
cyclo hexane
Water, CH.C!,—
evap. residue in
hexane
Water, direct in-situ
measurements (ad-
dition of
2-Propanol)
Water, hexane or
orCH,CI,— evap.
residue in hexane
Water, CH,C1,—
residue in hexane
Sediment, hexane or
CH,CI,— evap.
residue in hexane
Water, CCL— evap.
residue in hexane
Water, pentane
Sediment,
2-propanol/
cyclo hexane
(60:40 v/v)
Excitation
wavelength
(nm)
Varies with oil type
300
310
280
308
310
290
310
310
310, Fluorescence
contour maps also
used
290, Fluorescence
silhouette profiles
also used
310, Fluorescence
silhouette profiles
and synchronous
excitation also used
Synchronous
excitation
Emission wavelength
at which measurement
is taken (nm)
Varies with oil type
360
360
310
383
374
340
374
374
370
Emission maximum
between 300-540 nm
374
Varies with oil type
°n. Consequently, fractions of the petroleum are often analyzed as a
means of detecting oil pollution. The most common approach has
b«en to isolate, identify and quantitate the aliphatic and aromatic
factions ascribing their presence and composition to oil pollution.
T1>ese analyses are often complicated to interpret in areas previously
°r chronically contaminated.
The water soluble polynuclear aromatic hydrocarbons are often
'Myzed."i4) This approach of monitoring the aromatic fraction
(!>AH) has been used by numerous investigators examining tis-
>ues'-»i.".»i.i«« anfj sediments." However, caution has been advised in
Cecily ascribing PAH presence to oil pollution.14-" Aliphatic frac-
l'°ns are also analyzed to determine the presence of oil pollution"'1'
*nd some analysts examine both aromatic and alkane fractions.10 "
Further complicating the analysis of environmental samples (sedi-
"tents, tissues, water) for oil pollution, is the problem of background
c°ntamination in areas near pollution sources, other contaminants,
1d the natural occurrence of recent biogenic hydrocarbons. Several
?rit«ria are frequently used for identifying the presence of oil pollution
"acompiex matrix. "•"•"»
The identification of petroleum and fractions in sediments, tissues
•Id water after an acute or chronic event, often employs similar ap-
jlroaches. Analysis includes sample extraction, treatment of the ex-
a«t to remove or decrease background interferences, concentration
*ncl instrumental analysis. Multimethod approaches are frequently
esed analyzing both alkane and aromatic fractions. Most methods
internal standards or recovery data, and results are often re-
at the ug/kg level. Aspects of sample extraction and cleanup of
cts are subsequently addressed.
f **dimeni extraction. A common problem in analyzing sediments
6I Petroleum oil and fractions is the extraction of oil from the com-
e* sediment matrix. Several reports have addressed aspects of these
problems, their background and comparative sediment extraction
methods."'"•"'"•" A multimethod approach was commonly used.
The methods can be categorized as: (1) a caustic chemical digestion
process to remove recent biogenic hydrocarbons by saponification,
etc., (2) soxhlet extraction, (3) physical agitation-solvent extraction,
(4) headspace sampling, or (5) refluxing.
Caustic chemical digestion methods have employed various solvents
and-either KOHU'"'"-1!'" or NaOH." Soxhlet extraction procedures
predate digestion procedures and have also been used for sediment
analysis."'"1"'"" Extraction into methanol or benzene-methanol was
the most common method.
Various forms of physical extraction-solvent extraction have been
reported. Disruption and extraction of wet sediments in solvent has
been accomplished using a homogenizer" or ultrasonification.""
Other methods have used variations of a tumbling procedure in which
sediments are placed in a container with solvent and tum-
bled. ".".",110 l
A headspace sampling procedure has been used for analysis of the
volatile components in the sediments."1'" However, these procedures
used additional methods to obtain a total sample.1" Others concluded
that both solvent extraction and headspace methods yielded essentially
the same value for low levels of hydrocarbons but analyzed different
aspects of the oil (aromatics vs aliphatic).
A simple reflux procedure has been discussed which uses methylene
chloride and distilled water while stirring with a magnetic stirrer."
Results were considered good for PAH fractions and almost compar-
able to the digestion procedures.
Tissue extraction. Extraction and isolation of petroleum hydrocar-
bons from tissues is complicated by the biochemical content of the tis-
sue. The petroleum hydrocarbons may reside within the extra or intra-
cellular matrix or be bound to membranes. Complete analysis must,
therefore, ensure complete extraction of the organics from the tissues.
Extraction procedures are often coupled with a subsequent procedure
to decrease the levels of coextracted biotic hydrocarbons. Aspects of
these problems were addressed by Harrington1' and Pancirov el al."
Extraction procedures are based on either caustic digestion, soxhlet
extraction or physical disruption-solvent extraction. The most com-
monly used caustic in digestion procedures has been KOH with vari-
ous solvents.'4i1*'"'"'"'"1"11 Other analysts also have used digestion
approaches but substituted NaOH instead.11'10'1"'1" The efficiency of
these procedures is often enhanced if the tissue is prehomogenized.
Soxhlet extraction procedures also have been used, employing various
solvents and extraction times.10>1 '•''*•'!'
Other procedures of extracting petroleum hydrocarbons from tis-
sues have used a physical disruption of tissues—solvent extraction
approach. A method for naphthalenes and alkylnaphthalenes" simply
involved homogenizing the tissue in hexane, centrifuging and collect-
ing the hexane supernatant for sample cleanup. A similar procedure
was used to analyze clam tissues chronically oiled with Bunker C.4'
Extract cleanup. Extraction of petroleum hydrocarbons from envi-
ronmental samples invariably results in coextraction of recent biotic
hydrocarbons and contaminating organics. These background hydro-
carbons are often removed prior to instrumental analysis. There are
several approaches used to cleanup the extract including column chro-
matography using either alumina, silica gel, florisil or combinations,
TLC, solvent partitioning, removal of sulfur, or filtering the sample
through specific media.
Column chromatography is the most common procedure. These
procedures employ packed columns onto which the concentrated sam-
ple is charged, followed by multiple solvent elution to collect various
petroleum derived fractions for analysis. Columns have been packed
with silica gel, "•"•" alumina, "•"•'•• or more commonly with alumina
over silica gel.4-4'-"-" " Sample preparation also has been
achieved by merely adding small amounts of silica gel to sample ex-
tracts and stirring briefly."1" Other analysts have packed columns
with florisil for sample cleanup.'1"'"
Other procedures have employed TLC,'4'101'"" solvent partition-
ing,7'1' preconcentrating devices,11-"'" or filtration media."1"
Extraction of large amounts of sulfur is a common problem in the
analysis of sediments. The sulfur compounds will interfere with sev-
eral types of instrumental analysis and, consequently, are often re-
moved. The most frequent procedure is to percolate the extract
through a column of activated copper which removes the sulfur"1'"
or to include the copper as part of a multilayered column.1'
Ultimately, the sample extract cleanup procedure chosen should
enable the petroleum hydrocarbons or fractions to be identified and
211
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1979 OIL SPILL CONFERENCE
quantitated with the least interference from background contam-
inants.
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Teal, J. M., K. Burns, and J. Farrington, 1978. Analyses of aro-
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spills of No. 2 fuel oil in Buzzards Bay, Massachusetts. Journal
of the Fisheries Research Board of Canada. v35, n5, pp510-520
Thomas, M. L., 1978. Comparison of oiled and unoiled inter-
tidal communities in Chedabucto Bay, Nova Scotia. Journal OJ
the Fisheries Research Board of Canada. v35, n5, pp707-7J6
Thompson, S. and G. Eglinton, 1978. Composition and sourc6
of pollutant hydrocarbons in the Severn estuary. Marine PoM'
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Thruston, A. D. and R. W. Knight, 1971. Characterization 01
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ronmentalScience and Technology. v5, n64,p69 ,
U.S. Coast Guard, 1977. Oil Spill Identification System, F'n*
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U.S. Environmental Protection Agency, 1974. Methods ^\./
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D.C.
Van Vleet, E. S. and J. G. Quinn, 1977. Input and fate of petr°^
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Wakeham, S. G., 1977. Synchronous fluorescence sPeCtr°SC,jro-
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Walton, A. (ed), 1978. Methods for sampling and anaJX?1*^
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Warner, J. S.,, 1976. Determination of aliphatic and aro ^
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Warner, J. S., 1977. Summary Report on Characterizat'^6
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SENSING, MONITORING, AND ENFORCEMENT
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215
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Tt>\uoit>$ical unJ Emirontrwni Chemistry Reviews, 197H, Vol. 2, pp. 163-185
O I97S Gordon and Breach Science Publishers Ltd., 1978
Primal in Greul Hril.nii
A Review of Fluorescence
Spectroscopic Methods for Oil
Spill Source Identification
UWE FRANK
Industrial Environmental Research Laboratory-Ci, U.S. Environmental
Protection Agency, Edison, New Jersey 08817
(RwireilJuly 13. 1977)
A review and evaluation of analytical techniques and methods for oil spill source identifi-
cation by fluorescence spectroscopy is presented. The subject is discussed within the context
of a three dimensional system which permits the reader to readily visualize and comprehend
the many diverse approaches incorporated into the reviewed methods. The three dimensional
system utilizes the three interdependent variables inherent to fluorescence spectroscopy, i.e.,
excitation wavelength, emission wavelength and fluorescence intensity, and portrays the
fluorescence characteristics of petroleum oils as "total fluorescence spectra" that are
analogous to topographic maps of mountainous regions. Spectral information, derived by
each of the methods, is discussed in terms of appropriate portions of such total spectra.
The methods are categorized with respect to the approach used and are evaluated on the
basis of five criteria. These criteria address pertinent factors such as diagnostic ability,
weathering, quenching, scattering and solvent effects and their influence on a method's utility
for oil spill source identification. Conclusions are presented describing optimum techniques.
INTRODUCTION
Increased activity in the prevention and abatement of petroleum pollution
lias culminated in numerous and diverse analytical methods capable of
establishing the identity of the source or origin of an oil spill. The term
"Fingerprinting" is used to describe this type of methodology and involves
techniques whereby environmentally altered (weathered) petroleum oil,
found as waterborne surface slicks and shoreline residues, are matched
with virgin oils obtained from suspect sources. The magnitude and variety
of publications currently available on this topic have'fnade the selection of
appropriate methods difficult and, at best, time consuming. As a con-
sequence, several review papersli-Ji4 have been published on fingerprinting
216
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UWI I RANK
techniques. These reviews however, do not adequately address fingerprint-
ing techniques based on fluorescence speclroscopy and cite few of the
available methods. A statc-of-ihe-art review of methods for fingerprinting
oils by fluorescence speclroscopy is therefore presented to compensate for
this lack.
Photolumincscence includes both fluorescence and phosphorescence,
and is defined as the radiation emitted by a molecule, ion, or atom, that
has been excited by the absorption of ultraviolet or visible radiation. The
emitted radiation is characteristic of the electronic structure of the
emitting substance and serves as the foundation for the analytical utility of
luminescence. The two forms of luminescence, are of different analytical
importance and are distinguished by observing the lifetimes of their
excited stales: 10" 10 to 10~7 seconds for fluorescence, and KT-1 to 10
seconds for phosphorescence. Fluorescence techniques arc currently more
useful^ than phosphorescence techniques for fingerprinting petroleum oils
and are therefore exclusively reviewed.
A novel mode of presentation that incorporates a three dimensional
system of mutually orthogonal coordinale (cartesian coordinate) axes is
used in this paper to illustrate petroleum fluorescence and the appropriate
fingerprinting methods. This three dimensional system permits the reader
to readily visualize and compare the seemingly wide variety of fluores-
cence methods and compare basic similarities and subtle differences in the
approaches used for performing fluorescence measurements. Petroleum oil
fluorescence is characterized within the context of this system in the form
of "total fluorescence spectra". Figure 1, demonstrates this concept and
illustrates a total spectrum of an oil with three fluorescence maxima. The
three interdependent parameters inherent to fluorescence spectroscopy
(excitation wavelengths, emission wavelengths, and fluorescence intensity)
are simultaneously incorporated in these spectra and are represented by
the x, y and z axes, respectively. Total spectra of different oils exhibit
different maxima and generally resemble topographical maps of moun-
tainous regions.
The reviewed methods are divided into categories which serve to
differentiate the methods on the basis of the approach used for performing
fluorescence measurements and to highlight basic method differences and
similarities. The categories correspond to approaches which derive the
following spectral data: (1) excitation spectra, (2) emission spectra, (3)
synchronously excited spectra, (4) contour maps, and (5) silhouette
profiles. An additional category is used to describe techniques which
combine chromatographic procedures with determinations of the cited
spectral data.
The effectiveness of the methods for fingerprinting petroleum oils are
217
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FLUORESCENCE OIL SPILL IDENTIFICATION
FLUORESCENCE
INTENSITY
EMISSION
WAVELENGTH
EXCITATION
WAVELENGTH
X
FIGURE 1 Three dimensional presentation of the total fluorescence spectrum of ;in oil
with three maxima represented as mountain peaks.
evaluated on the basis of the five criteria described in Table I. Compliance
with these criteria affirms a method's utility for matching weathered, spill
derived, petroleum residues with oils from suspect sources.
Solvent media used for the fluorescence analysis of oils are also
examined and, because they perform an important function in all methods,
are discussed first.
DISCUSSION
Solvent media
Both liquids and solids have been used as dilution media for reducing
the effects of quenching phenomena encountered in the fluorescence
analysis of oils. Because quenching impairs spectral resolution and distorts
fluorescence spectra, the proper selection of solvent media is an essential
function. The criteria used for media selection are primarily based on
medium-oil interactions, the media's fluorescence characteristics, solvent
properties, and ease of application. Solvents that absorb in the 200-
218
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I'WH FRANK
TABLE I
Criteria used for contrasting the effectiveness of the reviewed techniques for fingerprinting
petroleum oils.
Criterion I — Ability "' discriminate between oil*.
A technique's effectiveness for measuring those parameters tli:it are unique In
each oil is thereby evaluated. Aromatic and polynuclear aromatic constituents of
oils primarily impart the characteristic fingerprint to oil fluorescence specli;i
and are therefore the parameters of concern.
Criterion 2— Ability in correctly mutch eiuironincntdlly weathered nils with corresponding
unaltered (source) oils.
Weathering effecls—evaporation, dissolution, photodecomposition, oxidation.
and biodcgradation- incurred by spilled oils and their influence on a
technique's fingerprinting ability arc thereby evaluated. Evaporation is the main
concern in the fluorescence measurement of oils and is responsible for a
concentration phenomenon that involves a build-up of principal fluorescing
compounds (e.g. polynuclear aromatic compounds) in weathered oil matrices
relative to corresponding unweathered source oils.
Criterion 3—Ability to cope with quenching phenomena.
This criterion evaluates a technique's ability to circumvent quenching
effects.Fluorescence quenching entails any process resulting in a decrease in the
true fluorescence efficiency of a compound and is responsible for a shift of
emission maxima to longer wavelengths. For oils the most common problem is
concentration quenching, characterized by the formation of combinations of
excited molecules and molecules in their ground state (excimers).
Criterion 4—Ability to resolve interferences caused by radiation scattering.
A technique's ability to avoid the effects of Rayleigh-Tyndall and raman scatter
is thereby evaluated. Rayleigh-Tyndall scatter emanates from reflections of
excitation radiation and produces intense and readily discernible peaks in
emission spectra at wavelengths corresponding to the applied excitation wave-
lengths. Raman scatter results from an interaction of the excitation radiation
with solvent molecules and appears in emission spectra at wavelengths charac-
teristic of the solvent used.
Criterion 5—Ability to be expediently and routinely applicable.
This criterion evaluates a technique's utility in oil spill incidents where
immediate analytical evidence is mandatory for establishing liability and for
enforcement of legislation. Complex and time consuming techniques are of very
limited utility.
400 nm region, or deteriorate when subjected to ultraviolet radiation are
usually excluded in fluorescence analysis.
Aliphatic hydrocarbon liquids such as cyclohexane, methylcyclo-
hexane, hexane and pentane are the most widely used solvent media for
obtaining fluorescence spectra of oils. They are relatively inert, non
219
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FLUORF.SCENCE OIL SPILL IDENTIFICATION
fluorescent, solubilize most oils and can be routinely applied. Fluorescent
impurities in solvents obtained from commercial sources often present a
problem, but extra pure "fluorometric grade" solvents are available or can
be readily prepared. Frank and Jeleniewski10 have demonstrated that
cyclohexane of sufficient purity can be prepared by simple distillation.
In addition to the aliphatic hydrocarbons, other liquids have been used
and were shown to be superior media for measuring oil fluorescence.
Savvicki27 demonstrated that high viscosity solvents tend to retard the
intramolecular twisting of polynuclear-aromatic petroleum components
and encourage their fluorescence. Frank4 demonstrated an increase in the
resolution of fluorescence spectra derived from oils in a solvent (dipro-
pylene glycol) 20 times more viscous at room temperature than
cyclohexane.
Solid media in the form of frozen liquids have also been used for
obtaining fluorescence spectra of oils. Procedures for preparing such
media involve the dissolution of oils in selected liquids and cooling the
solutions to 77 K. (cryogenic temperatures) with liquid nitrogen. The
liquids utilized are limited to those that form a transparent solid or
"glass" at that temperature. Drushel and Sommersh used mixtures of
methylcyclopentane-methylcyclohexane, and ether-isopentane-ethyl alcohol
(EPA) to prepare clear, transparent glasses at 77UK. Freegarde et «/."
also used EPA for the cryogenic fluorescence analysis of oils. Hornig and
Eastwood17 evaluated several solvents for the preparation of solid media
and concluded that methylcyclohexane results in the formation of an
"optimum glass". The last authors also compared fluorescence spectra of
crude and refined oils in both solid and liquid methylcyclohexane and
demonstrated dramatic improvements in the resolution of spectra derived
from the solid medium.
Excitation spectra
Excitation spectra of oils are obtained by exciting oil solutions with
continuously varying wavelengths of ultraviolet radiation and recording
the fluorescence intensity profiles of the emitted radiation. Instrumental
procedures for obtaining such spectra require two basic steps: (1) setting a
fluorescence spectrometer's emission monochromator at a constant wave-
length and (2) scanning the excitation monochromator. The spectra's
intensity profiles are often unique and can be used a,s fingerprints for the
comparison of oils.
Within the concept of the three dimensional system, excitation spectra
ol oils represent slices taken from total fluorescence spectra that are
parallel to the .v axis. Figure 2 illustrates three such spectra as slices taken
thnuisih the lotal fluorescence spectrum of Figure 1. The slices are a
220
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l'\\T. I KANK
FLUORESCENCE
INTENSITY
EXCITATION
WAVELENGTH
X
340 nm
380
420
EMISSION
WAVELENGTH
K1GURC 2 Three dimensional presentation of excitation spectra. The spectra represenl
slices taken al specific emission wavelengths through the total spectrum in Figure 1.
function of x (excitation wavelength) and z (fluorescence intensity) with y
(emission wavelength) constant.
The excitation spectra approach has not been used for developing any
major methods. However, it has been used in conjunction with other
approaches and some authors19'29 have advocated its potential utility as
an ancillary approach for expanding the discriminating ability of other
fluorescence methods.
Emission spectra
Emission spectra are derived by exciting oil solutions with a single
selected wavelength of ultraviolet radiation and recording the spectral
profiles of the emitted fluorescence radiation. This is accomplished by
fixing a spectrometer's excitation monochromator at a selected wavelength
and scanning the emission monochromator. Spectral envelopes of emission
spectra obtained by this procedure also serve as fingerprints for the
comparison of oils.
In the three dimensional system, emission spectra of oils represent slices
taken from a total fluorescence spectrum and are analogous in this respect
to excitation spectra. These slices however, are a function of v (emission
221
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FLUORESCENCE OIL SPILL IDENTIFICATION
wavelength) and r (fluorescence intensity) with x (excitation wavelength)
constant and are parallel to the y axis. Figure 3 illustrates three emission
FLUORESCENCE
INTENSITY
EXCITATION
WAVELENGTH
X
EMISSION
WAVELENGTH
Y 250 290 340 nm
I ICiLRh .' Throe dimensional presentation of emission spectra. The spectra represent slices
uiken at specific excitation \\a\elcngths through the total spectrum in Figure I.
spectra presented as slices of the three dimensional total fluorescence
spectrum of Figure 1. Each value of x represents an excitation wavelength
and generates a unique slice (emission spectrum).
Thruston and Knight20 initiated the use of emission spectra for
fingerprinting petroleum oils and used 340 nm as the excitation wave-
length: this represents the slice where x equals 340nm in the three
dimensional system in Figure 3. The authors developed a method based
on the use of emission spectra derived from oils dissolved in cyclohexane
at concentration levels of 10. 50 and lOOmg/1. Ratios of the spectra's
fluorescence intensities at 386 and 440nm served as fingerprinting indices
for the comparison of pollution and reference oils. The method's differen-
tiation capabilities were demonstrated with indices of several crude and
residual fuel oils.
Coakley" developed a similar method, also based on the single wave-
length excitation approach. Instead of exciting all oils at a constant
222
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l'\V! I RANK
wavelength, this author used wavelengths that produce maximum fluores-
cence intensities in excitation spectra of oils. The excitation wavelength for
each oil was determined by manually scanning the excitation monochro-
malor until a maximum emission response was achieved. The wavelength
that corresponded to this emission maximum then served as the excitation
wavelength for the oil being fingerprinted.
Coakley used only Ji single concentration of l()ing/l lor oil solutions
and correlated emission spectra by visual comparison of their profiles
instead of intensity ratios. He demonstrated the correlation of three
pollution samples to three source oils, in two separate oil spill incidents:
the excitation wavelengths used for these oils were 295 and 314 nin.
Freegarde et «/." proposed the use of a 250 nm wavelength for exciting
oils, which in the three dimensional system in figure 3, yields the slice
where x equals 250nm. These authors, however, presented only emission
spectra of four crude oils and did not develop a specific method for
fingerprinting oils.
Jadamec and Porro|y employed a similar wavelength and developed a
fingerprinting method based on an excitation wavelength of254mn. They
used solution concentrations of 10 and 100mg/l. and also correlated oils
by visual comparison of spectral profiles. They obtained emission spectra
of a large number of crude and refined petroleum oils and demonstrated
their method's fingerprinting ability in eight oil spill incidents.
Of the excitation wavelengths described thus far, it has been de-
monstrated that the lower wavelengths, such as 250nm, are superior in
discriminating power to wavelengths in the 280-350nm range. Jadamec
and Porro19 have shown that 254 nm produces a greater variability than
290 nm in oil emission spectra. Similarly, Gordon and Keizer13 de-
monstrated that excitation wavelengths in the 300 nm range produce only
slight variations in the emission spectra of ten oils examined. Goldberg
and Devonald12 compared excitation wavelengths of 200, 250, 300 and
350 nm and confirmed the diagnostic superiority of the 250 nm
wavelength.
Although a solution concentration of 10mg/l is used in all of the single
excitation wavelength methods, several authors suggested the use of
additional higher concentrations (e.g., 50 and 100mg/l) for utilizing the
maxima shifts, that accompany concentration quenching of oils, as identifi-
cation parameters. These higher concentrations however, are not com-
patible with weathered oils because quenching and weathering effects
produce similar spectral distortions.
Interferences caused by Rayleigh-Tyndall and raman scattering, upon
excitation at 290-340 nm, may significantly distort emission spectra as is
illustrated in figure 4, for the case of a crude oil. The difference between
223
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FLUORESCENCE on. SI-ILL IDENTIFICATION
:
53
UJ
z
g
55
Cfl
EXCITATION
AT 340nm
.OIL
ICYCLOHEXANE
I BLANK
EXCITATION
AT 290nm
OIL
ICYCLOHEXANE
,BLANK
'
340 380 290 320 350
EMISSION WAVELENGTH
I IGURE 4 Raman mailer imcrlc.x-iia: «nh I he emission spccira ol a crude oil. A
of 5ms ' 1 •' Rosa Crude in -M Mol ",,Pure cyclohcxane uas used.
the Rayleigh-Tyndall and raman peaks is constant in terms of frequency
bin the wavelength difference increases with increasing excitation wa
lengths In the three dimensional system, the combined etfec
scattering phenomena resemble an -interference wedge" that bisects a t,
oil .pectrum (Fiui.re 5). The use of the lower excitation wavelengths, such
as 251) and 254nm circumvents the distortions caused by the scattering
wedge and is therefore preferred. Figure 6 illustrates the extent ot
coincidence of the wedge with 340 and 250nm derived spectra.
-Ml of the methods based on the single excitation wavelength approac
can be expediently and routinely used for sample analyses.
Proportionately little time is expended lor the amount ol informal!.
obtained from oils by these techniques.
Synchronously excited spectra
Synchronously excited spectra are obtained by exciting oils with _con-
tinuously \ai-yinsi wavelengths of ultra\iolet radiation, while simul-
taneously scanning (he emitted radiation. This is performed by scanning
-------
I \VI I RANK
FLUORESCENCE
Z INTENSITY
-SCATTERING WEDGE
- TOTAL SPECTRUM
EXCITATION
WAVELENGTH
Y
EMISSION
WAVELENGTH
FIGURE 5 Three dimensional presenuuion of Rayleigh-Tyndall Raman "Scant-mi" wed-e"
and a lolal oil spectrum. Rayk'iuli-Tyndall and Raman scalier predominate in ihe la rue aild
small plane, respectively.
both the excitation and emission monochromators in synchronization and
separated by a wavelength interval. Each wavelength interval separating
the monochromators determines a unique spectrum, and is in that respect
analogous to an excitation wavelength. Like emission spectra, the en-
velopes of synchronously excited spectra are also used for fingerprinting
oils.
Figure '' illustrates this approach in the three dimensional system
Similar to emission spectra, synchronously excited spectra also represent
slices of total fluorescence spectra, but are oriented in planes at an angle
(0) to the x (excitation wavelength) and y (emission wavelength) axes, and
are a function of .v, y and :. Angle 0 is constant and equal to"45'.
Lloyd22 introduced a method based on this approach for fingerprintiim
trace quantities of petroleum derivatives encountered in forensic crimi-
nology. The author developed analysis proaJures for determininu spectra
of these products and evaluated the use of wavelength intervals ranging
from 10 to 50nm. Solution concentrations were based on the examined
products' fluorescence efficiencies but were selected to avoid concentration
225
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FLUORESCENCE OIL SPILL IDENTIFICATION
7 FLUORESCENCE
INTENSITY
— EMISSION SPECTRA
--RAYLEIGH/RAMAN
SCATTERING WEDGE
^ EXCITATION
WAVELENGTH
X
EMISSION
WAVELENGTH
,,,,,,„,„». Suspec, and source
e ,na,cn;,ls were correlated b, visual com-
a,,J for
as several emission spectra combine
al content as several
the duiiznosue ahilitv ot emission speed a.
226
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L'VVE I RANK
Z FLUORESCENCE
INTENSITY
EMISSION
WAVELENGTH
Although it has been
unwcathcrcd portions of oils has not
*
yet
Soutar- dlscussed the concentration
spectra and illustrated spectra
O.J-1 0.000 niE/1 The effects of
EXCITATION
WAVELENGTH
X
r
"
CXfClted
from
™
monochroma.or. Wulnn ,,,c ,llree d,raensiona|
is a,laloeous „ ., synchronously
227
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FLUORESCENCE OIL SPILL IDENTIFICATION
, FLUORESCENCE
INTENSITY
-SYNCHRONOUS
EXCITATION SPECTRUM
EMISSION SPECTRA
EXCITATION
WAVELENGTH
X
EMISSION
WAVELENGTH
1 !(.! Kl s Comparison of a synchronously c\uK-J -,pa:irum n- Uu> emission speeini. The
-;,ii,'hmnousU excited spectrum contains all of the maxima displayed In the emission
sIVCU'll.
interval set at zero and can therefore be totally avoided by selecting any
wavelength interval substantially greater than /ero. Raman scatter is
similarly avoided by judicious selection of wavelength intervals. Figure 9
demonstrates the feasibility of using a wavelength interval that entirely
circumvents interferences caused by the Rayleigh-Tyndall and raman
scattering wedge.
Synchronously excited spectra can be readily obtained by routine
procedures. An equivalent amount of time is required to derive syn-
chronously excited spectra as emission spectra.
Contour maps
Contour maps of oils are prepared by plotting equal fluorescence in-
tensities of oil emission spectra as a function of excitation and emission
wavelengths, and connecting the plotted points. A sulTicieniK large
228
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I'WI-. I RANK
z FLUORESCENCE
INTENSITY
\
SYNCHRONOUS
EXCITATION SPECTRUM
-SCATTERING
x WEDGE
EXCITATION
WAVELENGTH
\ I
EMISSION
WAVELENGTH
I- KM 'RE 9 Synchronously excited spectrum and Raylciph-Tvndall Raman "scatter]
toi^^^^J^&^ a — '""•' ««™ *—
number of emission spectra must be used, that encompass the entire
fluorescence range of an oil: this requires generally about 30 emission
spectra. Figure 10 illustrates this approach in the -three dimensional
system and demonstrates the derivation of the contour map of an <
having a very simple, inverted cone shaped total spectrum
Contour maps are a function of .v (excitation wavelength) and v
(emission wavelength); z (fluorescence intensity) is equal to zero
Comparison of simple maps can be performed visually. The majority of
matching "^ ^'^ ^ '°° C°mP'eX a"d require comP^
Several authors have recommended contour mapping for fingerprinting
oils and presented contour maps of various crude and refined oils but
uled to develop specific fingerprinting methods. Freeearde et'al."
initiated the use of this approach for oil analyses and demonstrated the
229
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FLUORESCENCE OIL SPILL IDENTIFICATION
FLUORESCENCE
INTENSITY -EMISSION SPECTRA
CONTOUR LINES
EXCITATION
WAVELENGTH
EMISSION
WAVELENGTH
EXCITATION
WAVELENGTH
— X
(A)
EMISSION
Y WAVELENGTH
(B)
l-Kll K\ ID I'lirce dimensional picscnuilum u
h.iMiii: an untried cone shapotl lolal spectrum.
l It i arc uhlained from live emission spectra (A).
llic dcrnaliiui u
I he daui pomiM
an oil
c niLip
preparation of a contour map from 20 emission spectra of a crude oil.
llomia'* demonstrated the feasibility of determining contour maps
••near"oil films and illustrated the variations caused by film thicknesses in
ihe maps of a fuel oil. Hornig and Brownrigg,1" and Horn.g et al.
discussed the advantaues of fingerprimmg oils by the contour mapping
approach and presented contour maps of various fuel and crude
dissolved in methylcyclohexane.
The utility of contour maps for fingerprinting weathered oils from spil
incidents has not been evaluated. In one experiment. Gordon and Kei/cr.
actually demonstrated that the contour mapping approach is
10 synchronous excitation in its abiht> 10 Imgcrprml a crude oil exposed
to simulated wealherine conditions.
The effects of quenching and radiation scattering on contour maps an
ihe combined effects of these phenomena on the emission spectra used as
data base. Precautions similar to ihose used in obtaining emission spectra
arc therefore necessary.
The preparation of contour maps is very time consuming and ted
when performed manually and requires specialized instrumentation when
230
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I'WT I RANK
computer services arc used. Correlation of maps also presents a problem
when compared visually and usually necessitates computer assistance As •,
Consequence, the contour mapping approach is primarily useful as
research too! and is less desirable than the previous, approaches for the
routine fingerprinting of spill samples.
Silhouette profiles
The use of silhouette profiles for fingerprinting o,l represents an approach
which combines the three dimensional character of oil fluorescence into -.
concise two dimensional format. Silhouette profiles of oils are obtained bv
plotting the maximum fluorescence intensities of oil emission speclri as M
function of excitation wavelengths and connecting the plotted points with
straight lines. Plotted oil profiles are amenable to direct visual comparison
and because of their format are also readily compatible with routine
computer software.
Figure 11 illustrates the derivation of a silhouette plot within the three
FLUORESCENCE
INTENSITY
-SILHOUETTE PROFILE
-TOTAL SPECTRUM
EMISSION SPECTRA
EXCITATION
WAVELENGTH
EMISSION
WAVELENGTH
240 280 320 360 400
nm
FIGURE 11 1 hrce dimensional prescniaiion of the derivation of a silhouette profile of •„
oil whose total spectrum contains two maxima. The profile is prepared bv plotting U,c
maximum fluorescence intensities of the emission spectra versus the excitation wavelength
and connecting the plotted points with .straight lines.
231
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FLUORESCENCE OIL SPILL IDENTIFICATION
«.
dimensional system. Silhouette profiles are a function of x (excitation
wavelength) and z (fluorescence intensity); y (emission wavelength) is
equal to zero.
Frank7 introduced a method based on this approach and de-
monstrated its utility for fingerprinting oils. In this method, silhouette
profiles are obtained by exciting 5 ppm solutions of oil in cyclohexane at
15 wavelengths in 20 nm intervals ranging from 220 to 500 nm. At each of
the 15 excitation wavelengths, a rapidly scanned emission spectrum is
obtained whose maximum intensity serves as a data point.
Frank demonstrated the utility of silhouette plotting for discriminating
between crude and refined petroleum oils and successfully correlated
artificially weathered oils with corresponding portions of unweathered oils.
Tanacredi,28 confirmed the fingerprinting capabilities of this approach
and demonstrated its utility for identifying weathered lubricating oils in
waste treatment effluents, surface waters and marine organisms. Silhouette
profiles from these studies were found to retain their identification
characteristics despite extensive weathering.
The effects of quenching and scattering on silhouette profiles are similar
to those of emission spectra. Frank7-8 performed several studies to
determine the extent of influence on profiles and discussed precautionary
procedures.
Silhouette plots of oils are rapidly obtained and can be routinely used
for fingerprinting spill samples. In contrast to contour mapping, this
approach circumvents the necessity of computer services for data handling
and comparisons, and still retains the essence of three dimensional total
spectra.
Individual oil component spectra
Fingerprinting oils by use of fluorescence spectra obtained from individual
petroleum compounds represents a multi-parameter approach which ex-
ploits the complexity of petroleum oil composition. A variety of chroma-
tographic procedures are used to first fractionate oils into simple mixtures
comprised of several similar type compounds. Fluorescence spectra of each
compound are then obtained and compared to reference spectra in order
io establish the compound's identity. This identification procedure is
possible because of the spectra's specificity and the ability to selectively
nbuiin spectra of individual mixture components. Correlation of oils is
consequently performed by comparison of identified compounds.
Although numerous authors have demonstrated the use of individual oil
component spectra for characterizing petroleum oils, this approach has
only recently been adopted as a fingerprinting tool. Drushel and Sommersh
232
-------
l'\VP I RANK
initialed the use of this approach and demonstrated the characteri-
zation of several petroleum distillates. A gas chromatographic (GLC'i
procedure was used by these authors for isolating simple multicomponent
fractions. They then identified many of these components as heterocyclic
aromatic compounds from their emission and excitation spectra and in
some instances from their phosphorescence spectra.
McKay and Latham-5'-" used this approach for characterizing hmh-
boiling petroleum distillates and adopted a technique utilizing a com-
bination of ion exchange, gel permeation and thin-layer chromatograph\
to isolate fractions of similar compound types. They then identified
numerous polynuclear aromatic hydrocarbons and helcrocyclic aromatic-
ring systems from emission and excitation spectra.
Lloyd24 demonstrated the use of this approach for fingerprinting
lubricating oils, gasolines and engine exhaust soots encountered in the
forensic investigation of crime. He used gradient clution chromatography
for the isolation of fractions and obtained emission, excitation and
synchronously excited spectra for the identification of individual com-
pounds. He also determined relative compound concentrations by com-
paring their fluorescence intensity yields to the intensity yield of an
internal standard, added to each isolated fraction. Polynuclear aromatic
hydrocarbons and alkylated benzothiophenes were identified and both
their identity and relative quantity served as fingerprinting parameters.
Jadamec et ctl.20 performed a preliminary study on the use of this
approach for fingerprinting petroleum oils. The authors used high pressure
liquid chromatography to separate major ultraviolet absorbing fractions of
four crude and fuel oils, and obtained emission and excitation spectra of
compounds in each isolated fraction. They identified a homologous series
of polynuclear aromatic hydrocarbons ranging from naphthalene to
coronene and suggest their use as fingerprinting indices.
In comparison to the other fluorescence approaches, the individual oil
component spectra approach exploits much of the potentially available
information that is otherwise obscured in the complexity of "whole" oil
spectra, i.e., usual fluorescence spectra are unresolved combinations of
individual spectra arising from a large number of fluorescent entities.
Several of the above authors,20-24 have demonstrated that this approach
enhances the ability to distinguish between very similar oils. Thus,
Lloyd24 used this approach to identify several oils that are indistinguish-
able by comparison of their emission spectra.
Jadamec20 and Lloyd24 developed the most applicable fingerprinting
techniques using this approach. Their method's utility, however, for
pairing weathered with corresponding unweathered oils has not yet been
demonstrated. It is believed that most of the isolated compounds used as
233
-------
FLUORESCENCE OIL SPILL IDENTII ICATION
kLniii'ication parameters are not significantly degraded by weathering but
thai ilteir relative concentrations in oil matrices are influenced.
:'.c;idy acceptance of this approach for the routine analysis of spill
samples is unlikely. The compound isolation and 'Spectra acquisition
poM-vdures for compound identification are excessively complex and time
tviBurning and are not amenable for the routine analysis of samples.
SUMMARY AND CONCLUSIONS
The reviewed methods are primarily based on five approaches which
derive spectral data that represent specific sections of total fluorescence
spoc'tra. Table 2 summarizes the spectral data obtained from these
methods and the corresponding approaches within the context of the three
dimensional system. Conclusions regarding the methods' relative effective-
ness for fingerprinting petroleum oils are based on the methods' com-
pliance with each criterion outlined in Table 1 and are as follows:
Criterion 1 (ability to discriminate between oils) identified several
methods as 'having superior diagnostic abilities over the other reviewed
methods. Data presented by authors of the following methods de-
monstrated their greater resolving power: excitation at 254nm,'9 syn-
chronous excitation21'22 contour mapping,11-18 silhouette plotting,7 and
fluorescence analysis of individual oil component spectra.20-24
Criterion 2 (ability to correctly match environmentally weathered oils
with corresponding unaltered oils) determined which methods have been
successfully applied to "weathered" oils and are suited for use in oil spill
incidents. Excitation at 254nm,19 and silhouette plotting7-28 were de-
monstrated by more than the original authors to correctly match weath-
ered with unweathered oils. Evidence has also been presented13-22 that
synchronous excitation is a potentially viable fingerprinting approach.
Criterion 3 (ability to cope with quenching phenomena) established that
some methods'y-2
-------
TABLF II
Summary of approaches, spectral data anil methods
ro
vn
Three dimensional representation of approach Spectral data Method (reference)
Description Coordinate function (F)
Slices parallel to x F(x, r):y=constant Excitation spectra 19,29.30
(excitation wavelength)
axis
Slices parallel to y F(y, z);x=constant Emission spectra 5. 11, 19. 20. 2<>
(emission wavelength)
axis
m
7
T*
to x (excitation wave-
length) and y (emission
wavelength) axes
Plotted "peripheries" of
total spectra
Plotted "shadows" of
total spectra
F(.v,z):y =
Synchronous
Contour maps
Silhouette profiles
21,22.23
11. 15. 16, 18
7.28
-------
FLUORESCENCE OIL SPILL IDENTIFICATION
*
Criterion 5 (ability to he expediently and routinely applicable) identified
those methods that can be readily implemented in spill incidents where
immediate analytical data is mandatory Tor legislative and damage assess-
ment purposes. Compliance with this criterion, therefore excludes methods
based on the contour mapping and individual component spectra
approaches.
I'xcilalion at 254 nm,19 synchronous excitation-1 2 and silhouette plot-
linn" most closely comply with the criteria in Table I. These methods
have been successfully used by several investigators,7' * which in
the case of synchronous excitation and silhouette plotting have presented
published data. Contrasting these three methods within the context of the
ihrce dimensional system demonstrates the variability of spectral infor-
mation obtained from each technique. Figure 12 illustrates the infor-
FLUORESCENCE
INTENSITY
••SILHOUETTE PROFILE
-TOTAL SPECTRUM
- SYNCHRONOUS EXCITATION
SPECTRUM
EMISSION SPECTRUM
EXCITATION
WAVELENGTH
Y
EMISSION
WAVELENGTH
-
K'l.'RF: 12 Comparison of emission spectrum, synchronously excited spectrum and
ill'iHicttc profile within I he content of a total oil spectrum. The emission spectrum includes
niv one maximum, and the synchronously excited spectrum includes two maxima. The
lHHieiie profile however includes all three maxima and represents the "shadow" of the total
spectrum.
236
-------
UWi; FRANK
million oblained by each method from an oil whose total spectrum is
shown as three peaks. In this example the single wavelength excitation
method derives the least detailed spectrum and includes only one of the
available three maxima. Synchronous excitation provides a more detailed
spectrum inclusive of two of the maxima. The silhouette profile method.
however, provides information that incorporates the essence of the total
spectrum, within a two dimensional format and includes all three maxima.
Silhouette profiles, therefore yield a fingerprinting parameter broader in
scope than either emission or synchronously excited spectra.
References
1. E. R. Adlarct, A review of the methods for the identification of persistent hydrocarbon
pollutants on seas and beaches. J. Inst. Petrol. 58, 63-74 (1972).
2. R. S. Becker, Theory and Interpretation of Fluorescence and Phosphorescence. Wiley
Interscience, New York, N.Y. (1969).
3. A. P. Bentz, Oil spill identification. Anal. Chem. 48, (6): 454-472 (1976).
4. J. V. Brunnock, D. F. Duckworth and G. G. Stephens, Analysis of beach pollutants. J.
Inst. Petrol. 54, 310-324 (1968).
5. W. A. Coakley, Comparative identification of oil spills by fluorescence spectroscopy
fingerprinting. Proceedings of Joint Conference on Prevention and Control oj Oil Spills,
pp. 215-222. Washington, DC: American Petroleum Institute (1973).
6. H. V. Drushel and A. L. Sommers, Combination of gas chromatography with
fluorescence and phosphorescence in analysis of petroleum fractions. Anal. Chem. 38 (1):
10-19 (1966).
7. U. Frank, Identification of petroleum oils by fluorescence spectroscopy. Proceedings of
Joint Conference on Prevention and Control of Oil Spills, pp. 87-91. Washington DC:
American Petroleum Institute (1975).
8. U. Frank, Effect of fluorescence quenching on oil identification. Analytical Quality
Control Newsletter, U.S. Environmental Protection Agency, Cincinnati, Ohio, Number
22(1974).
9. U. Frank, An improved solvent for fluorescence analyses of oils. Analytical Quality
Control Newsletter, U.S. Environmental Protection Agency, Cincinnati, Ohio, Number
21 (1974).
10. U. Frank and H. Jeleniewski, Solvent impurities and fluorescence spectroscopy.
Analytical Quality Control Newsletter, U.S. Environmental Protection Agency,
Cincinnati, Ohio, Number 18 (1973).
11. M. Freegarde, C. G. Hatchard and C. A. Parker, Oil spilt at sea: Its identification,
determination and ultimate fate. Laboratory Practice 20 (1): 35-40 (1971).
12. M. C. Goldberg and D. H. Devonald, Fluorescent spectroscopy, a technique for
characterizing surface films. J. Res. U.S. Geol. Survey 1 (6): 709-717 (1973).
13. D. C. Gordon. Jr. and P. D. Keizer, Estimation of petroleum hydrocarbons in seawater
by fluorescence spectroscopy: improved sampling and analytical methods. Fisheries and
Marine Services Technical Report Number 481. Environment Canada (1974).
14. D. M. Hercules, Fluorescence and Phosphorescence Analysis. Interscience Publishers,
New York, N.Y. (1966).
237
-------
FLUORESCENCE OIL SPILL IDENTIFICATION
15. -V W. Hornig, Identification, estimation and monitoring of petroleum in marine waters
!<> luminescence methods, NBS Special Publication 409. Proceedings of Symposium and
Workshop on Marine Pollution Monitoring (Petroleum), pp. 135-144. Gaithersburg,
Maryland (1974).
Ifi V W. Hornig and J. T. Brownrigg. Total luminescence spectroscopy as a tool for oil
identification. Abstracts of the Pittsburgh Conference on Analytical Chemistry and
lpplied Spectroscopy, Cleveland, Ohio (1975).
!'. \. W. Hornig and D. Eastwood, 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, DC (1971).
is. A. W. Hornig, H. G. Eldering and H. J. Coleman, The use of Total luminescence
i-.mtour spectra for oil identification. Abstracts of the Pittsburg Conference on Analytical
( lii'inistry and Applied Spectroscopy, Cleveland, Ohio (1976).
]'). J. R. Jadamec and T. J. Porro, Identification and fingerprinting of oils by fluorescence
spectroscopy. Abstracts of the Pittsburg Conference on Analytical Chemistry and Applied
Spcctrnscopy, Cleveland, Ohio (1974).
2li. .1. R. Jadamec, W. A. Saner and T. J. Porro, Identification of spilled petroleum oils by
combined liquid chromatographic and fluorescence spectroscopic techniques. Abstracts
i'/ ihr Pittsburg Conference on Analytical Chemistry and Applied Spectroscopy,
Cleveland, Ohio (1975).
1}. P. John and I. Soutar, Identification of crude oils by synchronous excitation spectro-
iluorimetry. Anal. Chem. 48 (3): 520-524 (1976).
22. J. B. F. Lloyd, The nature and evidental value of the luminescence of automobile engine
oil and related materials—I. Synchronous excitation of fluorescence emissions. J. Fores.
SV/. Sac. 11,83-94(1971).
2.V J. B. F. Lloyd, The nature and evidental value of the luminescence of automobile engine
oil and related materials—II. Aggregate luminescence. J. Fores. Sci. Soc. 11, 153-170
(1971).
24. J. B. F. Lloyd, The nature and evidental value of the luminescence of automobile engine
oil and related materials—III. Separated luminescence. J. Fores. Sci. Soc. II, 235-253
H971).
25. J. F. McKay and D. R. Latham, Fluorescence spectrometry in the characterization of
liigh-boiling petroleum distillates. Anal. Chem. 44 (13): 2132-2137 (1972).
-'*. J. F. McKay and D. R. Latham, Polyaromatic hydrocarbons in high-boiling petroleum
distillates. Anal. Chem. 45 (7): 1050-1055 (1973).
-~. E. Sawicki. Fluorescence analysis in air pollution research. Talunta 16, 1231-1266 (1969).
2\ J. T. Tanacrcdi, Petroleum hydrocarbons from effluents: detection in marine environ-
ment. JWPCF 49, 216-226 (1977).
2'J. A. D. Thruston, Fr. and R. W. Knight, Characterization of crude and residual-type oils
by fluorescence spectroscopy. Eno. Sci. and Tech. 5, 64-69 (1971).
238
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Bull. New Jersey Acad. Sci.
Vol. 24. No. 1, pp. 6-11, Spring 1979
OCCURRENCE OF EXTRACTABLE HYDROCARBONS
IN SEDIMENTS FROM RARITAN BAY, NEW JERSEY
DENNIS STAINKEN
U.S. Environmental Protection Agency
Oil and Hazardous Materials Spills Branch, lERL-Ci
Edison, New Jersey 08817
ABSTRACT. Sediments from 22 sires in the Raritan
Bay — Lower New York Bay complex showed that ex-
tractable hydrocarbons and percent volatiles increased
as the silt-clay content of the sediment increased. The
hydrocarbons appeared to be concentrated in the deeper
center of Raritan Bay and the Raritan Bay muds, head-
ing down the Bay towards Sandy Hook. Extractable
hydrocarbons ranged from 2.2-1098.2 pg/g of dry sedi-
ment and the percent volatiles ranged from 0.85-1139.
A peak within the n-Ca range and a large "unresolved
complex mixture" were characteristic of gas chromato-
grams. Mixed isomers of benz-pyrene and benz-anthra-
cence and unidentified 1-6 ring polynuclear aromatic hy-
drocarbons were present.
INTRODUCTION
Hydrocarbons may enter the estuarine environ-
ment by many routes including air pollution fall-
out, sewage, rivers, dredging and industrial out-
falls. The tidal nature of estuaries increases the
probability that some hydrocarbons will bs re-
tained and deposited. Hydrocarbons present in
an ecosystem can frequently be divided into two
components: a) those recently derived biogeni-
cally from flora, fauna, detritus, and b) those de-
rived from fossil hydrocarbons by anthropomor-
phic activities or seepage from the earth. Many
of the hydrocarbons are potentially dangerous to
human health and knowledge of hydrocarbon
content and distribution is necessary for manage-
ment decisions, e.g., dredging impacts, spill treat-
ment effects and alternatives, and use of biotic
resources (shellfish).
The Raritan Bay — Lower New York Bay
Complex receives hydrocarbons from municipal
and industrial wastes, both directly and from trib-
Manuscript received 3 Jul. 1978.
Revised ms received and accepted 16 Jan. 1979.
utary waters (Tanacredi, 1977). Unknown
amounts of hydrocarbons have entered the bay
from oil spills in the Arthur Kill and Raritan
River, yacht marinas, ship traffic, and dredging.
The Hudson River may also contribute hydrocar-
bons from the upper New York Bay and New York
City environs. DeFalco (1967) reviewed many
types and sources of effluents. The circulation
patterns (Patten, 1962) and flushing rates in the
Raritan Bay — Lower New York Bay Complex
tend to retain introduced pollutants.
DeFalco (1967) described general sediment
regions of the Bay but particle-size distributions
were not reported and extractable hydrocarbons
were not quantified with respect to sediment types.
Grain-size properties of the sediments may affect
the transport and preservation of organic com-
pounds and absolute amounts of sedimentary hy-
drocarbons considered without other information
on the physical properties of the sediments may
be of little value (Wehmiller and Lethen, 1975).
This study attempted to survey the grain-size dis-
tribution, percent volatiles and extractable hydro-
carbons of the sediments of the Raritan Bay —
Lower New York Bay Complex.
METHODS
Sediments were obtained from the Lower New
York Bay — Raritan Bay Complex during June,
1977 (Fig. 1). Samples were not taken near ship
channels or dredge spoil areas to avoid overt pos-
sibilities of contamination. Benthic surface sedi-
ments were sampled with a Petersen Grab (O.I
m3). Several grabs were taken per station and
composited. Sediments for chemical analysis
ERRATA: The illustrations for Figures 2 and 4 have been switched. The
chromatograras depicted in Figure 2 should have the legend of
Figure 4, and the chromatograms of Figure 4 should have the
legend of Figure 2.
239
-------
HYDROCARBONS IN RARITAN BAY SEDIMENTS
FIGURE 1. Location of sampling sites. Intertidal
samples were taken at OBI (near the Outerbridge
Crossing), FBI (Sequine Point), GK4 (a sand bar at
the mouth of Great Kills Harbor) and GK3 (below a
storm sewer outfalk). Sub-littoral samples were taken
at stations 1-14, GK1 and GK2 (in Great Kills Harbor),
PB2 (Princes Bay) and OKWD (off the outfall of the
Oakwood Sewerage Treatment Plant.
were placed in quart mason jars with tin lined
caps, refrigerated at 4°C, and analyzed within
two months. AH collection and storage materials
were washed with "distilled in glass" hexane
(Burdick and Jackson, Muskegon, Mich.). Sedi-
ments for textural characterization were subsam-
pled from the grab composite. Similar proce-
dures were utilized in the analysis of littoral
samples.
Particle-size analyses were performed by siev-
ing 100 g dry weight of sediment through a series
of U.S. Standard Wire-cloth sieves (4 mm, 2 mm,
0.5 mm, 0.24 mm, 0.125 mm, and 0.063 mm).
Macrodebris (i.e., shells, large rocks) were ex-
cluded from samples prior to weighing. Dried
congealed sediments (mud) were ground with a
mortar and pestle prior to sieving. The sediments
were sieved for 30-40 minutes. Sediment frac-
tions were then weighed and relative percentages
were calculated.
Percent volatiles of the sediments were meas-
ured by placing predried sediment samples in a
muffle furnace at 500-800°C for 24 hours. Dif-
ferences in weight were calculated as percent
volatiles. Total extractable hydrocarbons were
analyzed using a modification of the method of
Giger and others (1974). All material was pre-
washed with Freon 113 prior to use. Approxi-
mately 50 g of sediment from each sample was
dried to constant weight at 60°C for two days.
Sediments were then transferred to 250 ml screw
top Erlenmeyer flasks Csand sediments were sep-
arated with a spatula, mud sediments were ground
in an agate mortar). Fifty ml of Freon 113 was
added to each flask. Flasks were sealed with a
tinfoil lined cap and tumbled 10 minutes on a
wrist action shaker. The freon extract was then
filtered through glass wool into a 100 ml vol-
umetric flask. An additional 50 ml of freon was
added to the sediment in the Erlenmeyer flasks
and tumbled for another 10 minutes. This super-
natant was then filtered through glass wool and
added to the first. The combined extracts of
each sample were brought to volume and dried
with anhydrous sodium sulfate previously baked
at 150°C to remove impurities. The extracts
were analyzed by Infrared Spectroscopy utilizing
10 and 1 cm cells in a Perkin-Elmer Model 457
Infrared Spectrophotometer. The extracts were
scanned from 3200 cm'1 to 2700 cm-1 and quanti-
tated at 2930 cnr1 (Gruenfeld, 1973). Subsam-
ples (25 ml) of selected extracts were analyzed
by gas chromatography and fluorescence spectros-
copy.
The remaining freon extract in each flask was
treated with 2 g of 2% deactivated silica gel to
remove non-hydrocarbon interferences (Gruen-
feld and Frederick, 1977). The treated, extracts
were then re-analyzed by IR Spectroscopy similar
to the pretreated samples. All extracts were
quantitated by comparing the absorbances to a
reference mixture of iso-octane and hexadecane
in freon. To determine the types of hydrocarbons
present and the effects of silica gel treatment, se-
lect samples from oil contaminated sites (GK3
and OB 1) were analyzed by gas chromatography-
For gas chromatographic analysis, sample
were dried under carbon filtered air and brougM
to 100 fj.1 volume with cyclohexane (Fisher ?
Mo! %). A Perkin-Elmer (Model 900) gas
chrornatograph was used, equipped with a flam*
ionization detector and a six foot stainless steel
column packed with 3% OV-1 on 80/100 rnesb
of Chromosorb WAW. Operating conditions
240
-------
DENNIS STAINKEN
TABLE 1. Particle size classification (percent retained on sieves), percent volatiles, and extractable hydrocar-
bons before (BT) and after (AT) silica gel treatment Oig'g dry sediment).
Particle size (Wentworth)
Sampling
site
1
2
3
4
5
6
7
8
9
10
11
12
13
14
GK 1
GK 2
GK 3
GK 4
PB 1
PB 2
OB 1
OKWD
Granules
0.9
2.4
0.0
0.4
0.6
19.9
3.3
2.3
1.2
0.0
8.0
0.0
0.2
1.3
0.1
0.1
13.7
8.0
4.5
4.8
21.7
12.8
Very
coarse
sand
3.5
2.0
0.2
0.7
2.1
14.0
4.2
3.7
3.5
0.2
7.4
1.0
1.2
1.1
0.7
0.1
8.5
15.6
2.6
4.9
7.8
6.9
Coarse
sand
20.8
3.1
0.4
1.4
4.4
19.7
6.0
10.4
5.5
0.7
8.8
2.7
2.7
2.4
2.5
0.4
16.4
36.0
5.0
9.5
16.9
16.1
Medium
sand
66.6
14.6
1.1
1.9
5.3
31.4
20.9
16.5
13.8
2.3
33.1
5.2
4.4
10.3
4.3
1.1
29.7
38.4
36.6
39.6
42.8
42.3
Fine
sand
6.8
39.0
4.9
4.0
12.5
13.8
24.2
13.4
24.8
26.3
17.9
10.0
52.4
36.9
10.0
21.0
21.9
1.4
48.0
33.4
8.2
19.8
Very
fine
sand
1.1
34.2
65.3
55.9
42.7
0.6
24.7
32.7
27.7
10.0
12.8
28.7
23.2
43.4
31.9
42.5
6.2
0.3
2.9
5.0
1.3
1.8
Silt-
clay
0.2
4.1
27.6
33.9
30.4
0.3
14.7
20.4
22.7
59.8
11.4
52.2
14.7
4.5
50.1
34.1
3.5
0.2
0.4
2.8
1.0
0.3
Percent
volatiles
1.35
3.06
5.53
8.55
11.39
0.85
3.80
5.12
5.41
7.36
3.79
8.84
5.41
1.47
7.02
7.38
2.56
0.35
0.95
1.31
2.8
0.91
Extractable
hydrocarbons
(BT) (AT)
18.4
292.3
422.5
840.0
444.9
74.8
179.3
120.0
696.8
329.5
441.0
572.0
381.4
34.8
672.6
1194.3
715.2
7.2
14.5
109.4
737.8
19.9
11.5
269.2
395.7
768.3
414.3
55.1
179.3
120.0
636.7
307.2
412.9
549.5
332.9
30.4
655.6
1098.2
590.2
2.2
9.6
98.3
641.3
16.6
were: carrier gas; He2; column temperature;
60°C with a two-minute hold, programmed to
increase 8°C/minute to 300°C; injection sample:
1-2 microliters. Sample peak areas were identi-
fied by matching to alkane standards.
Sample volumes were then diluted to 5 ml with
cyclohexane and analyzed for polynuclear aro-
matic hydrocarbons (PNA) by synchronous ex-
citation fluorescence spectroscopy (Frank, 1978).
Confirmation was obtained by combination HPLC
and fluorescence spectroscopy. An ODS reverse
phase column (DuPont) and a mobile phase of
50:50 2-propanol/water was used to chromato-
graph the sample. The predominant constituent
was isolated and collected. Both single wave-
length (emission) and synchronous excitation
spectra were then obtained by fluorescence anal-
ysis in a 10 mcl cuvette. Solutions of known
PNA standards were similarly analyzed. Reten-
tion times on chromatograms and fluorescence
spectra were used as identification parameters for
the chromatographed sample constituent.
RESULTS
The results of textural analysis, determination
of percent volatiles and quantitation of extract-
able hydrocarbons are presented in Table 1.
Values of hydrocarbons are reported before and
after silica gel treatment to illustrate the large
residual amount of hydrocarbons remaining after
treatment. Silica gel treatment removed an aver-
age of 14.4% of non-hydrocarbon material from
extracts. The extractable hydrocarbons (after sil-
ica gel treatment) ranged from 2.2 to 1098.2
mg/g of dry sediment.
Percent volatiles and /*g extractable hydrocar-
bons (after silica gel treatment) were examined
to relate their quantitative values to the percent
silt-clay fraction of the sediment. Table 2 indi-
cates that the percent volatiles and jug extractable
hydrocarbons apparently increase as the silt-clay
fraction increases.
Relationships between the silt-clay fraction,
percent volatiles and /tg of extractable hydrocar-
bons were analyzed by linear regression. A cor-
241
-------
HYDROCARBONS IN RARITAN BAY SEDIMENTS
TABLE 2. The percent volatiles and /ig extractable
hydrocarbons (recorded as mean values ± S.E.) deter-
mined sediments with different silt-clay fractions.
% Silt-clay
fraction
0.1- 1.0
1.1-10.0
10.1-20.0
20.1-30.0
30.1-40.0
40.1-50.0
50.1-60.0
n
5
1
'
I
•'.
0
3
% Volatiles
0.88 ± 0.16
2.1 ± 0.42
4.3 ± 0.54
5.3 -t-0.13
10.0 ± 1.42
7.7 ± 0.56
ng extractable
hydrocarbons
19.0 •+- 9.31
247.0 ± 124.9
30X.4 •+- 68.2
384.1 ± 149.3
591.3 ± 177.0
504.1 ± 103.1
relation coefficient of 0.64 was found for the re-
gression of the percent silt-clay fraction vs. ^§ of
hydrocarbons. A correlation coefficient of 0.73
was calculated for the regression of the percent
volatiles vs. /*g of hydrocarbons. However, the
calculated standard error of the regression for
both of the above indicated considerable scatter
around both calculated regression lines. The
calculated correlation coefficient (0.85) of the
regression between the silt-clay fraction and the
percent volatiles indicated a closer relationship.
In calculations, data from sites OBI and GK2
were excluded because of several previous spill
events of petroleum oil (OBI) and sewage
(GK2).
To determine types of hydrocarbons present
and the effects of silica gel treatment, select sam-
ples (OBI, GK3, Sites 1, 4, 5, 6, 8, 11 and 12)
were analyzed by gas chromatography. Figure 2
illustrates chromatograms of samples from sites
OBI and GK3, before and after silica gel treat-
FIGURE 2. Gas chromatograms of Sites OBI (A) and
GK3 (C). Chromatograms B and D are sites OBI and
GK3 after treatment with silica gel. The baseline is
drawn below chromatogram D.
FIGURE 3. Gas chromatograms of Light Arabian
Crude oil (A) and No. 2 fuel oil (B).
ment. Although some material was removed by
the silica gel, the occurrence of a large "unresolved
complex mixture" in the chromatograms indicated
the hydrocarbon material was not recently bio-
logically produced (Gruenfeld and Frank, 1977).
The chromatogram of site OBI illustrates residual
oils in the sediment after several spill incidents
of crude and fuel oils over a period of years. The
chromatograms of GK3 indicate the presence of
well weathered oil, higher boiling materials and
possible sewage derived hydrocarbons from the
storm sewer outfall which was located approxi-
mately 40 feet from the site. Gas chromatograms
of a light Arabian Crude oil and a No. 2 fuel oil
are presented in Figure 3 to illustrate non-weath-
ered oil chromatograms.
Figure 4 contains chromatograms from Sites
1, 4, 5 and 6. A large indigenous hydrocarbon
content is evident in the chromatograms of Sites 4
and 5. Site 4 was near the "Raritan Bay-West
Reach Ship Channel" and the Site 5 sample was
from a large mud bank. Sites 1 and 6 were sandy
areas and the chromatograms indicate low levels
of hydrocarbons present.
Synchronous fluorescence spectra were ob-
tained for samples from Site OBI and GK3 (be-
fore silica gel treatment; (Frank, 1977) and
Sites 1, 5, 6 and 8. In analyzing the spectra of
OBI and GK3, benz-pyrene (mixed isomers)
were the predominant compounds with possible
242
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DENNIS STAINKEN
FIGURE 4. Gas chromatograms of Site 1 (A), Site 4
(B), Site 5 (C) and Site 6 (D).
4 and 6 ring compounds present. In the Site 1
spectra, two ring compounds were the most prev-
alent (with possible three ring compounds pres-
ent), five ring compounds exhibited a well re-
solved peak and a six ring compound peak was
partially resolved. In the Site 5 spectra, benz-
anthracence was predominant with benz-pyrene
and other five ring compounds prevalent. The
peaks for benz-anthracence and benz-pyrene were
well resolved. In the spectra of Site 6, three ring
compounds were prevalent, probably anthracence.
Five ring compounds were next in prevalence
with six ring compounds indicated in the spectra.
The spectra of Site 8 indicated a predominance
of three ring compounds with 5 and 6 ring com-
pounds present and partially resolved.
DISCUSSION
The particle-size distributions found in this
study generally conformed to those found by
DeFalco (1967). This indicates that the general
bottom sediment distribution is a relatively stable
feature of the Bay.
The particle-size distribution and percent vola-
tiles reported here indicates a pattern of move-
ment of sediments in the Bay complex. The sedi-
ments entering the Bay from various locations
move progressively toward the area bounded by
Sequine Point, Great Kills, Keyport and Keans-
burg. The pattern conforms with earlier descrip-
tions by DeFalco (1967) and Greig and McGrath
(1977). The distribution of extractable hydro-
carbons reflects this sediment movement with
relatively high values at Stations 4, 5, 7, 8, 9, 10
and 11. Generally, sample sites with high per-
cent volatiles were areas with large silt-clay and
fine-sand fractions.
Several of the values of Mg of extractable hydro-
carbons after treatment were higher than others
and reflect the area sampled. Site GK2 was sam-
pled from the center of Great Kills Harbor and
receives inputs of petroleum hydrocarbons from
boat marinas, storm sewers and dredging. Sites
GK1 and GK3 also contain hydrocarbons found
within semi-enclosed Great Kills Harbor. The
hydrocarbon material found at Site OBI reflects
several previous oil spills within the Arthur Kill.
This beach was predominantly medium and
coarser sands, and oil appeared in bootprints
while sampling. When taking sediments, a light
crude or fuel type oil appeared at the bottom of
the hole. Site 4 was near the "Raritan Bay-West
Reach Ship Channel" and the higher hydrocarbon
values may reflect settled dredge spoil or ship de-
rived hydrocarbons.
Gas chromatograms frequently exhibited a
large 'unresolved complex mixture' indicative of
pollution derived hydrocarbons. The Bay re-
ceives large amounts of sewage from many
sources, and both Hatcher and others (1977),
and Goodfellow and others (1977), have re-
ported frequent contamination of sediments by
sewage derived coprostanol (an n-C28 sterol).
Some material eluting in the chromatographs in
this study in the n-C28 region may also be sewage
derived contaminants. However, Wehmiller and
Lethen (1975) reported that Delaware estuary
sediments showed a significant 'unresolved com-
plex mixture,' and many chromatograms also con-
tained a mixture that peaked in the n-C28 to
n-Cao region. They termed these "high boiling"
mixtures which were frequently observed in coarse
grained sediments.
The lack of resolution in the chromatograms
of this report indicates that a substantial amount
of hydrocarbon material is present. The fluores-
cence spectra indicated that many aromatic com-
pounds were present. Mixed isomers of both
benz-pyrene and benz-anthracene were frequently
found with other 1-6 ring compounds present.
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HYDROCARBONS IN RARITAN BAY SEDIMENTS
Kites and others (1977), and Tanacredi (1977),
have also reported the occurrence of PNA hydro-
carbons in sediments. Although Tanacredi re-
ported that sewers in Jamaica Bay were discharg-
ing PNA hydrocarbons, the derivation of aromatic
hydrocarbons in this study is not known.
LITERATURE CITED
DEFALCO, P. 1967. Report for the conference on pol-
lution of Raritan Bay and adjacent interstate waters,
third session. Federal Water Poll. Contr. Admin.,
New York, pp. 15-865.
FRANK, U. AND GRUENFELD, M. 1978. Use of sychro-
nous excitation fluorescence spectroscopy for In situ
quantifications of hazardous materials in water.
Proc. 1978 Natl. Conf. Haz. Mater. Spills, Informa-
tion Transfer Inc., Rockville, MD., pp. 119-123.
FRANK, U. 1977. Adsorption of fluorescing oil compo-
nents onto silica get. Analyt. Qual. Cont. Newsl.,
U.S. EPA, Ctnn., Ohio. 33:4.
GIGER, W.; REINHARD, M.; SCHAFFNER, C.; AND STUMM,
W. 1974. Petroleum derived and indigenous hy-
drocarbons in recent sediments of Lake Zug, Swit-
zerland. Environ. Sci. & Technol., 8:454-455.
GOODFELLOW, R. M.; CARDOSO, J.; EOLINTON, G,; DAW-
SON, J. P.; AND BEST, G. A. 1977. A faecal sterol
survey in the Clyde Estuary. Mar. Pollut. Bull.,
8:272-276.
GREIO, R. A. AND McGRATH, R. A. 1977. Trace metals
in sediments of Raritan Bay. Mar. Pollut. Bull.,
8:188-192.
GRUENFELD, M. 1973. Extraction of dispersed oils
from water for quantitative analysis by infrared
spectrophotometry. Environ.- Sci. and Technol.,
7:636-639.
GRUENFELD, M. AND FRANK, U. 1977. A review of
some commonly used parameters for the determina-
tion of oil pollution. Proc. 1977. Oil Conference,
Amer. Petrol. Inst., Wash, D.C., pp. 487-492.
GRUENFELD, M. AND FREDERICK, R. 1977. The ultra-
sonic dispersion, source identification, and quantita-
tive analysis of petroleum oils in water. Rapp.
P.-v. Reun. Cons. int. Explor. Mer. 171, pp. 33-38.
HATCHER, P. G.; KEISTER, L. E.; AND MCGILLIVARV,
P. A. 1977. Steroids as sewage specific indicators
in NY Bight sediments. Bull. Environ. Contain. &
Toxicol., 17:491-498.
KITES, R. A.; LAFLAMME, R. E.; AND FARRJNGTON, J. W.
1977. Sedimentary polycyclic aromatic hydrocar-
bons: the historical record. Science 198:829-831.
PATTEN, B. C. 1962. Species diversity in net phyto-
plankton of Raritan Bay. J. Mar. Res., 20:57-75.
TANACREDI, J. T. 1977. Petroleum hydrocarbons from
effluents: detection in the marine environment. J-
Wat. Pollut. Contr. Fed., 49:216-226.
WEHMILLER, J. F. AND LETHEN, M. 1975. Saturated
hydrocarbon material in sediments of the Delaware
estuary as determined by gas chromatographic anal'
yses. Univ. Delaware, Coll. Mar, Stud., CMS-
RANN-3-75, pp. 3-87.
U. S. GOVERNMENT PRINTING OFFICE: 1979 — 657-060/5551
244
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