PUBLICATIONS AND ARTICLES RELATING
                  TO THE
  CHEMICAL ANALYSIS OF OIL POLLUTION
      EMISSION
      WAVELENGTH
                  FLUORESCENCE
                  INTENSITY
                               EXCITATION
                              WAVELENGTH
      U.S. ENVIRONMENTAL PROTECTION AGENCY
INDUSTRIAL ENVIRONMENTAL RESEARCH LABORATORY-Cl
   OIL AND HAZARDOUS MATERIALS SPILLS BRANCH
            EDISON, NEW JERSEY 08817

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COVER ILLUSTRATION  is A THREE- DIMENSIONAL REPRESENTATION OF A TOTAL
FLUORESCENCE SPECTRUM,  FOR FURTHER INFORMATION SEE SECTION I:
" DETERMINATION OF PETROLEUM OILS IN SEDIMENTS BY FLUORESCENCE
SPECTROSCOPY AND NMR " ,

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                              SECTION I

                      FLUORESCENCE PUBLICATIONS



                       AQC Newsletter Articles

ANALYSIS FOR CRANKCASE OIL IN WATER BY FLUORESCENCE SPECTROPHOTOMETRY
                  U. Frank
      PASSIVE TAGGING OF OILS BY FLUORESCENCE SPECTROPHOTOMETRY
                  U. Frank
              A METHOD FOR QUANTITATING OIL DIRECTLY IN
               WATER BY FLUORESCENCE SPECTROPHOTOMETRY
                  U. Frank
        SOLVENT IMPURITIES AND FLUORESCENCE SPECTROPHOTOMETRY
                  U. Frank, H. Jeleniewski
       PASSIVE TAGGING OILS BY FLUORESCENCE SPECTROPHOTOMETRY
                  U. Frank
        AN IMPROVED SOLVENT FOR FLUORESCENCE ANALYSES OF OILS
                  U. Frank
                     RECLAIMING A WASTE SOLVENT
                  U. Frank
       EFFECT OF FLUORESCENCE QUENCHING ON OIL IDENTIFICATION
                  U. Frank
    IDENTIFICATION OF PETROLEUM OILS BY FLUORESCENCE SPECTROSCOPY
                  U. Frank
          SYNCHRONOUS EXCITATION FLUORESCENCE SPECTROSCOPY
                  U. Frank, L. Pernell
          SYNCHRONOUS EXCITATION FLUORESCENCE SPECTROSCOPY
                1  U. Frank, M. Gruenfeld

                           Research Papers

    IDENTIFICATION OF PETROLEUM OILS BY FLUORESCENCE SPECTROSCOPY
                  U. Frank
            DETERMINATION OF PETROLEUM OILS IN SEDIMENTS
                BY FLUORESCENCE SPECTROSCOPY AND NMR
                  U. Frank, M. Gruenfeld
              A REVIEW OF SOME COMMONLY USED PARAMETERS
               FOR THE DETERMINATION OF OIL POLLUTION
                  M. Gruenfeld, U. Frank
          PETROLEUM HYDROCARBONS FROM EFFLUENTS:  DETECTION
                        IN MARINE ENVIRONMENT
                  J. T. Tanacredi

                            Bibliography

               BIBLIOGRAPHY OF RECENT METHODS FOR THE
               FLUORESCENCE ANALYSIS OF PETROLEUM OILS
                  U. Frank

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 NEWSL   ETTER
   01 TON
                    Analytical
     U  S   ENVIRONMENTAL  PROTECTION  AGENCY
ENVIRONMENTAL  MONITORING  AND  SUPPORT  LABORATORY
             CINCINNATI , OHIO   43268
               PHONf : 513-684- 7301
                     SELECTED  REPRINTS

                        FLUORESCENCE
These selected reprints describe work that was performed at  the

Industrial Environmental Research Laboratory - Ci.,  Oil and  Hazardous

Materials Spills Branch, Edison, New Jersey 08817.

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Analytical  Quality Control Newsletter  (U.S.  EPA),  No.  13,  April 1972
Analysis for Crankcase Oil in Water by Fluorescence Spectrophotometry
Potential environmental damage from repeated applications of spent
crankcase oil onto unpaved roadways, for dust control, is under investi-
gation at Edison.  Our laboratory recently examined a suspected "run-off"
water sample, collected in the vicinity of such a road, for traces of
crankcase oil.  After the usual extraction and concentration steps,
analysis was attempted by gas chromatography (GC),  using a flame ioni-
zation detector.  An unresolved chromatogram was obtained, in which the
presence of crankcase oil could not be established.  This difficulty
was attributed to excessive interfering contaminants in the sample ex-
tract, that masked any GC profile resulting from traces of crankcase
oil.  This analysis was successfully performed, however, by using a
modified version of the fluorescence spectrophotometric method previously
reported by Thruston and Knight (Env.  Science and Tech., 5_, 64-69, 1971)
We excited the sample extract at 290 my instead of the recommended
340 my, because we had previously established that this change considerably
improved differentiation between the spectrum of a known crankcase oil
and the spectra of ten other common oils that were examined (crudes and
crude oil fractions).  The entire sample extract was dissolved in the
minimum cyclohexane volume needed to fill a 1 cm rectangular cell.  A
Perkin Elmer MPF-3 fluorescence spectrophotometer was used for this
analysis.  The water sample was found to contain traces of crankcase oil.
The pollutant was identified through the shape of its emission spectrum,
and the wavelength of its maximum emission (329 my).  It matched in these
spectral properties a known crankcase  oil, but differed considerably from
all the other oils examined.   Fluorescence spectrophotometry therefore
shows unique promise as a method for characterizing oils in situations
that are not amenable to analysis by gas chromatography.  fU. Frank)

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     Analytical Quality Control Newsletter  (U.S. EPA), No. 15, October 1972
  PASSIVE  TAGGING  OF  OILS  BY  FLUORESCENCE  SPECTROPHOTOMETRY

Fluorescence  spectrophotometry  is  currently  under evaluation as
a method for  passive  tagging  of oils.   The potential usefulness
of this technique  was  previously reported  by Thruston and
Knight  (Env.  Science  and Tech., 5_,  64-69,  1971),  but diffi-
culties were  encountered while  applying some of  their recommenda-
tions.  Misleading emission spectra were obtained from dilute
solutions  of  crude and processed oils  in cyclohexane (0.5
mg/liter)  when  these  were  excited  at the recommended wavelength
of 340 my.  All the resulting spectra  were essentially identical,
consisting of a pronounced maximum at  380  my surrounded by a
variable background envelope.   These spectra were more fully
resolved by examining  several solutions  containing different
oil concentrations, at various  excitation  wavelengths in the
range 290  my  -  400  my.  Two distinct emission spectra re-
sulted; the maximum that was  previously  noted at  380 my,  was
due to the cyclohexane solvent  Raman C-H stretch  band,  while
the background  envelope was due to  oil.  These conclusions
were confirmed  by  separately  exciting  at 340 my pure cyclo-
hexane and the  oils as tnin solvent-free films.   A decrease
in dissolved  oil concentration  increased interference by the
Raman band.   Several other solvents  were also excited at
340 my and yielded  interferring Raman  bands:   hexane,  pentane
and isooctane were  tested.  In  this  study  290 my  was selected
as the excitation wavelength  of choice.  It  yielded improved
spectral separation, with the emission maxima of  the oils
occurring between  330  my - 360  my and  the  Raman band of cyclo-
hexane occurring at 320 my.   (U. Frank,  201-548-3347)

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    Analytical Quality Control Newsletter, (U.S. EPA), No.  18, July 1973

           A METHOD FOR QUANTITATING OIL  DIRECTLY  IN WATER
           	BY FLUORESCENCE SPECTROPHOTOMETRY	

 A preliminary evaluation of fluorescence  spectrophotometry  for  the
 quantitation of oil directly in water has been completed.   Since
 in this approach the oil is not extracted from water,  two big advan-
 tages over other methods are realized:   shorter analysis time and
 small sample volume requirements.  As little as 3 ml  suffices for
 analysis; the usual methods require 1000 ml sample  volumes.  In
 the present study, standard solutions of oils were  prepared for
 fluorescence measurement by vigorously mixing 2 yl  -  60 yl portions
 of the oils with 100 ml portions of distilled water.   Ten ml
 aliquots of the emulsions were then mixed with 5.0  ml  of 2-
 Propanol.  This solvent acts in an intermediary capacity to solu-
 bilize oil in water.  A weathered West Texas Sour Crude Oil, an
 unweathered No. 4 fuel oil, and an unweathered South Louisiana
 Crude Oil were used to prepare the standard solutions.  The fluo-
 rescence intensities of the solutions were measured at 340 my in
 10 mm path length cells, by exciting at 290  my.  The resulting in-
 tensity vs. concentration plots were linear, and passed through
 the origin.  This confirms the usefulness of the method for single
 point analysis.  The influence of oil weathering on the method  was
 evaluated by exposing portions of the South Louisiana Crude Oil and
 No. 4 fuel oil, as thin films, to environmental conditions for  30
 days.  The fluorescence intensities of these oils in the water-
 alcohol solvent system remained unchanged.  Quantitative results
 obtained with this method were also compared to those obtained  with
 the IR extraction method described in the AQCL Newsletter #15,
 October 1972.   Both methods were found to yield identical re-
 sults.   (U. Frank, FTS 201-548-3510, Coml. 201-548-3347)


      SOLVENT IMPURITIES  AND FLUORESCENCE  SPECTROFHOTOMETRY

 Solvents  that  are  used  for  the  analysis  of oils  by fluorescence
 spectrophotometry  often  contain impurities which give rise  to
 interfering  emission spectra.   "Spectroanalyzed"  and "99  Mol%
 Pure"  cyclohexane  (Fisher Scientific Co.)  were  tested at  room
 temperature  by  exciting  from 200  -  400 nm in 10  mm path length
 cells.  The  "Spectroanalyzed"  cyclohexane was  found to contain
 impurities which cause a significant contribution  to typical oil
 emission profiles.   "99 Mol%  Pure"  cyclohexane  displayed  only the
 characteristic  Raman band  (described in  the  AQCL Newsletter  #15),
 and is therefore the solvent  of choice.   Similarly,  methylcyclo-
 hexane obtained from two different  sources,  Fisher Scientific Co.,
 and Eastman  Kodak  Co., was  examined.  This solvent is commonly
 used  for fluorescence studies of  oils at  cryogenic temperatures
 (77K), where it forms a transparent glass.   Both  the Fisher and
Eastman Kodak methylcyclohexane yielded  emission spectra  of  im-
purities when excited at this temperature.   The  Eastman Kodak
product was  found  to be a more  suitable  solvent  for excitations
at low wavelengths, while the impurities  in  the  Fisher product
contributed  less to emission  spectra when excited  at wavelengths
abouve 290 nm.   (U. Frank/H.  Jeleniewski, FTS 201-548-3510, Coml. 201-548-3347)

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   Analytical Quality Control Newsletter (U.S.  EPA), No. 20,  January 1974
       PASSIVE TAGGING OILS_BY_FLUORF.SCENCE SPECTROMETRY

A question has been raised by several investigators about  the
stability of fluorescing components in oils exposed to  environ-
mental conditions, especially sunlight.  A preliminary  evaluation
of the reliability of fluorescence spectra for passive  tagging
oils was carried out by performing a limited weathering study.
Six oils  (No. 2, No. 4 and No. 6 Fuel Oils; and South Louisiana,
W. Texas Sour, and Bachaquero Crude oils) were placed as surface
slicks on ocean water, contained in wide mouth jars, and exposed
on a building roof for 30 days.  Similarly, four other  oils  (one
light Arabian ar^ four Persian Gulf Crude oils) were subjected
to outdoor weathering for 50 to 300 hours in 500 gallon tanks
provided with constantly circulating salt water drawn from Casco
Bay, Maine.  After the indicated weathering, the oils were re-
covered from the water surface and solutions were prepared
having concentrations of 1 and 10 mg/1 oil in solvent,  using
99 mol % rjure cyclohexane.  Solutions of the corresponding un-
weathered oils were prepared in a similar manner.  Solvent
selection was in accordance with AQC Newsletter #18, "Solvent
Impurities and Fluorescence Spectrometry".  Analyses were  per-
formed by exciting the solutions at 290, 320, 340 and 360  nm
and scanning each emission spectrum in the range of 220 to 600
rim.  The resulting spectral envelopes, which are used for  passive
tagging oils, were found to retain their characteristic shapes
despite extensive weathering, although their intensities were
slightly reduced.

    (U.  Frank; FTS 202-548-3510,  Coml. 202-548-S347)

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 Analytical Quality Control Newsletter (U.S. EPA), No.  21, April 1974
 AN  IMPROVED  SOLVENT FOR FLUORESCENCE ANALYSES OFViOILS
                                                  
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  Analytical Quality Control Newsletter (U.S. EPA), No.22, July 1974
     Effect of Fluorescence Quenching on DM  I dent If I .cat Ipn

Questions  have  been  raised  by several  Investigators  about  the
effects of  quenching  on  fluorescence  spectra  when   used  for
passively   tagging   oils.    Quenching   as   defined   for  the
fluorescence analysis of oil, occurs at high   concentrations  and
Is  characterized  by supresslon of the oil's  emission  radiation.
The exact concentrations at which quenching becomes apparent  was
determined  for four oils.  Solutions having  known concentrations
of the oils In 99 Mol % pure cyclohexane were  prepared.   Emission
spectra were obtained In the range 220-600 nm, by exciting  at  290
and  31*0  nm.   Graphs  were   then   constructed   by    plotting
fluorescence  Intensities  versus  concentrations.  The  points at
which the plots began to deviate from linearity marked  the  "onset
of  quenching."  Those  points  corresponded   to  the    following
concentrations:

     01 L              CONCENTRATION OF "ONSET  OF QUENCHING"
                                       mg/1

                            Ex 290 nm          E.x 340 nm

#2 Fuel  Oil                     30             No Emission
#6 Fuel  Oil                      6                   7
Bachaquero Crude                 6                  18
Iran - Gach Crude               16                  25

By  Inspection  of the spectral  envelopes  It was found  that above
the onset of quenching the shapes of the  envelopes  varied with
concentratIon/  while  below  the  onset (region of llnarlty)  the
shapes remained constant.   !n addition,  at  concentrations   above
100   mg/1   the   envelopes   were  sometimes  distorted   beyond
recognition.  It Is therefore vital  to prepare oil solutions with
concentrations  below  the  onset  of  quenching  when  passively
tagging   oils by the fluorescence technique.    (U, Frank.  FTS 201-
5U8-3510,  Coml .  201-5U-33U7)

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Analytical Quality Control Newsletter (U.S.  EPA), No.24,  January 1975
 IDENTIFICATION  OF  PETROLEUM  OILS  BY  FLUORESCENCE SPECTROSCDPY

A simple and  rapid method  for  fingerprinting  petroleum oils by
 taking advantage of  the  three  dimensional  character   of  their
fluorescence    spectra   has   been   developed.    Our   approach
 involves excitation  of  the oils   at   15   wavelengths,   in  the
range  of   220-500   nanometers  (nm),  at  20 nm  intervals.  The
emission monochromator  is  rapidly scanned at  each   excitation
wavelength  to  obtain  the emission  maximum.  These maxima  are
then plotted  versus  the  excitation  wavelengths   to  derive
"silhouette profiles", which are  used  to  fingerprint  the oils.
We  tested  our  method by matching  weathered with unweathered
portions of nine  petroleum  oils,   and   discriminating  among
them.    Each  oil  yielded a unique  and different profile that
remained  substantially  unchanged   despite  weathering.    (U
Frank,  FTS 201-5U8-3510, Coml. 201-51*8-33^7)

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 Analytical Quality Control Newsletter (U.S. EPA), No. 31, October 1976
      Synchronous Excitation  Fluorescence Spectroscopy

A preliminary evaluation of the utility of synchronous excitation
fluorescence spectroscopy for the quantitative determination  of
petroleuai oil and phenol has demonstrated that by the use  of
this technique Rayleigh-Tyndall and Raman scatter interferences
are totally eliminated.  These scatter interferences, which
are usually encountered in emission spectra obtained by  con-
ventional constant wavelength excitation, techniques, were  pre-
viously described in AQC Newsletter No. 13.  Since the scatter
interfer^nces are avoided by synchronous excitation, this  tech-
nique offers two advantages over constant wavelength excitation:
(1) shorter analysis time because solvent blank determinations
and corrections are not necessary, and (2)  increased sensitivity
because spectral interferences are avoided.  In our study  we
compared synchronous excitation spectra to emission spectra of
oil and phenol solutions, and of appropriate solvent blanks.
Synchronous excitation spectra were obtained by simultaneously
scanning the excitation and emission monochromators with the
emission monochromator leading by a 20 nm interval.  Emission
spectra were obtained at 290 nm for oil and 270 nm for phenol.
(U. Frank, FTS 342-7510, Coml 201-548-3347/L. Perneli, FTS
342-7517  Coml 201-548-3347)

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  Analytical Quality Control Newsletter (U.S. EPA), No.32, January 1977
      Synchronous  Excitation  Fluoregcence Spectroscopy

Use o t" synchronous excitation fluorescence spectroscopy for the
quantification  of  petroleum oils  and phenol was previously de-
scribed in AQC  Newsletter  No.  31,  October 1976.  Use of this tech-
nique has now been extended to aniline,  benze.ne,  benzolic acid,
cresol, ethyl benzene,  naphthalene,  phenol, toluene, and xylene.
These materials are part of more  than 300 toxicants that may be
designated "hazardous"  by  anticipated legislation (40 CFR Part 116)
Detection limits obtained  by  the  synchronous excitation tech-
nique varied between  0.5 racg/1 (ppb)  -  0.1 mg/1 (ppm),  using
100 ml cyclohexane for  extracting  1  liter water.   A preliminary
evaluation of interference by commonly  present materials in
brackish lake and  marsh waters, was  also performed.   No inter-
ference was found.   (U. frank,  FTS 34G-6626, Coml.  201-321-6626/
M. Gruenfeld, FTS  340-6625, Coml.  201-321-6G25)

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              Reprinted  from:   Proceedings  1975  Conference  on Prevention and  Control
              of  Oil  Pollution,  March  25  -  27,  1975,  San Francisco, API, Wash.,  DC
                     IDENTIFICATION  OF  PETROLEUM  OILS
                      BY  FLUORESCENCE  SPECTROSCOPY
                                                      Uwe Frank
                                      Industrial Waste Treatment Research Laboratory
                                           U.S. Environmental Protection Agency
                                                  Edison, New Jersey
ABSTRACT

   A simple and rapid method for the identification of weathered
petroleum  oils (passive tagging) by fluorescence spectroscopy is
described.  The  approach  used takes  advantage of the  three-
dimensional character of the oil fluorescence spectra. Oil identifica-
tion methods of other investigators that use fluorescence spectro-
scopy are also reviewed within the context of the three-dimensional
system. Our  method involves excitation of the oils at  15 wave-
lengths, between 220-500 nanometers (nm), at 20-nm intervals. The
emission  monochromator is  rapidly scanned at each excitation
wavelength to obtain an emission spectrum. The maximum emission
intensities  are then plotted versus the^ excitation  wavelengths  to
derive silhouette profiles. These are used as fingerprints for passive
tagging petroleum oils. The influence of weathering, quenching, and
solvent effects on our method are also examined.
INTRODUCTION

   The discharge of petroleum products into the marine environ-
ment has caused extensive environmental damage in the past. The
current increasing transportation and offshore production of petro-
leum is,  therefore, of vital  concern to the U.S.  Environmental
Protection Agency. Effective legislation, with adequate analytical
support for enforcement, should reduce this damage, and method-
ology for  the identification of the source of discharged oil is needed.
   Fluorescence spectroscopy is a rapid and promising tool for the
source identification of weathered oil (passive tagging). While in the
past  few  years many  laboratories have  used this tool for passive
tagging both  crude and refined petroleum products, few innovations
in the technique have come about. Although three  parameters are
inherent to the fluorescence technique, only two of them have been
commonly used. This can be explained more clearly within  the
context of a three-dimensional system. Because fluorescence spec-
troscopy  entails three  parameters (excitation wavelength, emission
wavelength, and fluorescence intensity), the total spectrum of an oil
can be presented as a topographical map of a mountainous region.
Figure 1 depicts the total fluorescence spectrum of an oil with three
fluorescence  maxima;  the excitation wavelength, emission wave-
length, and fluorescence intensity represent  the x, y,  and z axes,
respectively.
   Most publications on the  identification of oils by  fluorescence
spectroscopy do not mention  the three-dimensional character of oil
fluorescence spectra. Their authors  limit themselves by using only
two of the  three  parameters. Within the  context of the three-
dimensional system, the  approach  of these investigators can be
described  as  taking "cuts" through  figure  1 in planes that  are
parallel to the y axis and at specific points along the x axis. Figure 2
illustrates  this; each cut is an emission spectrum at  one excitation
wavelength.
Figure 1.  Three-dimensional  presentation of the total fluorescence
spectrum of an oil

   Thus, Thruston and Knight  [1] utilize 340 nanometers (nm) as
the excitation wavelength for oil solutions in cyclohexane at three
concentration levels.  They then ratio  the 386-nm  and 440-nm
emission maxima to one another. A similar approach is taken by
Coakley [2]  who  excites each  oil at a discrete wavelength.  He
selects  for each oil the excitation  wavelength that  yields the
maximum emission for  that  oil. His wavelengths are usually in the
range 290-320 nm. Jadamec [3], in a more recent method, uses 254
nm as the excitation wavelength.  He generates emission spectra for
passive tagging 8 oil spill samples.
   All of these methods utilize the same basic approach. The oils
are excited at specific wavelengths and their  emission  spectra  are
obtained.  The methods differ only in their wavelength of excitation.
   Other fluorescence methods are available or have been proposed
for passive tagging oils, but  their utility for this purpose has not
been established. Lloyd [4,5]  describes a method whereby cross-
sectional  cuts are taken at 45 angular planes to the  x  and y axes.
This is accomplished by simultaneously scanning the excitation and
emission monochromators of the fluorescence instrument. Another
method by  Freegarde et al. [6]  proposes the analysis of oils by
constructing contour maps. While this approach somewhat simulates
a  three-dimensional system, it is extremely time  consuming and
requires computer services for data manipulation.
                                                         87

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88
CONFERENCE ON PREVENTION AND CONTROL OF OIL POLLUTION
                                                                                                 EMISSION
                                                                                             Z   INTENSITY
                                                   EXCITATION
                                                  WAVELENGTHS
     EMISSION
   WAVELENGTHS
 Figure 2. Three-dimensional presentation of the emission spectra of
 the oil in figure 1 excited at three wavelengths

    Our method circumvents  these limitations; sample analysis and
 data handling are rapid and commensurate with the  other com-
 monly  used fluorescence techniques  [1,2,3,].  We  use  all  three
 fluorescence parameters, and thereby exploit the advantages that
 derive from the three-dimensional system that is illustrated in figure
 1. We obtain in our method a silhouette  profile of an oil's total
 three-dimensional spectrum as it is observed in a plane that parallels
 the x axis. Figure 3 illustrates how a simplified silhouette profile of
 an  oil  is obtained  from the  three-dimensional  spectrum that  is
 illustrated in figure 1. In actual practice, we excite each oil at 20-nm
 wavelength  intervals between 220 nm and  500  nm. The  emission
 monochromator is rapidly scanned  at each excitation  wavelength,
 and the maximum emission intensity of each scan is recorded. These
 maximum intensities are then plotted manually versus the excitation
 wavelengths, and the silhouette profiles of the oils are obtained by
 connecting the points with straight lines.
   We evaluated the ability of the method to identify weathered
 oils, i.e., to correlate weathered oils with unweathered portions of
 the same oils (passive tagging). Pertinent environmental factors such
 as photodecomposition, evaporation,  dissolution, and  biodegrada-
 tion were considered separately and in combination. Quenching and
 solvent effects that may influence the accuracy of our method were
 also examined.

 Weathering effects

   Previous investigators indicate, that weathering may drastically
 degrade petroleum oils. Thiuston and  Knight [1], Coakley  [2], and
 Freegarde  et  al.  [6]   surmised  that significant  changes in the
 intensity and shape of the emission spectrum can occur when oils
 aie exposed to sunlight. Two studies  were  therefore conducted in
 order to establish our method's ability to cope with weathering
 effects. The first study dealt primarily with photodecomposition
 effects, and the  second study  examined the combined effects of
 water, radiation, heat, and bacteria.

 Quenching effects

   Thniston and Knight [1] found  that the intensity and  shape of
oil fluorescence spectra substantially depend on the concentrations
of the solutions  that  are measured.  Fluorescence quenching is a
phenomenon that occurs at high solution concentrations and  is
                                                                                                               ^ SILHOUETTE PtOMLE


                                                                                                               	EMISSION SPECTRA
                                                                                                                    EXCITATION
                                                                                                                   WAVELENGTHS
                                                             EMISSION
                                                           WAVELENGTHS
                                                          Figure  3.  Three-dimensional  presentation of the derivation of the
                                                          silhouette profile from the oil :n figure 1

                                                          characterized by the formation of excimers. These are combinations
                                                          of excited  molecules. Excimer formation  and  their  effect on
                                                          fluorescence spectra was first noted by Forster and Kasper [7], who
                                                          demonstrated that while a polynuclear  aromatic compound (PNA)
                                                          yielded a fluorescence emission maximum at 390 nm, a hundredfold
                                                          increase in the  concentration of this compound shifted its fluores-
                                                          cence maximum to 480 nm. Other PNAs, exhibit  similar behavior.
                                                          Because other investigators [8,9]  have demonstrated that petroleum
                                                          oil fluorescence is primarily attributable to PNAs, we maintain that
                                                          the distortion of high concentration oil fluorescence spectra is due
                                                          to excimer formation.
                                                             Excimer  formation is considered  important  to our  method
                                                          because its effects can combine  with or can be confused with the
                                                          effects of  weathering. Also,  losses of volatiles by   spilled  oils
                                                          invariably result  in  increased  concentrations of  PNAs in  the
                                                          weathered residues; the PNAs are higher boiling  materials that do
                                                          not readily  volatilize.  As  a  consequence, excimer formation  can
                                                          become quite prominent  and can cause ambiguities during passive
                                                          tagging analyses. Therefore, the oil solution concentrations at which
                                                          substantial quenching appears  must be  known before  our method
                                                          can be successfully used.

                                                          Solvent effects

                                                             Cyclohexane, our solvent of choice, presents two problems: (1)
                                                          interference by Raman Scatter, and (2)  interfering emissions by sol-
                                                          vent impurities. Raman scatter is caused by the  interaction of ex-
                                                          citation radiation with the bonds of the cyclohexane molecules. It
                                                          reveals itself as an emission band that appears  0.3 microns""1 from
                                                          the Rayleigh band in cyclohexane [10]. Also, impurities in  cyclo-
                                                          hexane yield spectra that can coincide with those of oils. The effects
                                                          of these interferences on our method  are also  examined in this
                                                          paper.

                                                          Experimental

                                                             Apparatus. A Perkin-Elmer model MPF-3 Fluorescence  Spectre-
                                                          photometer,1 with a constant temperature cell bath (P.E.  Part No.
                                                          220-1419) was  used. The cell (10 mm pathlength, quartz;  P.E. Part
                                                          No. 990-2711) was maintained at 20   0.5C.

                                                          'Mention of  trade  names  or commercial  products does  not
                                                           constitute endorsement by the U.S. Government.

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                                                                                                        MONITORING
                                                            89
   Procedure. The silhouette profile of a typical oil was obtained in
the following manner:
   A stock  solution  with an  accurately  known  concentration
approximating  1000  mg/1 oil  in  cyclohexane was  prepared.  An
aliquot  of  this  solution  was  then  diluted  to obtain  a final
concentration of 5 mg/1. This was measured in  a cell  which was
placed into  the constant temperature cell holder for a five-minute
equilibration period.  The  analysis was  performed at  a  constant
temperature of 20 C.
   In order  to prevent the recorder pen from going off scale during
a run, the excitation wavelength that yielded the maximum emission
resonse  was  determined. This was performed by manually scanning
the excitation and emission monochromators until a maximum pen
deflection was obtained. Next, the instrument settings were adjusted
to  yield  ca.  90% recorder scale deflection  at this  excitation
wavelength.
   The  oil was then excited at  15  wavelengths, 20 nm apart, in the
range 220 to 500  nm  (i.e. 220, 240, 260, , 480, 500 nm), to
obtain  the   maximum  emission  at  each wavelength.  This  was
accomplished by  setting  the excitation  monochromator at each
excitation  wavelength  and rapidly  scanning the emission mon-
ochiomator over  the  entire fluorescence spectral region of the  oil.
Fifteen  compressed emission spectra  were  thereby  obtained.  A
cyclohexane "blank"  was similarly analyzed by exciting at  the 15
wavelengths to determine its contributions to the oil spectra. None
were found at the instrument settings used.
   The  silhouette profiles were prepared by manually plotting  the
maximum emission intensity of each spectrum versus its excitation
wavelength  and then connecting these points  with  straight lines.
Two criteria were used to compare  one profile with another:  (1)
shape, and (2) the excitation wavelength of the maximum emission
intensity.

   Weathering effects. Photodecomposition effects were examined
by placing four oils (No. 2 and No. 6 fuel oil; South Louisiana and
Bachaquero crude  oil)  as  surface  slicks  in wide-mouth, quart-size
jars, containing ocean water. The jars were exposed to the elements
on a building roof during the entire month of August.
   The other environmental effects were examined  by subjecting
five other crude oils to weathering conditions which simulate  typical
oil spill incidents.  The weathering was performed by the Depart-
ment of Environmental Protection, State of Maine, at the facilities
of the TRIGOM laboratory, South Portland, Maine, as part of a U.S.
Environmental Protection Agency  (EPA) sponsored grant. The oils
were  weathered  by  spilling  one pint  of oil on  salt water  in
500-gallon fiberglass tanks. Seawater drawn from Casco Bay, Maine,
was  constantly circulated through the tanks whose geometry was
such that the outlets were located at the bottom and the inlets at
the top.  All  weathering  was performed under  ambient outdoor
conditions,  during  the  summer months,  and for periods of 7-14
days. The weathered oils  were skimmed from the water surface,
dissolved  in cyclohexane, and centrifuged  in order to separate  the
oil from paiticulate matter and residual water. The solutions were
then decanted, and the solvent  stripped off with the aid  of an air
stream  at room temperature. Silhouette  profiles  of the weathered
and unweathered oils were then prepared and compared.

   Quenching effects The concentration at which quenching phe-
nomena became apparent was determined for four  representative
crude and refined oils. A No. 2 fuel oil (a low-viscosity distillate), a
No. 6 fuel oil (a high-viscosity residual), a South Louisiana crude oil
(a low-viscosity  crude), and a Bachaquero crude oil (a  medium-
viscosity crude) were examined.  Solutions having accurately-known
concentrations of these oils in cyclohexane were prepared. Emission
spectra  in the range 220-600 nm were obtained by exciting  at 290
nm and 340 nm, and fluorescence intensity versus  concentration
plots were prepared. The maximum concentration within the linear
region of each plot  was designated  at the onset of quenching  (figure
4).

   Solvent effects. The emission spectra of ten oils were examined
in order to demonstrate the effects of solvent Raman scatter on the
fluorescence spectra of the oils. Solutions having concentrations of
1  mg/1 and 10 mg/1 oil in  cyclohexane were excited at 290 and 340
nm. A cyclohexane blank measurement was obtained concurrently
 z
 o
     70
     60
     SO
     40
     30
     20
     10
                        I
                         \_
ONSET OF

QUENCHING
              
             10   20   30    40   SO   60   70

                   CONCENTRATION (ng/1)

Figure 4.  Emission intensity versus concentration plot of No. 2 fuel
oil  in  cyclohexane demonstrating the onset of quenching.   The
maximum concentration within  the  linear region is  30 mg/liter


with each oil measurement using the same instrument settings. The
emission spectra  of  two solvents  (Fisher Scientific Co., Spectro-
analyzed and 99  Mol % Pure  cyclohexanes) were also checked for
the presence of fluorescing impurities.

Results and discussion

   Weathering  effects.  The criteria that are used  to judge  the
efficacy of a method for passive tagging oils are:  (1) whether the
method can correctly correlate weathered and unweathered portions
of the same oils,  and (2) whether it  can  distinguish between oils.
Our first weathering study, i.e., the study of photodecomposition
effects, demonstrates that the  profiles of four oils remain relatively
unchanged despite weathering  (figure  5) and  that  the method
distinguishes  between the  oils (figure 6). Four discrete silhouette
shapes and excitation wavelength maxima were obtained, yet the
profiles of the weathered and unweathered portions of each oil were
quite  similar.  We therefore conclude that  our  method not  only
discriminates between  oils but also that exposure of the oils to
sunlight did not adversely affect our method.
   The results  of our second  weathering  study, i.e.,  the study of
other  environmental effects, also support the contention that our
method is adequate for passive tagging oils.  The silhouette profiles
of the weathered and unweathered portions of five crude oils are
shown in  figure 7. Each weathered oil  is again uniquely  matched
with its unweathered counterpart.

   Quenching  effects.  The optimum  solution concentration for
fluorescence  measurement  was found to be 5 mg/1  oil in cyclo-
hexane. Table 1 illustrates  our reasons for selecting this concentra-
tion by listing the concentrations of the oils at which the onset of
quenching  occurs (figure  4). Oil  solutions  that exceeded  this

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90      CONFERENCE ON  PREVENTION AND CONTROL OF OIL POLLUTION
   100


    90


    80



 f  7
 l/l
 |  60


 2  50
 C/)
 to

 S  40


    30


    20


    10
          NUMBER 2 FUEL
                                                	 UNWEATHERED OIL
                                                	 WEATHERED OIL

                                              NUMBER 6 FUEL
                                                                          S. LOUISIANA CRUDE
                                                                                                            BACHAQUERO CRUDE
                               _lJ    I
     240      320       400       480    240      320       400      480     240      320       400      480     240      320     400      480

                                                       EXCITATION WAVELENGTH (rim)

Figure 5. Silhouette profiles of weathered and unweathered portions of four crude and processed oils
                                                 NUMBER 2 FUEL OIL

                                                 S.LOUISIANA CRUDE

                                                 NUMBER 6 FUEL OIL

                                                 BACHAQUERO CRUDE
      240      260      320      360     400     440      480      520
                          EXCITATION WAVELENGTH (nm)

Figure 6.  Overlay of the silhouette profiles of the unweathered oils
in figure 5


concentration yielded fluorescence spectral envelopes that changed
with solution concentration. The spectral envelopes of less concen-
trated solutions remained  constant despite concentration changes.
                                                                     We, therefore, suggest that it is vital to  use solution concentrations
                                                                     that are within the linear portion  of  figure 4  to avoid  spectral
                                                                     distortion by quenching effects.

                                                                        Solvent  effects.  Raman  scatter  can  be  quite prominent in
                                                                     cyclohexane  at concentrations below 10 mg/1  oil in solvent.  Its
                                                                     impact depends on the excitation wavelength that is used and on
                                                                     the fluorescence efficiency (emission intensity)  of the oil. Typical
                                                                     Raman  band interference  with  a spectrum of oil  is illustrated in
                                                                     figure 8. Two excitation  wavelengths are used,  and they produce
                                                                     different  effects.  This  figure  demonstrates the  importance  of
                                                                     measuring  neat  solvent  prior to oil analysis.  Fortunately, most
                                                                     petroleum  oils  have such  a  high  fluorescence efficiency that  this
                                                                     solvent  Raman  band  overlap does  not  handicap our  ability to
                                                                     achieve oil identification.
                                                                        The  presence of fluorescing impurities in  the solvent  is a factor
                                                                     that must also  be taken into account.  Two grades of cyclohexane
                                                                     were examined: Spectroanalyzed and 99  Mol % Pure cyclohexane
                                                                     (Fisher  Scientific Company).  Impurities in  the  Spectroanalyzed
                                                                     grade solvent yielded substantial interfering fluorescence envelopes,
                                                                     but  the 99 Mol % Pure solvent  was essentially free of these
                                                                     interferences. We  successfully purified  the Spectroanalyzed cyclo-
                                                                     hexane, however, by simple distillation [11].
                                                                     Summary

                                                                        We  have  described the  three-dimensional  character  of oil
                                                                     fluorescence spectra and reviewed within this context the pertinent
                                                                     work of previous investigators.  We have also presented a straight-
                                                                     forward method for identifying the source of discharged petroleum
                                                                     oil, i.e., correlating a weathered oil correctly with an unweathered
                                                                     portion  of  the  same oil (passive tagging). Our method utilizes the
                                                                     three-dimensional aspect of fluorescence spectra, and requires only
                                                                     simple  data manipulation. In  order to validate our method for
                                                                     passive tagging  oils, we tested its ability to match weathered with
                                                                     unweathered  portions  of  nine  petroleum oils and to discriminate
                                                                     among them. Each oil yielded a  unique and different profile in these
                                                                     tests,  and  the  profiles remained substantially unchanged  despite
                                                                     weathering.  We  also demonstrate that  some common phenomena
                                                                     such as fluorescence quenching, Raman  scatter, and solvent impuri-
                                                                     ties do not handicap our method.

-------
                                                                                                         MONITORING
                                                                                                 91
                                                           UWEATHERED OIL
                                                           WEATHERED OIL
              USD TRECO CRUDE
                                LIGHT ARABIAN CRUDE
                                                                                                               HEAVY I HAH I AH CRUDE
         240     32     40    480   240     320     400     480    Z40     320     400
                                                                                 240     320    00     480   240     320    400
                                                            EXCITATION WAVELENGTH nm
Figure 7. Silhouette profiles of weathered and unweathered protions of five crude oils
          Table 1. Concentrations of oil in cyclohexane
      at which the onset of quenching appears for 4 crude and
                          refined oils
       OIL




    Number 2 Fuel Oil

    Number 6 Fuel Oil

    Bachaquero Crude

    Iran   Gach Crude
CONCENTRATION   (mg/1)


  Ex    290  nm       Ex    340 nm
     30

      6

      6

     16
No Emission

     7

    18

    25
ACKNOWLEDGMENTS

   The author is  grateful  to Mi. Michael Gruenfeld, Supervisory
Chemist of this laboratory, for many helpful discussions and for his
assistance in preparing this  manuscript, and to Mr. Henry Jeleniew-
ski for the data acquisition that has made these findings possible.
REFERENCES

 1. Thruston, A.D., and Knight, R.W. 1971. Environmental science
      & technology, 5:64.
 2. Coakley, W.A. 1973. Proceedings of Joint Conference on the
      Prevention and  Control  of Oil  Spills,  p. 215. Washington,
      D.C.: American Petroleum Institute.
 3. Jadamec,  J.R. 1974.  Abstracts  of Pittsburgh Conference  on
      Analytical Chemistry  and Applied Spectroscopy. Cleveland,
      Ohio.
 4. Lloyd, J.B.F. 1971. / Forens. Sci. Soc.  11:83.
 5. Ibid., p. 153.
 6. Freegarde,  M.;  Hatchard,  C.G.;  and Parker,  C.A. 1971. Lab.
      Practice, 20(1):35.
 7. Forster, T., and Kasper, K. 1955. Z. Elektrochem. 59:976.
 8. McKay, J.F., and  Latham,  D.R. 1972. Analytical Chemistry
      13:2132.
                                                                                  EXCITATION AT 340nm
                                                                                                              EXCITATION AT 290nm
OIL
                                              340
                                                     380
                                                                         290  320
                                                             EMISSION WAVELENGTH
                                                                                   350
                                     Figure 8.   Raman band interference with the emission spectra of a
                                     La Rosa crude oil. The concentration 1  mg/l oil in 99  Mol % Pure
                                     cyclohexane is used


                                      9.  McKay, J.F., and  Latham,  D.R. 1973. Analytical Chemistry
                                           7:1050.
                                     10.  Parker, C.A.  1959. Analyst 83:446.
                                     11.  Frank, U. 1974. EPA  Analytical Quality Control Newsletter
                                           21:11.

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NO. 400
                              Presented  at  the
                     1977  Pittsburgh  Conference On
                       Analytical  Chemistry  And
                          Applied  Spectroscopy
                      February  28  - March  4,  1977
                               Cleveland,  Ohio
                     DETERMINATION OP PETROLEUM  OILS IN SEDIMENTS
                        BY FLUORESCENCE SPECTROSCOPY AND NMR
      U.  FRANK AND M. CRUENFELD, Oil 8 Hazardous Material! Spills Brnch,  Ind. Env.
         Res.  I.ab.-Ci, U.S. Environmental t'otection Agency, Edison,  N.  J.   08817
The use of several  fluorescence methods and  one  NMR procedure were evaluated  for monitor-
ing the presence  of petroleum oils in sediments.  Analyses were performed  on  sediments
from a mangrove swamp  in Puerto Rico that was  impacted by a major oil  spill in  1973, and
on sediments from a proximate area that had  no known history of petroleum  oil pollution.
The latter sediments served as control samples.
The following fluorescence techniques, that  are  believed to be most cCHiunonly  used for
oil spill source  identification, were examined:  (a) Single Wavelength  Excitation^^>^^;
(b) Synchronous Excitation^; and (c) Derived Silhouette Profiles".  These  techniques
are compared and  contrasted within the context of a three dimensional  system, using the
three interdependent variables that are inherent to fluorescence Spectroscopy,  i.e., ex-
citation wavelength (x), emission wavelength  (y) , and fluorescence intensity (z).  With-
in this context,  the fluorescence characteristics of petroleum oils are  presented as
"total fluorescence spectra" and the spectral  information that is obtained by each  tech-
nique is discussed  as  an appropriate portion of  such spectra.  Figure  1  shows a hypo-
thetical three dimensional total oil spectrum  (illustrated as mountains  by solid lines),
and the spectral  information that Is available.  This figure also highlights  the rather
limited information used by techniques (a)  and (b).  Technique (a) measures only the
slice of the total  spectrum that parallels  the y axis; technique (b) measures only  the
slice of the total  spectrum that lies at a  45   angle to the x and y axes.  Technique (c)
differs, however.   This method measures the  total spectrum's silhouette  profile as  pro-
jected onto the plane  bordered by the x and  z  axes.  Unlike methods (a)  and (b), silhou-
ette profiles of  method (c) incorporate the  essence of three dimensional fluorescence
spectra, within a  two  dimensional format. A comprehensive discussion  of method (c)  is
available in Reference 6.  In addition to Its  greater informational content,  this method
is rapid and easily used.  It was therefore  selected for the mangrove  sediment analyses.
Use of NMR spectroscopy for confirming the presence of petroleums in the mangrove sedi-
ments was also evaluated.  The NMR procedure is  based on the absorption  of aromatic
compounds in the  6.5-8.0 ppm spectral region,  relative to tetramethylsilane at 0 ppm;
substantially elevated levels of aromatics  in  sediments are thought to indicate the
presence of petroleums.  These absorptions were  observed in the spill  impacted sediments
but not in the'control sediments.  Figure 2  illustrates a composite section of the NMR
spectra of the contaminated sediment (upper),  and the control sediment (lower).  The
fluorescence and  the NMR data agree on the presence of petroleums in the spill impacted
sediments.
 FIGURE I
             	 Method (a)1'2'3'4
             	 Method (b)S
             	.	 Method (c)6
                                                  FIGURE 2
                                               10
                                                References:

                                                1.
                                                              8
                                                            P-P-
                                                                     Hatchard,  and
                                                                     Practice,  20  (1)
4.  J.R.
5.  P  J
6.  II. F
    CA. ,
 Jndamec
ohn,  and
rank,  Pr
 A.P.I. ,
                                   M. Freegard, C.G.
                                   C.A. Parker, Lab.
                                   35-40 (1971)
                                   A.D. Thruston and  R.W.  Knight, Env.
                                   Sci . and Tech.,  5_,  64  (1971)
                                   W.A. Coakley, Proc.  of  Joint Conf.
                                   on Prevent. 6 Contr. of Oil Spills,
                                   Nash., D.C., A.P.I., 215  (1973)
 and T.J. Porro, Abstract Pittsburgh Conference  (1974)
 I. Soutar, Anal. Chem. ,  4^,  520  (1976)
ic. of Joint Conf. on Prevent.  S  Contr. of Oil Spills,  San Pranclsco,
 87 (1975)

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Reprinted  from:  Proceedings  1977  Oil  Spill Conference,  March 8-10
1977,  New  Orleans,  API,  Wash.,  DC
             A  REVIEW  OF  SOME  COMMONLY  USED PARAMETERS
                     FOR  THE DETERMINATION  OF  OIL  POLLUTION
                                                  Michael Gruenfeld and Uwe Frank
                                              Oil and Hazardous Materials Spills Branch
                                      Industrial Environmental Research Laboratory-Cincinnati
                                                U.S. Environmental Protection Agency
                                                      Edison, New Jersey 08817
      ABSTRACT

        A state-of-the-art review is provided describing specific parameters of
      petroleum oils that are used b\  various investigators to demonstrate the
      presence of oil pollution in water, sediments, and biological tissues. Several
      representative publications are discussed with regard to the techniques used
      for distinguishing between petroleum h\drocarbons and organics that are of
      recent biological origin. The techniques include chromatographic proce-
      dures using alumina and silica gel for separating hydrocarbons from other
      organics, followed by instrumental methods such as gas chromatography,
      fluorescence spectroscopy. ultraviolet absorption specfroscopy, et al. The
      various oil parameters thai are used to demonstrate the presence  of petro-
      leum oils are discussed, and the  most effective ones are recommended. In
      addition, a recent studv is also described in which several of the parameters
      were used to demonstrate the presence of oil pollution in sediments from a
      mangrove swamp In Puerto Rico.
      INTRODUCTION

        Petroleum oils that are spilled or otherwise discharged into the aqueous
      environment migrate in all directions. Spilled oil floats on water and is
      carried onto shorelines. Oil also migrates downward through the water
      column and contacts fish,  benthic sediments, animals and plants.  Several
      kinds of chemical analyses are commonly performed to monitor the presence
      of oil pollution. "Fingerprinting" methods are used to locate the source of
      oil discharge. Methods of quantitation are used  to measure dispersed oil
      levels in water, and oil levels that are incorporated by benthic sediments and
      tissues of animals and plants. While most fingerprinting methods require
      gram quantities of oil and are therefore restricted to oil rich environmental
      samples  such as surface slicks and shoreline residues, the quantitation
      methods are used to measure ppm (mg/kg) or smaller amounts of oil. These
      methods are emphasized in the following discussion. Descriptions of highly
      satisfactory fingerprinting  methods are available, however.2
        Although hydrocarbons are the major components of petroleum oils, they
      are also  produced  by living marine organisms. Methods for measuring
      petroleums in sediments, plants and animals, and methods for measuring
      sub-ppm  levels of oils in water consequently select characteristic petroleum
      oil parameters that differentiate between recent biologically produced hy-
      drocarbons and petroleum derived hydrocarbons. The following discussion
      reviews some of these parameters and illustrates their use for confirming the
      presence  of petroleum oil  in samples.
      Unresolved complex mixture

        Petroleum is an extremely complex mixture of thousands of different
      hydrocarbons and related compounds. Hydrocarbons, according to Ander-
      son, et at.1, are the most numerous and abundant organic compounds
comprising crude oils and refined petroleum products. Comprehensive dis-
cussions of petroleum oil composition are available in the preceding refer-
ence, and in reports by Zafiriou,  el al..20  Bieri, et al.,3 and the U.S.
Department of Commerce."
  When injected into a gas chromatograph (GC), petroleum oils exhibit an
inverted "cup and saucer effect" (Figure 1). This description of a rather
characteristic feature of GC profiles of petroleum oils was obtained from the
Department of Commerce  report. This report also describes this configura-
tion as a "smear of unresolved hydrocarbons", Zafiriou, et al.'l call it an
"unresolved envelope", while Farrington and Medeiros" describe it as due
to an "unresolved complex mixture (UCM)." The presence of this configu-
ration results from the inability of gas chromatographic methods to separate
all petroleum oil components from one another. According to Blumer and
Sass,J this unresolved envelope is characteristic of the  homologous and
isomeric hydrocarbons in fossil fuels. According to Clark and Finley,"'8 the
presence of this  large unresolved envelope below discrete n-paraffin peaks
(discussed below) strongly suggests the presence of petroleum oils in  en-
vironmental samples.
  Environmental samples that contain only recently biologically produced
hydrocarbons exhibit less complex chromatograms. According to Ander-
son, et al.', living organisms use  rather specific biosynthetic pathways
which favor the  production of hydrocarbons in preferred size ranges. Such
chromatograms exhibit a few exceptionally large peaks and often show some
degree of baseline resolution.
n-alkane homologs

  Crude oils  and most refined petroleum products contain a homologous
series of n-alkanes. These appear as discrete peaks above the unresolved
envelope of the GC profiles of oils (Figure 2). According to Zafiriou, etal*
n-alkanes from Ci-Ceo are present in petroleum oils with adjacent members
occurring in similar quantities. Zafiriou contrasts this with the distribution of
n-alkanes in petroleum-free plants, organisms, recent sediments, and shales,
all of which  yield GC profiles showing only a  limited range  of normal
                 BACHAQUERO CRUDE OIL
                -INCREASING TEMPERATURE
                                                BEGIN TEMP.
                                                 BASELINE
Figure 1. Gas chromatogram of a Bachaquero crude oil exhibiting the
presence of an unresolved complex mixture
                                                                  487

-------
488
1977 OIL SPILL CONFERENCE
                                               DEFINITION  OF  TERMS
                                                                                               PROGRAM  START
                                                   INCREASING  TEMPERATURE
                                                                                                      INJECTION
             Figure 2. Gas chromatogram of a No. 2 fuel oil exhibiting the presence of n-alkane homologs, and the isoprenoids
                                                           pristane and phytane
 alkanes (mostly C-is-C-js), and a strong predominance of odd-numbered
 over even-numbered compounds. The exceptionally broad distribution of
 n-alkanes (Ci-Cso)  in crude oils and most refined products is  also em-
 phasized by Anderson, el at.' These authors find a distinct predominance of
 odd carbon number versus even carbon number n-alkanes in petroleum-free
 environmental samples (i.e., samples containing only recent biologically
 produced hydrocarbons). While odd-to-even n-alkane carbon number ratios
 in  petroleum oils are approximately  I.O, GC profiles of environmental
 samples containing only recently produced  hydrocarbons  often show  a
 strong predominance of one or two n-alkanes over all others. Use of GC
 profiles of environmental samples to demonstrate petroleum incorporation is
 also suggested by Clark and Fmley.' According to these authors, the first
 indication of petroleum uptake is often seen by the presence of a large
 unresolved envelope below the n-alkane peaks, and  by  the presence of
 n-alkane peaks in the range Cr2-C22 which have the same  order of mag-
 nitude. The n-alkanes are quite susceptible to microbial catabolism. and may
 not appear in GC profiles of substantially weathered oils. Such chromato-
 grams should retain their characteristic inverted cup and saucer appearance.
 however.
 Isoprenoids

   Gas chromatograms of petroleum oils often contain major peaks that
 appear in addition to those of n-alkanes. Two isoprenoids. pristane (2. 6. 10,
 14-tetrame'hylpentadecane) and phytane (2.  6,  10,  14-tetramethylhexa-
 decane) are of particular interest in evaluating the presence of petroleums in
 samples.  When relatively  non-polar stationary  phases are  used  (e g..
 Apiezon L. OV-1, O V- IOI, or SE-30), pristane and phytane yield GC peaks
 that adjoin and are  usually only partially separated from the Ci? and Ci
 u-alkane peaks (Figure 2) This characteristic four peak configuration is
 common to many oils and indicates the likely incorporation of petroleums by
 marine environmental samples. Pristane  is commonly  produced by marine
 organisms, however, and consequently does not confirm petroleum incorpo-
 ration. But. according to Anderson. I'lul.' and Ehrhardt and Hememann."1
 phytane has not been found as a natural component of marine organisms. Its
 presence in samples therefore suggests oil incorporation.
 Fluorescing components

   Aromatic hydrocarbons are abundant in most crude and refined petroleum
 products, and their presence in marine samples is often thought to indicate
                                                            petroleum  incorporation.  For example.  Margrave and  Phillips15  use  the
                                                            presence of triaromatic and greater substituted  aromatic compounds as a
                                                            useful indication of petroleum pollution. The U.S.  Department of Com-
                                                            merce19 report emphasizes that aromatic hydrocarbons in marine waters are
                                                            currently believed  to be from petroleum sources.  Similarly,  Brown and
                                                            Huffman7 cite evidence suggesting that marine  organisms do not  produce
                                                            aromatic hydrocarbon mixtures.  Conversely, Blumer  and  Youngblood6
                                                            describe forest fires and prairie fires as a likely source of many polynuclear
                                                            aromatic hydrocarbons in marine  sediments.  Similarly, Anderson, ci a/.'
                                                            indicate that although aromatic hydrocarbons have not been isolated from
                                                            plankton, they may be produced by some marine organisms. Despite these
                                                            possibilities, elevated levels  of  polynuclear aromatic  hydrocarbons  in
                                                            marine samples are widely thought to indicate the presence of petroleums.
                                                              Several publications describe methods for measuring aromatic hydrocar-
                                                            bons in  marine  samples  Fluorescence  spectroscopy  is the most highly
                                                            recommended technique.  According  to Gordon.   <;  /.''  fluorescence
                                                            methods are rapid, sensitive, and  simple. While  these authors believe
                                                            fluorescing materials to be aromatics. they describe the uncertainty of actual
                                                            compound  identity as a major drawback. Gordon and Keizer1'1  describe
                                                            several fluorescence methods for measuring aromatics  in water,  while
                                                            Zitko,21 and Hargrave and Phillips1'' provide some useful fluorescence data
                                                            describing  petroleum  incorporation  by  animals and  sediments.  Several
                                                            fluorescence spectra of oils are illustrated in  Figure 3.
                                                                                                   FOSTERTON CRUDE

                                                                                                   VENEZUELAN BUNKER C U0.33)
                                                                                                   .REDWATER CRUDE
                                                                                                   KUWAIT CRUDE
                                                                                                   GUANIPA CRUDE
                                                                                                   PEMBINA AND
                                                                                                   LEDUC CRUDE
                                                                       340        380       420

                                                                           EMISSSION WAVELENGTH (nm)
                                                             Figure 3. Fluorescence spectra of several petroleum oils13

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                                                                              OIL SPILL BEHAVIOR AND EFFECTS
                                                                                     489
Naphthalenes and substituted naphthalenes

  The toxicity of higher boiling aromatics such as napthalenes, to fish, is
well established according to Anderson, el al.'  In fact, these authors feel
that the toxicity of naphthalenes to fish may even exceed that of lower boiling
aromatics such as benzene, toluene, and xylene. Naphthalenes are  more
soluble in water than most other high boiling aromatic hydrocarbons, and
consequently have a greater tendency to migrate from  petroleum oils into
marine waters, sediments, and animals.  Discussions of these properties and
of the unique uptake and depuration characteristics of naphthalenes, and
their known toxicity to marine organisms are presented by Rossi, el ill.. "
and also in several other papers by Anderson that are cited by these authors
   Naphthalene, methyl naphthalene, and dimethyl naphthalene, according
to the preceding authors, are the major high-boiling aromatic hydrocarbons
that transfer from oils into the water  column  and consequently  contact
marine organisms. In addition, napthalenes are readily absorbed by  the
organisms, but only slowly depurated. As a consequence of their enhanced
availability, high toxicity, rapid uptake and slow depuration, napthalenes
are often viewed with special interest during  oil pollution  studies. These
aromatics are apparently not produced by marine organisms, and con-
sequently serve to indicate petroleum incorporation. Naphthalenes are read-
ily measurable,  using the ultraviolet spectroscopic method  in Neff and
Anderson."
 Use of parameters

   The preceding discussion addressed several readily measurable and com-
 monly used parameters for monitoring and confirming the presence  of
 petroleum oils in waters, sediments and animals of the marine environment.
 Oil  pollution is indicated whenever samples from oil  spill or discharge
 impacted areas  are found to contain petroleums, while samples from proxi-
 mate non-impacted  areas  do  not contain  oil.  Several  parameters  are
 suggested for monitoring the presence of petroleum oils in marine environ-
 mental samples:
   I. gas chromatograms exhibiting the presence of an inverted "cup and
      saucer eff'ect"-unresolved complex mixture
   2. gas chromatograms  exhibiting the  presence of n-alkane homologs
      covering  a  broad range of molecular weights, and  with adjoining
      n-alkane  peaks of essentially equal size
   3. gas chromatograms exhibiting the presence of the isoprenoid phytane,
      most often as a partially resolved peak adjoining the n-Ci alkanepeak
   4. the occurrence of substantial fluorescence spectra, following excita-
      tion at specified wavelengths; aromatic components are thereby indi-
      cated
                          5.  the  presence of  napthalene,  methyl  naphthalene, and dimethyl
                             naphthalene; measurement can be accomplished by ultraviolet absorp-
                             tion spectroscopy
                          Measurement  of  these  parameters  is  usually  accomplished  after
                        chromatographic separation of hydrocarbons  from other organics, using
                        silica gel, alumina, or florisil chromatographic techniques. The occurrence
                        of any one of these parameters  may suffice to  establish the presence of
                        petroleum oils. Of course if more than one is found, then petroleum is shown
                        to be present with a higher degree of certainty.
                        Sample  analysis

                          Application of these parameters is illustrated by discussing a recent study
                        in which some of the parameters were used to establish the presence of oil
                        pollution in aquatic sediments from a mangrove swamp in Puerto Rico. The
                        area was impacted  by a major oil spill in 1973. General ecological aspects of
                        this project are discussed in greater detail by Nadeau  and  Bergquist."
                          Gas  chromatography (GLC), fluorescence spectroscopy,  and  nuclear
                        magnetic resonance (NMR) spectroscopy were used to analyze extracts of
                        sediments  from the spill site, and from an area having no known history of
                        petroleum  oil pollution.  Extractions were  performed by triturating 20 g
                        portions of air dried sediment with four  50 ml portions  of carbon tet-
                        rachloride. Separation  of  hydrocarbons from  other  organics was ac-
                        complished as  in  Blumer,  el  al.,5 using silica gel and  alumina  packed
                        columns. The eluates were evaporated to dryness, and the residues analyzed
                        by the following procedures.

                            GLC. The analysis was performed with a 50ft OV-101 support-coated
                          open tubular(SCOT) column, using a flame ionizationdetector(FID) and
                          a temperature program of 75-275C at 6/minute. Program activation was
                          initiated to coincide with solvent peak elution.

                            NMR. Spectra were obtained using a 60 Megahertz (MHz) instrument.
                          All peak field positions were referred to tetramethylsilane (TMS) at 0
                          ppm, using the  delta scale.

                            Fluorescence. Measurements were  made according to Frank,12 using
                           15 different excitation wavelengths between 220-500 nanometers (nm) at
                          20 nm intervals. This procedure differs somewhat from that illustrated in
                          Figure 3.
                          Analysis results are  summarized in  Figures 4-6.  GC profiles  of the
                        contaminated sediments  exhibit the typical inverted "cup  and  saucer ef-
                        fect"  This unresolved complex pattern does not appear in GC profiles of the
                        clean  sediments,  however (Figure 4).  The n-alkane  homologs and  the
                          40
  30                    20
RETENTION  TIME  (min.)
10
         Figure 4.  Gas chromatograms of sediment sample extracts: (upper line) sediment from an oil spill impacted area; (lower line)
                                                     sediment from a non-impacted area

-------
490
1977 OIL SPILL CONFERENCE
   80 -
 m
 i-
   60
 HI
 O
 r
                                                                                                                         IMS
                  *^u/K^y^i/^
     10
                                                                                                                           1  0
                                                               p.p.m.(5)
        Figure 6.  NMR spectra of sediment sample extracts: (upper) sediment from an oil spill impacted area; (lower) sediment from
        a non-impacted area; peaks between 0.5-2.0 ppm and 6.5-8.0 ppm are in absorption regions characteristic of aliphatic and aromatic
               hydrocarbons, respectively; the aromatic absorption region is emphasized by increasing instrument sensitivity

-------
                                                                              OIL SPILL BEHAVIOR AND EFFECTS
                                                             491
      in aquatic organisms. Proceedings of Join! Conference on Preven-
      tion  and Control of Oil Spills.  American Petroleum Institute,
      Washington, D.C.
 9  Clark, R. C. and J. S. Finley, [974, Analytical techniques for isolating
      and  quantifying  petroleum  paraffin  hydrocarbons  in  marine  or-
      ganisms. Marine Pollution Monitoring (Petroleum). NBS Special
      Publication 409. Proceedings of a Symposium and Workshop held at
      National Bureau of Standards, Gaithersburg, Maryland, May  13-17
10.  Ehrhardt, M. and J. Heinemann.  I974. Hydrocarbons in blue mussels
      from the Kiel bight  Marine Pollution Monitoring (Petroleum) NBS
      Special  Publication 409. Proceedings of a Symposium and  Work-
      shop held at National Bureau of Standards, Gaithersburg, Maryland.
      May  13-17
II.  Farrington, J. W. and G  C.  Medeiros,  1975. Evaluation of some
      methods  ot  analysis for  petroleum  hydrocarbons  in  marine  or-
      ganisms. Proceedings of Joint Conference on Prevention und Con-
      trol of Oil Spills.  American Petroleum Institute,  Washington, D.C.
12.  Frank,  U.,  1975. Identification  of petroleum oils by fluorescence spec-
      troscopy Proceedings of Joint Conference on Prevention and Con-
      trol of Oil Spills.  American Petroleum Institute,  Washington, D.C.
13.  Gordon, D.  C.  and P. D.  Keizer,  1974. Estimation of Petroleum
      Hydrocarbons in Seawaterby Fluorescence Spectroscopy: Improved
      Sampling and Analytical Methods. Technical Report No 481. Envi-
      ronment Canada, Fisheries and Marine Service
14.  Gordon, D C., P. D.  Keizer, and  J.  Dale,  1974. Estimates using
      fluorescence spectroscopy of the present'state of petroleum  hydro-
      carbon contamination in the water column of the northwest Atlantic
      Ocean.  Marine Chemistn-. v2, pp251-261
15.  Hargrave,  B. T. and G. A.  Phillips, 1975. Estimates of oil in aquatic
      sediments by fluorescence spectroscopy. Environmental Pollution.
      v8. pp193-21 I
16.  Rossi, S. S., J.  W. Anderson.  andG. S. Ward,  1976. Toxicity of water
      soluble  fractions of four test oils for the polychaetous annelids,
      Meanthes arenaceod?ntala and Capttella capitata.  Environmental
      Pollution. vlO,  pp9-18
17.  Nadeau, R. J. and E. T. Bergquist, 1977. Effects of a  Major Oil Spill
      Near Cabo Rojo, Puerto Rico  on Tropical Marine Communities
      Proceedings, 1977 Oil Spill Conference. American Petroleum Insti-
      tute, Washington, D.C.
18.  Neff, J. M. and J. W. Anderson, 1975. An ultraviolet spectrophotomet-
      nc  method   for  the determination of  naphthalene  and  alkyl-
      naphthalenes in the tissues of oil-contaminated marine  animals.
      Bull,  of Environ. Contain. To.iicol  v!4, pp!22-128
19.  U.S. Department of Commerce, 1976. Measurement and Interpretation
      of Hydrocarbons in the Pacific Ocean. Dept. of Commerce Report
      AID.6BA. 76/EPR.3EX.76. Final report on Contract No. 4-35266.
      April  1976
20.  Zafiriou, O., M. Blumer, and J. Myers, 1972.  Correlation of Oils and
      Oil Products by Gas Chromatography. Woods Hole  Oceanographic
      Institution. WHOI-72-55
21.  Zitko, V., 1971. Determination of residual  fuel oil contamination of
      aquatic animals. Bull. of Environ. Contain. Toxicol. v5, pp 559-564

-------
 Petroleum hydrocarbons
 from  effluents:
 detection  in  marine environment
 John T. Tanacredi
 Hunter College, New York, N. Y.
  The marine  environment has  become the
 primary disposal ground  for  an increasing
 quantity of petroleum wastes.  Mushrooming
 demands for petroleum products and the lack
 of economic incentive to recycle waste oil will
 increase the  concentrations  of  detrimental
 petroleum  hydrocarbons  in the  marine  en-
 vironment.
  Although a continuous, low-level discharge
 of  waste  petroleum hydrocarbons into  the
 marine environment may not be  as dramatic
 as a major oil spill, the consequences could be
 more devastating over an extended period. As
 noted by Blumer,1 earlier interpretations of the
 environmental effects of  oil must now  be re-
 evaluated in the light of recent evidence of its
 effects on marine organisms  2~5 and its environ-
 mental persistence,  which  resembles that  of
 DDT, PCBs, and other synthetic materials.6-9
  Of the 4.73  mil m3 (1.25 bil gal) of new
 oils purchased annually by  the automotive in-
 dustry,10 an  estimated 68  percent will leave
 automobile engines as waste. Of this 3.22 mil
 m3  (850 mil gal), it is estimated11 that 0.7 to
 1.5 mil m3 (200 to 400 mil gal )are recycled
 annually, with only 0.4 mil m3 (100 mil gal)
 actually being  refined.  In the New  York
 metropolitan area, of the  estimated 0.3 mil m3
 (94 mil gal) of automotive and industrial waste
 petroleum  recorded  annually only 40 percent
 is being reprocessed.12  The sources of waste
 petroleum  in municipal  wastewater systems
 and their  receiving waters range  from indi-
 viduals who change the oil in their automobiles
 and indiscriminately dump  the wasted  crank-
 case oil into a nearby sewer to large establish-
 ments that sporadically  discard accumulated
 stocks  of  waste oils.   On many occasions,
 especially  during periods  of plant  by-pass,
 large "oil slicks" pass through water pollution
 control facilities undetected and  untreated.13
 A major portion of this waste petroleum will
 eventually  be discharged to a receiving body
 of water because most hydrocarbons are more
resistant to degradative processes than other
compounds commonly found in wastewater,14
and because skimming and setting operations
fail to  remove a major portion of petroleum
hydrocarbons  which are finely dispersed.15
  The  current energy  dilemma makes it im-
perative to consider this waste not simply as a
pollutant  but also as a potentially  re-usable
source of  energy.  This  paper will attempt to
determine:
  1.  Whether waste automotive petroleum hy-
drocarbons are present in the treated effluents
discharged by water pollution control facilities
into Jamaica Bay,
  2.  Whether there is a sufficient quantity of
petroleum-derived hydrocarbons present in the
Jamaica Bay waters to warrant immediate at-
tention,
  3.  Whether  a  significant portion of this
waste petroleum persists in the surface waters
of the Bay, and causes a chronic  exposure of
this  ecosystem  to  petroleum-derived  hydro-
carbons.
  4.  Whether petroleum hydrocarbons are be-
ing incorporated in biological tissue  of a Bay
marine organism, Mya arenaria.

ANALYTICAL APPROACH
  Adlard 16 showed that a variety of analytical
techniques should be used in a multi-parameter
approach to oil analysis.  Because  of the com-
plex chemistry of the oil, each oil sample lends
itself to  differentiation from  others.   This
passive-tagging  approach establishes specific
qualitative parameters for oil samples, in the
form  of "profiles" or "fingerprints" to be com-
pared to a "standard profile."  Thus, positive
correlations for environmental samples are
either established or not established with stan-
dards depending upon those portions  of the
petrochemical waste that exhibit themselves in
fingerprints and remain  stable under environ-
mental conditions.
  Jamaica Bay, one of the few viable wetland
ecosystems in the New York Metropolitan area,
216  Journal WPCF
      The  author  conducted the  chemical analyses  for  this  report  at
a facility of  the U.S. Environmental Protection  Agency, Industrial
Environmental  Research Laboratory-Ci,  Edison, New Jersey.   Use  of
these laboratory facilities is provided  in order to  encourage grad-
uate level environmental  research.

-------
                                                                  Petroleum Hydrocarbons
was chosen as the study site because its unique
hydrological characteristics afford a long resi-
dence time  for treated or untreated  effluents
(Figure  1).  The sampling  scheme  involved
collections  of  weekly  (September   7,  1973
through November 5, 1973) final effluent sam-
ples from the. four major water pollution con-
trol facilities emptying into  the Bay, surface
water samples of the  Bay (0.61 m (2 ft) below
surface  by  Kemmerer  water sampler),  and
marine organism samples.   My a  arenaria were
collected from an area considered to be of high
pollution potential  (HPP)  (Mya II)   and an
area of low  pollution potential  (LPP) (Mya
III).

METHODOLOGY
  Water samples  were collected  in  980  ml
wide-mouth, glass Mason jars with Teflon-lined
caps.  Five ml  of 1:1 sulfuric acid and 20 ml
of carbon tetrachloride  (CC14) were added to
all water samples to retard bacterial action and
for extraction purposes.  All  samples  were  re-
frigerated  after  collection.  Glassware  was
detergent washed  and oven  heated at 120C
to ensure removal of possible contaminants.17
The quantity of total extractable hydrocarbons
in water  sample extracts was  determined by
use  on  an infra-red  (IB)  method tentatively
accepted  for  oil  in water  analysis  by  the
EPA's  Industrial Waste Treatment Research
Laboratory, Edison,  N.  J., with  some  slight
modifications that were  necessary because of
sample concentration factors.18   Carbon tetra-
chloride extracts were jet-air evaporated, con-
centrated, and  residues weighed  and  brought
to volume in  hexanes for ultraviolet  (uv)-
fluorescence and  gas chromatographic   (GC)
analysis. Organism extraction required shucked
clams to be homogenized in a blender  with 50
ml  of n-hexane, mixed with three times wet
tissue weight  of anhydrous Na2So,j,  and re-
frigerated for 24 h.  The tissue extract mix was
Soxhlet extracted in n-hexane for 6 h.  These
extracts  were   divided into subfractions  by
hexane/benzene/methanol  elution through a
3 percent  water deactivated silica  gel/alumina
column at a flow rate of 120 ml/s.  The three
analytical  methods  used for sample  analyses
were gas   chromatography,  uv-fluorescence
spectroscopy and cc-mass spectroscopy.
         B R o
    FIGURE 1.  Tri-phase sampling  scheme  for  Jamaica Bay,  New York.   Organism
                 samples  were designated Mya II and  Mya III  representative  of  high
                 pollution potential   (HPP)  and  low  pollution   potential  (LPP),
                 respectively.
                                                                     February 1977   217

-------
Tanacredi
                       INCREASING TIME AND TEMPERATURE
FIGURE 2.   Gas chromatographic profiles of
              "standard"  petroleum  entities:
              (A)   waste automobile crank-
              case   oil,  (B)  10W/30 virgin
              motor   oil,   (C)   transmission
              fluid.  (D)  Arabian  light crude
              oil. (E)  No. 2  fuel  oil,  (F)
              standard  Ce-C36  hydrocarbon
              "spike"  used for retention  time
              and  relative peak height cor-
              relations.
  Gas chromatographic  method.   Hydrogen-
flame gas chromatography with flame  ioniza-
tion  detector and a  15.24 m X  0.51  mm  (50
ft  X 0.02  in.)  ID,  stainless  steel,  support-
coated,  open-tubular column (SCOT  OV-101;
non-polar silicone oil*) operated with nitrogen
carrier gas with a flow rate at the column out-
let of 240 ml/s.  Samples  were temperature
programmed from 75 to 300 C at  6C/min
with  isothermal operation  at  300C for  12
min.
  Waste crankcase oil GC profiles exhibited a
large envelope  area above  the baseline at-
tributable to polynuclear and  polycyclic aro-
matic ring compounds; a "light-end" of hydro-
carbons with carbon numbers under C12; C17-
pristane/C18-phytane peak-pairs; various peaks
atop  the   envelope  portion   indicative  of
branched paraffinic,  olefinic, and hetero-com-
pounds  over a wide range  of boiling  points.
The  chromatograms generated by refined petro-
leum products differed from  those of  crude or
fuel  oils (Figure 2).  GC-retention times and
relative  peak heights  were  used to  identify
petroleum hydrocarbons in environmental sam-
ples.  A Cfl through C36 n-paraffin hydrocarbon
"spike"   (Figure 2F)  was added to  samples
after noting  the original samples' profiles and
retention times for resolved  peaks.   Increases
in peak heights  of  previously  noted  sample
components indicated its presence in  the sam-
ple  extract.   Organism  sample  sub-fractions
were analyzed by retention times generated in
the GC-MS system.
  uv-fluorescence  spectroscopic  method.   Re-
cent investigators 19  have exhibited the ability
of fluorescence  spectroscopy to detect trace
quantities of petroleum-derived hydrocarbons
in oceanic waters.   Goldberg and Devonald 20
have been   able to  differentiate between  a
lubricating  oil  and  a  crude  or  fuel  oil using
fluorescence  spectroscopic   techniques.   All
petroleum  products  fluoresce  when  excited
by uv light because of the presence of aromatic
hydrocarbons with  multi-ring  configurations
such  as fused  ring  polynuclear  aromatics.21
A uv-fluorescence  spectrophotometer with two
independent monochromators (150 watt xenon
arc light source),  and a constant temperature
cell bath that maintained a 10 mm path length,
quartz  cell  at 20  0.5C  were used for all
fluorescence  analyses.  Each standard oil was
excited  at 290 m/j, while  scanning the emission
spectrum from 240 to  540 m^.  Thurston and
Knight22 had used  340 m^ as the excitation
wave length for the characterization  of petro-

  "Perkin-Elmer Corp.
218   Journal  WPCF

-------
                                                                    Petroleum Hydrocarbons
 leum entities; however,  it has  been  recently
 demonstrated 23 that Raman scattering at that
 frequency obscures  the  fluorescence  profiles,
 making  sample differentiation difficult.
   A  relatively  new fluorescence  technique 24
 was  also used  to excite  each sample at suc-
 cessive excitation wavelengths from 240 m^u, to
 440  uifj.  (at  20-mp,  intervals) while  scanning
 for the maximum fluorescence emission (Figure
 3).   Each maximum peak was used as a point
 to be plotted graphically;  this process gener-
 ated  a  "fluorescence maxima profile"  (FMP)
 for each sample.  Correlation was  determined
 by visually comparing the maxima profile plots
 of known oil standards to  the maxima profile
 plots of environmental samples.  When these
 maxima profiles "fit" (Figure 4)  in  addition to
 exhibiting the other qualitative  characteristics
 established by standards, detection was estab-
 lished  for  the particular  sample  under  in-
 vestigation.
   When the profile  for  a sample  met  the
 qualitative criteria established by standard oils,
 it was recorded in a "correlates" column. When
 profiles  generated were not totally consistent
 with  the qualitative  criteria for a correlation, it
 was recorded in a  "slight-correlation"  column.
 Slight correlations were felt to be the result of
 mixtures  of petrochemical entities.   It  should
 be emphasized that  the correlation criteria  are
 essentially qualitative in that source identifica-
 tion,  (such as gas stations or individuals dump-
 ing crankcase oil into a sewer), of the  detected
 waste petrochemical cannot be directly estab-
 lished by  this technique.  This  method  was
                                    100-
                                    90-
                                    80-
                                     70-
                                    60 
                                                                    KEY
                                                                   > = WCCO Standard
                                                                    = 26th. Ward
                                                          A (mn)

                                 FIGURE 4.  Fluorescence   Maxima   profile
                                               (FMP)  "fit"  for waste  crank-
                                               case oil with  pollution control
                                               facility  final  effluent  extract
                                               samples collected on September
                                               7, 1973.
        260  280  300  320 340
                                380  400   420
              EXCITATION FREQUENCY
FIGURE  3.
Fluorescence  emission maxima
peaks  for  the  standard  waste
automobile  crankcase  oil.
originally intended to match spilled weathered
petroleum products with already-known stock
oils.  When  a positive  correlation  exists in all
fluorescence   categories,  it  is  an  extremely
strong  indication  that  a  waste  automotive
petroleum product  is  present; however, the
correlation arrays  are based solely upon gener-
ated profiles  and fluorescence analysis of stan-
dard oils.
   cc-mass spectroscopic method.    The  final
analytical phase of this project used  a com-
puterized  cc-mass  spectrometer  combination
with  1.52 m X 2 mm  ID glass, packed with
3  percent OV-1 on a Chromsorb  W column.
Samples were temperature programmed from
100  to  280C at  6C/min with  isothermal
operations at 280C for  approximately 10 min.
Helium carrier  gas  at 90  ml/s was  measured
'at  the  column outlet.   Only  organism  sub-
                                                                       February 1977  219

-------
 Tanacredi
 fractions were analyzed for specific petroleum-
 derived aromatic compounds by this method.

 RESULTS
   When  a  waste petroleum product  is  dis-
 carded  and enters the environment, it  is sub-
 jected to  a  variety of weathering phenomena.
 Volatiles are lost as a result of evaporation and
 bacteria sequentially degrade different hydro-
 carbons.25   To  see  if  detection  parameters
 established for standard oils would be appre-
 ciably affected  by  weathering,  standard  oils
 were  weathered in  filtered sea water for  a
 period of 32 days.  Profiles generated indicated
 no significant changes in fluorescence detection
 criteria  even  though  some profiles exhibited
 decreases  in intensity because of concentration
 factors.26  Waste crankcase oil chromatograms
 did lose "light-ends"; however, weathering had
 little effect on other  detection parameters such
 as the unresolved envelope portion.
   Treated wastewater samples.   Thirty-nine
 treated  effluent samples were analyzed for total
 CC14  extractable  hydrocarbons  (Table  I).
 Regular and spiked (C6 through C38 n-paraffm
 mix)  GC runs  were conducted and qualitative
 characteristics and retention time data of re-
 solved peaks were noted.   In all cases, chro-
 matograms indicated an unresolved envelope
 portion  above  200C.  Spiked  samples  ex-
 hibited  increases in  peak heights  for a major
 portion  of previously  resolved peaks.   Those
 peaks that occurred between standard peaks
 were  tentatively labeled as being  a series  of
 isomeric or branched chain compounds.27
   Clrpristane, C18-phytane hydrocarbon peak
 pairs,  indicative of  petroleum contamination,
 were observed in some effluent chromatograms,
 but  not  in all cases.
   uv-fluorescence analysis  and correlation re-
 sults for treated  effluent extracts are shown in
 Table II.  Figure 5 exhibits fluorescence maxi-
 mum profiles for wastewater effluents and their
 relation  to  standard waste crankcase  oil and
 No. 2 fuel oil fluorescence maxima profiles.
   Surface water samples.  By using a similar
 solvent extraction method, average background
 levels of petroleum  hydrocarbons in  oceanic
 waters have been established at approximately
 2 fj.g/1;  greater  than average,  or excessively
 high levels  of  petroleum  hydrocarbons  for
 oceanic  waters,  ranged  between  10 and 20
 iug/l.  Table  III reveals that the values ob-
 tained for the' Jamaica Bay surface waters are
 significantly above normal background  oceanic
 levels of hydrocarbon concentrations.   Non-
 petroleum, biologically derived compounds are
 extracted with CC14 and are not all lost during
 the  jet-evaporation step.   Extensive  care and
 consideration was taken to prevent contamina-
 tion from sample containers, extraction opera-
 tions,  solvents  or  from  the research  vessel
 during collection.  The January 8  Jamaica Bay
 surface water  extracts (Figure 6) were an-
 alyzed by gas chromatography using  the  same
 sample  injection quantities   and   attenuation
 conditions in  order  to observe a  gradient re-
 sponse of increased petroleum hydrocarbons as
 one  moves in and through the Bay.
   Table IV exhibits fluorescence results on sur-
face water  samples  and  reveals  the  presence
 of petroleum fractions in those samples taken
from the Bay proper.  Again, the degree of
weathering  to  which  the oil had been  sub-
jected probably dictated  the  intensity of the
fluorescence response, yet in  no instance did
profiles correlate with other types of petroleum
 entities  such as fuel  or crude oils.
   Organism samples.  Mya II (HPP)  and Mya
III (LPP) extracts were  analyzed by GC, uv-
fluorescence  and  GC-MS methods  outlined
previously.  Results of analyses clearly demon-
strate the presence of petroleum-derived hydro-
carbons from Mya arenaria tissue extracts con-
sidered to be of high pollution or contamination
potential. The aromatic portion of the Mya II
TABLE I. IR Quantification of Total Extractable Hydrocarbons from Treated Effluents.
Water Pollution
Facility
Coney Island plant
26th Ward plant
Jamaica plant
Rockaway plant
9/10
1.50
29.7


9/15
16.4
20.0
10.7

9/17
7.1
34.9
12.0f
4.9
9/24
2.0
28.9f
7.2
1.3
9/29
3.5
22.9
5.3
4.7
Date
10/1
15.6
12.3
4.7
0.5
10/8
3.0
19.2
9.6
lO.Oj
10/15
39.8f
19.1
4.6
13.8
10/22
10.5
9.3
9.4
8.5
10/29
416

14.2
7.7
11/5
8.6

18.8
3.2
* All values in mg/1.
t = Gas chromatographic analysis.
220   Journal WPCF

-------
                                                                   Petroleum Hydrocarbons
TABLE II.  Effluent Sample Correlation Data.

                       Fluorescence Maxima
                           Profile Fit.
                                      Maxima Region Fit
                                                                      332 HIM Peak Fit

Date               Correlates     Slight     Correlates     Slight     Correlates     Slight
1973     Sample   With WCCO  Correlation  With WCCO  Correlation  With WCCO  Correlation
  7 Sept.



 10 Sept.

 15 Sept.


 17 Sept.



 24 Sept.



 29 Sept.



  1 Oct.



  8 Oct.



 15 Oct.



 22 Oct.



 29 Oct.


 5 Nov.
26th Ward
CIP
Jamaica
Rockaway
CIP
26th Ward
26th Ward
CIP
Jamaica
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
26th Ward
CIP
Jamaica
Rockaway
CIP
Jamaica
Rockaway
CIP
Jamaica
Rockaway
extract  (Figure  7A) was shown  to  contain a
wide range of aromatic compounds with boiling
ranges above  250C.  Mass spectral analysis
of this aromatic portion indicated the presence
of  substituted  akyl-benzenes,  a  group  of
aromatic  hydrocarbons  found  in   extremely
small  amounts or not at all in marine organ-
isms.28' 29   Chromatograms  of the  Mya  II-
saturated portion revealed the presence of iso-
                                     meric compounds  or other members  of the
                                     homologous  series.   The  Mya  III aromatic
                                     portion  (Figure 7B)  was  considerably  less
                                     complex than  the  Mya II aromatic fraction;
                                     this suggested that there was less  exposure of
                                     the organisms  to petroleum hydrocarbons  out-
                                     side the Bay than  within.  Brown and Huff-
                                     man 80 showed this when they observed lower
                                     concentrations of  aromatic hydrocarbons in
                                                                      February 1977  221

-------
 Tanacredi
         tu
         I-
o
- No. 2 Fuel Oil
                                                              x = 9/7/73 26th Ward
                                                              o= 9/7/73 Jamaica
                                                              * = 9/7/73 Rockaway
                                                              o-9/7/73 Conev Is.
                                                              a = 9/24/73 Rockaway
                                                               - 9/24/73 Jamaica
                  240   260        300   320   340 .  360   380   400
                                                 X (mju)
                                                                      420   440  460
     FIGURE  5.
          UV-Fluorescence Maxima profiles  for pollution control  facility extracts
          of September 1973 in correlation with standard crankcase oil and No. 2
          fuel oil.
ocean waters  than  would  be  found  during
petroleum pollution incidents.
   The results  of  the fluorescence analysis  on
Mya  II extracts, (Table V)  indicate that sam-
ples collected  from the Bay proper  correlated
excellently with standard  crankcase  oil waste;
however, the Mya III extract did not correlate
successfully with  these  standards.  When the
Mya  III  extract was excited  at 290  m/n,  an
additional peak of greater  intensity occurred at
370 niju,.   This peak  could  be attributable to
contamination  from other  petroleum  pollutants
such  as residual fuel oil, which peaks between
350 and  400  m/x.31   Further investigation is
needed to determine whether or not  similar
findings are obtained  from non-petroleum ma-
terials in  such  samples.


DISCUSSION
   Chromatograms  generated  by waste auto-
motive lubricating oil and  refined petroleum
have  been shown  to be characteristic and dif-
                                       ferentiable from chromatograms of other petro-
                                       leum  entities.   Once  a waste  oil  enters  the
                                       environment,  it seems that weathering phe-
                                       nomena such  as evaporation and  bacterial  de-
                                       gradation will have little effect upon the  less
                                       soluable aromatic and  higher molecular weight
                                       components;   thus,  detection parameters  are
                                       TABLE III.  Jamaica Bay Surface Water Total
                                                    Extractable Hydrocarbons.*
Sample
Site
NYN16
NYN09A
NYJ01
NYJ02
NYJ03
NYJ05
NYJ07
7 November
1973
0.94
1.20
2.10
1.17
3.10
0.50
1.08
8 January
1974
1.13
0.88
2.16
2.20
5.10
1.50
1.40
                                        ' All values in mg/1.
222   Journal  WPCF

-------
                                                                   Petroleum Hydrocarbons
preserved.  The wide-boiling range, variety of
substituents separated, and the unresolved en-
velope portions of chromatograms indicate the
presence of crankcase oil, although  they are
not conclusive.   These chromatographic  data
may be considered as  preliminary findings for
crankcase oil in wastewater and will require a
more quantitative investigation.
  Though  the specific sources of the detected
waste petroleum  could not be established, the
accumulated   evidence   from   all  analyses
strongly indicates a crankcase oil origin.  Gas
chromatograms of pollution  control plant ex-
tracts did show  a wide range of hydrocarbon
compounds above C2o, a characteristic of lube
oils.29  They did exhibit  unresolved  envelope
portions with some samples revealing the C17-
pristane/C18-phytane   peak-pairs.   Gas  chro-
matograms  generated by organism  extracts
strongly  exhibited  the presence of  aromatic
compounds in body tissue.  Tentative GC-MS
identification of organism subfractions indicated
the presence of alkyl-substituted benzene struc-
tures, which are highly toxic substances indica-
tive of  petroleum  contamination.   uv-fluo-
rescence  analysis  furnished dramatic  evidence
for  the presence  of crankcase oil in environ-
mental samples and greatly  strengthened the
other  analytical  results  obtained.   Emission
spectra of  environmental  samples consistently
demonstrated  the  presence  of  polynuclear
aromatics  (PNA),  compounds  which could only
FIGURE  6.   Gas  chromatographic  profiles
              generated by Jamiaca Bay sur-
              face water samples collected  on
              January 8, 1974.
TABLE IV.   Fluorescence Correlation Data for Jamaica Bay Surface Water Samples.

                          Fluorescence Maxima
                              Profile Fit         Maxima Region Fit       332 mM Peak Fit
Date
7 November
1973





9 January
1974





Slight
Correlates Corre- Correlates
Source With WCCO lation With WCCO
NYN16 + +
NYN09A + +
NYJ01 + +
NYJ02 + +
NYJ03 + +
NYJ05 + +
NYJ07 + +
NYN16 + +
NYN09A + +
NYJ01 + +
NYJ02 + +
NYJ03 + +
NYJ05 + +
NYJ07 + +
Slight Slight
Corre- Correlates Corre-
lation With WCCO lation
+
+
-f
+
+
+
+
+
-).
+
-f
+
+
+
                                                                      February  1977  223

-------
 Tanacredi
 FIGURE 7.  Gas  chromatograms   of  (A)
              Mya II  aromatic sub-fraction,
              and (B)  Mya III aromatic sub-
              fraction.
 be attributable to petroleum pollution.32  Due
 to  crankcase  oil's  unique  fluorescence  and
 characteristic profiles, its presence  in environ-
 mental  samples was  tentatively  established,
 even though  the fluorescence techniques used
 for this  project were originally to  be used for
 the comparative identification of unknown oils
 to standard oils.
   The results presented give a strong  indica-
 tion  that  hydrocarbons that  are  discharged
 with  treated wastewater into Jamaica Bay and
 that ultimately accumulate in biological tissue
 are of a waste  automotive petroleum  origin.
 Whether this is from intentional or  accidental
 dumping into sewers,   cannot  be  determined
 here.  Because of the unique characteristics of
 the study area and the  fact that the  only fresh
 water input   comes  from  pollution   control
 plants, there is a good  indication that a major
 portion  of detected  hydrocarbons originate
                              from combined wastewater effluents.  Further
                              investigations are necessary to determine  the
                              contributions of hydrocarbons from atmospheric
                              washings,  recreational  boating,   and  biotic
                              sources.

                              CONCLUSION
                                 The  results  of  this  project  indicate   the
                              existence  of  an  unusually  large  hydrocarbon
                              burden in Jamaica Bay.  Continued addition of
                              these hydrocarbons can only lead to a further
                              deterioration of this ecosystem.  Tidal-wetland
                              areas provide food  and shelter for a variety of
                              indigenous and migratory  wildlife,  and thus
                              provide critical support to marine food  chains
                              reaching all  the way  to man.  It seems that
                              problems  of  aquatic pollution  in  Jamaica Bay
                              will  be magnified in  the future  even though
                              portions  of the Bay  may be included  in  the
                              Gateway National Park.  Added pollution from
                              new housing, continued land-fill operations, and
                              off-shore oil  drilling will continue to pollute,
                              perhaps  irreversibly,   this   coastal area.   In-
                              creased efforts should be  aimed  at restoring
                              these waters  so that they may support a much
                              greater variety  of  marine  life.   The idea  of
                              fostering a shell-fishery  in  Jamaica  Bay is a
                              good one.
                                 The analytical results  of  this study suggest
                              that  appreciable quantities  of hydrocarbons  at-
                              tributable to waste automotive petroleum prod-
                              ucts  are present in treated wastewater effluents
                              entering Jamaica Bay.  The  discharge of petro-
                              leum hydrocarbons in the  effluent is chronic.
                              Significant  quantities   of detectable hydrocar-
                              bons  remain  in  solution in  the surface waters
                              of the Bay, while aromatic hydrocarbons from
                              waste petroleum are found in tissue extracts of
                              marine benthic organisms collected in the Bay.
                              The  establishment of  a national waste oil  re-
                              cycling program would  not only reduce  the
                              pollution already developing in the  Bay, but
                              would be  an  exemplary step towards the most
                              efficient use of U. S. energy sources.
TABLE V.  Fluorescence Data for MYA Extracts.
                                 Fluorescence Max
                                      Profile
      Date
                   Source
                                Corre-
                                lation
                          Slight
                       Correlation
  Max Region
     Fit

       Slight
Corre-  Corre-
lation  lation
  Excitation
   290 HIM

        Slight
Corre-   Corre-
lation   lation
     12/3/73
     12/3/73
Mya Ilf
Mya IIIJ
f Collected at Diamond Point, Jamaica Bay.
t Collected at Rockaway point, Atlantic Ocean.
224   Journal  WPCF

-------
                                                                      Petroleum Hydrocarbons
ACKNOWLEDGMENTS
  Credits.   George  Kupchik, Jack  Foehren-
bach,  Michael  Gruenfeld,  Uwe  Frank  and
Michael Alavanja reviewed and commented on
this  project.  This project was made possible
through the cooperation of the Environmental
Protection Agency, Region II,  Industrial Waste
Treatment Research  Laboratory,  Edison, New
Jersey;  the  Department  of  Environmental
Health  Sciences,  Hunter  College, of the  City
University of New York; and the New York
City  Department of  Water   Resources.   B.
Dudenbostel conducted the GC-MS analyses.
  Author.  John  T.  Tanacredi was a lecturer
with the Department of Environmental Health
Sciences at Hunter College, New York, N. Y.
He is currently Environmental Protection Ad-
ministrator with the U. S. Coast Guard, N. Y.
REFERENCES
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      Marine Bio., 5,  198 (1970).
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      Dispersions and Water-Soluble Extracts  of
      Crude and Refined Oils and their  Toxicity
      to Estuarine Crustaceans and Fish."  Marine
      Bio., 27, 75 (1974).
 4. Clark, R. C.,  et al, "Acute Effects of Outboard
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      Env. Sci. and Tech., 8,  1009 (1974).
 5. Zitko, V., and Tibbo, S. N., "Fish Kill Caused
      by an Intermediate Oil  From Coke  Ovens."
      Bulletin  Env. Cont. and Toxicology,  6, 24
      (1971).
 6. Chan, G. L., "A Study of the Effects of  the
      San Francisco  Oil Spill' on Marine Organ-
      isms."  Proc. of Joint Con/, on the  Preven-
      tion and Control of Oil Spills, Washington,
      D. C., 741 (1973).
 7. Ehrhardt,  M., "Petroleum  Hydrocarbons  in
      Oysters from  Galveston Bay."   Env. Poll.,
      3, 257 (1972).
 8. Kasymov, A. G., and Alier, A. D.,  "Environ-
      mental Study of the Effect  of Oil on Some
      Representatives  of Benthos  in the  Caspian
      Sea."  Water, Air and Soil Poll,  2, 235
      (1973).
 9. Morrow, J.  E.,  "Oil-Induced Mortalities  in
      Juvenile Coho and Sockeye  Salmon."  Jour.
      of Mar. Res.,  31,  135 (1973).
10. Lederman, P. B., and Weinstein, N. J., "Sales
      in Lubricating Oils and Greases for 1969."
      U. S. Dept. of Commerce  Series  MA-29C
      (69)-lB and  MH-29C  (71)-1, 2 (1973).
11. Lederman, P. B., and Weinstein, N. J., "Sales
      in Lubricating Oils and Greases for 1969."
      U. S. Dept. of Commerce  Series  MA-29C
      (69)-lB and MH-29C  (71)-1,  3  (1973).
 12. Maltezou, S., "Waste Oil Generation, Disposal
      and Management Data for  the  New York
      Metropolitan Area."   Manuscript  of speech
      presented at International Conf.  on  Waste
      Oil Recovery and Reuse 3 (1974).
 13. Personal  interviews with personnel  and plant
      superintendents in October 1973.
 14  Farrington, J.,  and Quinn, J., "Petroleum Hy-
      drocarbons and  Fatty Acids in Wastewater
      Effluents."   Jour. Water Poll. Control Fed.,
      45, 705 (1973).
 15. Loehr, R. C.,  and  DeNavarra,  C. T., "Grease
      Removal at a Municipal Treatment Facility."
      Jour.  Water Poll Control  Fed., 41, 142
      (1969).
 16. Adlard, E. R.,  "European Experiences  in the
      Identification of Waterborne Oil."  Proposed
      paper  for presentation at Meeting of Na-
      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 SpectroscopyA  Technique for
      Characterizing Surface Films."  Jour. Res.
      U. S. Geol. Survey, 1, 714 (1973).
21. Riecher,  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  and  personal  communications  with
      Dr. B.  Dudenbostel, Region II, Surveillance
      and Analysis Lab., EPA,  Edison,  N.  J.,  on
      "Computerized  Gas  Chromatography/Mass
      Spectroscopy" presented October  9, 1973.
                                                                          February  1977   225

-------
 Tanacredi
 28. Meinschein.  W.  G..  "Origin of Petroleum."
       Bull Am.  Assoc.  Petrol  Gcol.  43.  925
       (1959).
 29. Meinschein, W. G..  "Hydrocarbons:  Saturate.
       Unsaturated  and  Aromatic."  In  Elington.
       G,,  and Murphy,  M, I.  J. (.Eds,1!  Organic
       Geochemistry ^Nevr York:  S^ineer-Yerla^V
       346 (1969)."
 30. Brown, R. A.,  and Huffman, H. L.. "Hvdro-
      carbons In Open  Ocean "\Yaters."  Science.
      191.  S47 (1976).
31.  Zitko. V., "Determination of Residual Fuel Oil
      Contamination of  Aquatic Animals."   Bull.
      Env.  Contain, and Tox.. 5, 560 (1971).
32.  Wedge\vod.  P., and  Cooper, R. L.,  "Detec-
      tion and Determination of Traces of Poly-
      nuclear Hydrocarbons in Industrial Effluents
      and Sewage."  Analyst. SO. 651 (19551
226   Journal \YPCF

-------
              BIBLIOGRAPHY  OF RECENT METHODS FOR THE FLUORESCENCE

                         ANALYSIS OF PETROLEUM OILS
                               Uwe  Frank
             Industrial  Waste  Treatment  Research Laboratory
                 U.S.  Environmental  Protection Agency
                           Edison, New Jersey


I.   Qualitative Methods

     1.  Thruston,  A.D., Jr.,  and Knight,  R.W. 1971. Characterization  of  Crude
            and Residual-Type  Oils by Fluorescence  Spectroscopy.  Env.  Sci.  and
            Tech. 5:64-69.

     2.  Coakley, W.A.  1973.  Comparative Identification  of Oil Spills  by  Fluor-
            escence Spectroscopy Fingerprinting. Proceedings  of Joint  Conference
            on Prevention and  Control of Oil Spills, pp.  215-222. Washington,
            D.C.: American Petroleum Institute.

     3.  Jadamec, J.R.,  and Porro, T.J.  1974. Identification  and  Fingerorinting
            of Oils by Fluorescence  Spectroscopy. Abstracts of the Pittsburgh
            Conference on Analytical Chemistry and  Applied Spectroscopy.
            Cleveland, Ohio.

     4.  Lloyd, J.B.F. 1971.  The Nature  and Evidental  Value of the Luminescence
            of Automobile Engine Oil and Related Materials -  I. Snychronous  Ex-
            citation of Fluorescence Emissions. J.  Fores. Sci. Soc. 11:83-94

     5.  Lloyd, J.B.F. 1971.  The Nature  and Evidental  Value of the Luminescence
            of Automobile Engine Oil and Related Materials -  II.  Aggregate  Lu-
            minescence.  J. Fores.  Sci. Soc. 11:153-170.

     6.  Lloyd, J.B.F. 1971.  The Nature  and Evidental  Value of the Luminescence
            of Automobile Engine Oil and Related Materials -  III. Separated Lu-
            minescence J. Fores.  Sci, Soc. 11:235-253.

     7.  Drushel, H.V.,  and Sommers , A.L.  1966. Combination of Gas Chromato-
            graphy with Fluorescence and Phosphorescence  in Analysis of Petroleum
            Fractions. Anal.  Chem.  38(1):10-19.

     8.  McKay, J.F.,  and Latham,  D.R. 1972. Fluorescence Spectrometry in the
            Characterization of High-Boiling Petroleum Distillates. Anal. Chem.
            44(13):2132-2137.

     9.  McKay, J.F.,  and Latham,  D.R. 1973. Polyaromatic Hydrocarbons in High-
            Boiling Petroleum Distillates. Anal. Chem. 45 (7) :1050-1055.

     10. Frank, U. 1972. Analysis  for Crankcase Oil in Water  by Fluorescence
            Spectrophotometry. Analyt. Quality Contr.  Newsl., U.S. Environmental
            Protection Agency, Cincinnati, Ohio' 13:3.

-------
11.  Frank,  U.  1972.  Passive  Tagging  of  Oils by  Fluorescence  Spectrophoto-
       metry.  Analyt.  Quality Contr.  Newsl.,  U.S. Environmental Protection
       Agency, Cincinnati, Ohio 15:4-5.

12.  Frank,  U.  1974.  Passive  Tagging  of  Oils by  Fluorescence  Spectrophoto-
       metry.      Analyt.  Quality Contr.  Newsl., U.S. Environmental Pro-
       tection Agency, Cincinnati, Ohio  20:8.

13.  Frank,  U., and Jeleniewski, H. 1973.  Solvent Impurities  and Fluores-
       cence Spectrophotometry. Analyt.  Quality Contr. Newsl., U.S. Environ-
       mental Protection Agency, Cincinnati,  Ohio 18:10-11.

14.  Frank,  U.  1974.  An Improved Solvent  for Fluorescence Analyses of Oil.
       Analyt. Quality Contr. Newsl., U.S. Environmental Protection Agency,
       Cincinnati, Ohio  21:10.

15.  Frank,  U.  1974.  Effect of Fluorescence Quenching on Oil  Identification.
       Analyt. Quality, Contr. Newsl.,  U.S. Environmental Protection Agency,
       Cincinnati, Ohio 22:5.

16.  Sawicki, E. 1969.  Fluorescence Analysis in  Air  Pollution Research.
       Talanta 16:1231-1266.

17.  Gruenfeld, M. 1973. Identification  of Oil Pollutants:  A Review of
       Some Recent Methods.  Proceedings  of Joint Conference  on Prevention
       and Control of  Oil Spills, pp. 179-193.  Washington, B.C.: American
       Petroleum Institute.

18.  Freegarde, M., Hatchard, C.G., and  Parker,  C.A.  1971. Oil Spilt at
       Sea:  Its Identification, Determination  and  Ultimate  Fate. Labora-
       tory Practice 20(1):35-40.

19.  Frank,  U. 1975. Identification of Petroleum Oils by Fluorescence Spec-
       troscopy. Proceedings of Joint Conference on Prevention and Control
       of Oil Pollution, pp. 87-91.  San Francisco,  California: American
       Petroleum Institute.

20.  Hornig, A.W. and Brownrigg, J.T. 1975. Total Luminescence Spectroscopy
       as a Tool for Oil Identification.  Abstracts  of the Pittsburgh Con-
       ference on Analytical Chemistry  and Applied  Spectroscopy, No. 400.
       Cleveland, Ohio.

21.  Jadamec, J.R., Saner, W.A., and  Porro, T.J. 1975. Identification of
       Spilled Petroleum Oils by Combined Liquid Chromatographic and Fluo-
       resence Spectroscopic Techniques.  Abstracts  of the Pittsburgh Con-
       ference on Analytical Chemistry  and Applied  Spectroscopy, No. 456.
       Cleveland, Ohio.

22.  Hornig, A.W., and  Eastwood, D. 1971. Development of  a Low Temperature
       Molecular Emission Method for Oils. Progress Report,  Program No.
       16020 GBW, U.S. Environmental Protection Agency, Water Quality
       Office. Washington, D.C.

-------
II.  Quantitative Methods

     1.  Goldberg, M.C.,  and Devonald,  D.H.  1973.  Fluorescent  Spectroscopy,
            A Technique for Characterizing  Surface Films.  J. Res.  U.S.  Geol.
            Survey 1(6):709-717 .

     2.  Gordon,  B.C.,  Jr., and Keizer, P.D.  1974. Estimation  of Petroleum
            Hydrocarbons  in Seawater by Fluorescence  Spectroscopy:  Improved
            Sampling and  Analytical  Methods.  Fisheries  and Marine  Services
            Technical Report No.  481. Environment  Canada.

     3.  Frank, U. 1973.  A Method for Quantitating Oil  Directly in Water by
            Fluorescence  Spectrophotometry.  Analyt. Quality Contr . Newsl.,
            U.S.  Environmental Protection Agency,  Cincinnati,  Ohio 18:9-10.

     4.  Keizer,  P.D.,  and Gordon, D.C., Jr.  1973. Detection of Trace Amounts
            of Oil in Sea Water by Fluorescence Spectroscopy.  Journal of the
            Fisheries Research Board of Canada 30(8) :1039-1046.

     5.  Zitko, V., and Tibbo, S.N.  1971. Fish Kill by  an  Intermediate  Oil
            From Coke Ovens. Bulletin of Environmental  Contamination and
            Toxicology 6(l):24-25.

     6.  Zitko, V., and Carson, W.V. 1970.  The Characterization of Petroleum
            Oils and Their Determination in the Aquatic Environment. Fisheries
            Research Board of Canada Technical Report No.  217.

     7.  Zitko, V. 1971.  Determination  of Residual Fuel Oil Contamination of
            Aquatic Animals. Bulletin of Environmental  Contamination and Toxi-
            cology 5(6) :559-564.

     8.  Hornig, A.W. 1974. Identification,  Estimation  and Monitoring of Petrole-
            um in Marine Waters by Luminescence Methods. NBS  Special Publication
            409.. Proceedings of  Symposium and Workshop  on  Marine Pollution Mon-
            itoring  (Petroleum) ,  pp, 135-144. Gaithersburg, Maryland.

     9.  Cretney, W.J., and Wong, C.S.  1974. Fluorescence  Monitoring Study at
            Ocean Weather Station P. NBS Special Publication  409.  Proceedings
            of Symposium and Workshop on Marine Pollution  Monitoring (Petroleum) ,
            pp. 175-177. Gaithersburg, Maryland.

-------
                            SECTION  II

                  OTHER ANALYTICAL  PUBLICATIONS
                     AQC Newsletter Articles

   EXTRACTION OF OIL FROM WATER FOR QUANTITATIVE ANALYSIS BY IR
                 M.  Gruenfeld
          COMPARISON OF HYDROCARBONS IN MARINE ORGANISMS
             FROM UNPOLLUTED WATER WITH PETROLEUM OILS
                 B.  F.  Dudenbostel
          PREPARATION OF HEAVY OILS FOR INFRARED ANALYSIS
                 M.  Gruenfeld
                QUANTITATIVE ANALYSIS OF OIL BY IR
                 M.  Gruenfeld
STORAGE AND TRANSPORT OF OIL CONTAINING SAMPLES IN PLASTIC BOTTLES
                 B.  F.  Dudenbostel, M. Gruenfeld
     A TLC METHOD TO FACILITATE THE QUANTITATION OF OIL BY IR
                 U.  Frank
  ULTRASONIFICATION FOR PREPARING STABLE OIL IN WATER DISPERSIONS
                 M.  Gruenfeld, F. Behm
           USE OF GAS CHROMATOGRAPHIC PEAK HEIGHT RATIOS
               FOR PASSIVE TAGGING OF PETROLEUM OILS
                 B.  F.  Dudenbostel
             STORAGE AND TRANSPORT OF OILS IN SOLVENTS
                  FOR QUANTITATIVE ANALYSIS BY IR
                 M.  Gruenfeld, J. Puglis
 CALCULATION OF ABSORBANCE FROM IR ORDINATE EXPANSION MEASUREMENTS
                 M.  Gruenfeld
      GLASSWARE CLEANING FOR THE QUANTITATION OF OIL IN WATER
                 J.  Puglis, M. Gruenfeld
               SOLVENT EXTRACTION OF OIL FROM WATER
                 U.  Frank
               SULFUR INTERFERENCE IN U.V. ANALYSIS
                 M.  Gruenfeld, J. Lafornara
          REMOVAL OF CHARRED OIL DEPOSITS FROM GLASSWARE
                 H.  Jeleniewski, U. Frank
                     SOLVENT FOR OIL ANALYSIS
                 M.  Gruenfeld
           EVALUATION OF A PORTABLE IR SPECTROPHOTOMETER
                 M.  Gruenfeld, U. Frank
     IDENTIFICATION OF MILLIGRAM QUANTITIES OF PETROLEUM OILS
                 M.  Gruenfeld, R. Frederick
         SEPARATION OF PETROLEUM AND NON-PETROLEUM OILS
                 M.  Gruenfeld
                   REPLICATE OIL CHROMATOGRAMS
                 M.  Gruenfeld, M. Urban
              DETERMINATION OF OIL IN SEDIMENT BY NMR
                 U.  Frank, M. Gruenfeld

-------
                            Research Papers


  IDENTIFICATION OF OIL POLLUTANTS:   A REVIEW OF SOME RECENT METHODS
                   M.  Gruenfeld
       EXTRACTION OF DISPERSED OILS  FROM WATER FOR QUANTITATIVE
                ANALYSIS BY INFRARED SPECTROPHOTOMETRY
                   M.  Gruenfeld
           QUANTITATIVE ANALYSIS OF  PETROLEUM OIL POLLUTANTS
                     BY INFRARED SPECTROPHOTOMETRY
                   M.  Gruenfeld
PRELIMINARY OBSERVATIONS ON THE MODE OF ACCUMULATION OF NO.  2 FUEL OIL
                  BY THE SOFT SHELL  CLAM,  MYA ARENARIA
                   D.  Stainken
         THE ULTRASONIC DISPERSION,  SOURCE IDENTIFICATION,  AND
           QUANTITATIVE ANALYSIS OF  PETROLEUM OILS IN WATER
                   M.  Gruenfeld, R.  Frederick
    THE EFFECT OF A NO. 2 FUEL OIL AND A SOUTH LOUISIANA CRUDE OIL
        ON THE BEHAVIOR OF THE SOFT  SHELL CLAM, MYA ARENARIA L
                   D.  Stainken
       A DESCRIPTIVE EVALUATION OF THE EFFECTS OF NO.  2 FUEL OIL
         ON THE TISSUES OF THE SOFT  SHELL CLAM, MYA ARENARIA L
                   D.  Stainken

                             Bibliography

            BIBLIOGRAPHY OF PETROLEUM OIL ANALYSIS METHODS
                   M.  Gruenfeld

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                     SELECTED  REPRINTS

               OTHER ANALYTICAL PUBLICATIONS
These selected reprints describe work that was performed at the

Industrial Environmental Research Laboratory - Ci., Oil and Hazardous

Materials Spills Branch, Edison, New Jersey 08817.

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          Analytical Quality Control Newsletter #12 January 1972
Extraction of Oil from Water for Quantitative Analysis by IR   A
preliminary study was made to compare the extraction efficiency of
two solvents that are commonly used to separate oil from water.  This
study also examined the changes in extraction efficiency that result
from additions of acid and salt.  Trichlorotrifluoroethane (Freon 113)
and carbon tetrachloride (CC14) were the solvents of interest, and
both are usable for the quantitative analysis of oils by infrared
spectrophotometry.  Carbon tetrachloride exposure to laboratory
personnel can be extremely hazardous.  This material is highly toxic
when  inhaled  (10 ppm TLV), or  when absorbed  through  the  skin,  and
should  be handled with  gloves  in well ventilated  laboratory  hoods.
Freon 113 is  much safer (1,000 ppm TLV)  and  is better, particularly
in situations where adequate ventilation may be  lacking,  such as  in
mobile  laboratory and field use.  The solvents were  found to be
about equally efficient for extracting  a broad range of  oils from
water;  i.e.,  light and  heavy;  processed and  unprocessed  oils.  Freon
113 is  therefore  generally recommended  as  the  solvent  of choice.
Tests were  performed with 1 liter emulsions  prepared from tap water
and No.  2 and No. 6 fuel  oils,  which are light and heavy processed
oils, and South  Louisiana and  Bachaquero crudes,  which are  light
and moderately heavy crude oils.  Addition of  acid  and salt  dramat-
ically  improved  extraction efficiency.   When acid and  salt were not
present,  complete separation of the  oils was not  achieved even after
fifteen 25  ml solvent extractions; however,  complete separation was
achieved  with only four extractions  when 5 gms sodium chloride and
5  ml  50%  sulfuric acid  were added to the 1 liter  synthetic  samples.
Additional  salt  yielded no further  improvement.   During  the  latter
determinations,  more than 90%  of  each emulsified  oil was separated
in the  first  extract.   Quantitative  analyses were performed  by
measuring the oil absorbance band  intensity  at 2,930 cm"-'- in the
infrared  spectral region, using 10 nun and  100  mm path length po-
tassium bromide  cells and near infrared silica cells.   (M.  Gruenfeld)

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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)

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           Analytical Quality Control Newsletter #15 October 1972
          PREPARATION OF HEAVY OILS FOR INFRARED ANALYSIS

 Although  Freon is  adequate for extracting dispersed oil from
 water,  it is  not usable for preparing IR standard solutions of
 heavy oils.   Bachaquero Crude and No. 6 Fuel oils do not readily
 dissolve  in Freon.   Satisfactory "simulated" standard solutions
 were prepared by mixing 2.0 ml carbon tetrachloride containing
 known amounts of oil with  98 ml of Freon in a 100 ml volumetric
 flask.  The preferred amounts of oil  for measurements in 10 mm
 and 100 mm cells are 1 - 40 mg and 0.2 - 2.5 mg,  respectively.
 The El%cm values (absorbances in 10 mm cells normalized to 1%
 w/v dissolved oil)  of No.  2 Fuel and  South Louisiana Crude oils
 in 98%  Freon  - 2%  carbon tetrachloride are virtually identical
 to these  values  in  Freon.   All the Ei%cm values of the oils in the
 solvents  are  listed below.   This parameter may merit inclusion in
 passive tagging  profiles;  e.g.   Bachaquero Crude  oil is clearly
 distinguishable  from the other oils by virtue of  its diverging
 Ei%cm value.

                         Carbon        Freon    2% - 98%
                       Tetrachloride     113    Solvent Mix

 No. 2 Fuel Oil              23.3         21.5      21.5

 South Louisiana             25.3         22.5      23.1
 Crude Oil

Bachaquero Crude Oil        17.8                 16.5

No. 6  Fuel Oil              26.2                 23.7

                                (M.  Gruenfeld,,  201-548-3347)

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                QUANTITATIVE ANALYSIS OF OIL BY IR

 A-preliminary evaluation of infrared spectrophotometry for
 quantitating petroleum oils nas been completed.  No. 2 and
 No.  6 fuel oils and South Louisiana and Bachaquero Crude oils
 were examined.  Solutions naving known concentrations of these
 light and heavy, processed and unprocessed oils in carbon
 tetrachloride, Freon 113, and mixtures of these solvents were
 prepared.  Trichlorotrifluoroethane and Freon-TF are synony-
 mous with Freon-113 which is obtainable from E. I. DuPoat de
 Nemours, Co.  Oil absorbances were measured at 2930 cm"1 in
 10 mm path lengths silica cells at ordinate expansions 1 and 5.
 Absorbance versus concentration plots derived from the measure-
 ments were linear and passed through the origin (Lambert-Beer
 Law).  Concentrations of 0.2 - 2.5 mg/100 ml and 1-40 mg/100
 ml were preferred for measurements at ordinate expansion 1 in
 100 mm and 10 mm cells, respectively.  The detection limit of
 these oils by IR at ordinate expansion 5 using 100 mm cells was
 estimated to be 0.05 mg/100 ml.  This represents 0.05 mg/liter
 oil in water when using the extraction method described in the
 Analytical Quality Control Laboratory Newsletter #12, January
 1972.  A final volume of 100 ml was selected to facilitate
 direct correlation with the previous method.

                                  (M. GruenfeZd, 201-548-3247)


STORAGE AND TRANSPORT OF GIL CONTAINING SAMPLES IN PLASTIC BOTTLES

  HALAR plastic bottles manufactureed by Vanguard Plastics, Inc.
  (104-118 Wagaraw Road, Hawthorne, New Jersey 07506) were tested
  for storing and transporting oil containing samples for labora-
  tory analysis.  Preliminary findings indicate that these bottles
  are generally usable for this purpose.  Hexane, chloroform,
  carbon tetrachloride, and Freon 113, used as solvents for quan-
  titative analysis of oil by gravimetric and infrared spectro-
  scopiq methods, did not extract any materials from the plastic
  that interfere with:  (a)  infrared analysis of oil at concen-
  trations exceeding 2 mg/liter water (slight interference was
  found at lower levels),  or (b) gravimetric analysis of oil.
  Exposing the plastic bottles to methanol and acetone yielded
  similar results.   The HALAR bottles prevented volatile oil com-
  ponents from escaping and thereby assured sample integrity as
  effectively as glass stoppered Pyrex bottles.  They also cleansed
  free of oil, for reuse,  as easily as Pyrex containers.  A slight
  "memory" effect for polar solvents was noted, however.  Traces
  of  methanol and acetone  were not as readily removed as from Pyrex.
  Following exposure to polar solvents the plastic bottles should
  be  carefully cleansed before reuse.  These findings are based on
  the very limited number  of plastic bottles that were available
  for testing and that were repeatedly exposed to the solvents.
  The reported findings and conclusions  should therefore be con-
  sidered preliminary.   (B.  F.  Dudenbostel/M.  Gruenfeld. 201 -548-XZ47)

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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,  201-548-3347)

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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 100F) was examined by gas chromatography,
and  by  infrared, ultraviolet,  and  fluorescence  spectrophotometry.
These measurements were made  after ultrasonification  at  23C  -
36C in tap water, solvent  extraction,  and recovery after  solvent
stripping.  Comparison was  made to the  chromatogram and  spectra
of a portion of this oil not  exposed  to ultrasonification,  but
otherwise  subjected to the  same treatment.   No  significant
spectral  or chromatographic changes were  evident.  Stability  of
oil  in  water dispersions, appears  to  diminsh with increasing  oil
viscosity.  The South Louisiana crude oil yielded 1%  dispersions
that remained  stable for more  than four days.   But a  more  viscous
Bachaquero crude oil  (1,070 centistokes at 100F) yielded  less
stable  dispersions.  A model  W185  Sonifier Cell Disrupter,  sold
by Heat Systems-Ultrasonics,  Inc., Plainview,  L.I., N.Y.,  was
used.
                               (M.  Gruenfeld/F.  Behm,  201-548-3247)

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           Analytical Quality Control Newsletter #18 July 1973
            USE  OF  GAS CHROMATOGRAPHIC PEAK HEIGHT RATIOS
                FOR PASSIVE TAGGING OF PETROLEUM OILS

A  novel  GC  data handling system is under investigation as a simple
method to utilize  both the quantitative and qualitative aspects of
gas  chromatograms  for passive tagging of petroleum oils, both
weathered and unweathered.  Samples are compared by using only the
"heart fraction" of the gas chromatograms, eliminating volatile
components  and  heavy-end components from consideration to reduce
the  possible effect of environmental change, such as weathering.
A  well-resolved GC peak, present in all samples under consideration,
is chosen as the reference and the other peak heights in the
"heart fraction" are ratioed to this reference peak height.  By
comparison  of the  data obtained in this fashion on all samples
under consideration, it appears possible to "match" an environ-
mental sample with possible source mathematically to those obtained
from the environmental sample and the closest match determined.
This approach has  been shown to give almost identical results on
an authentic No. 2 fuel oil and a sample of the same oil that had
been naturally  weathered in the Sandy Hook area for one month.  The
method was  also successful in identifying the source gasoline
sample  (from among several possibilities) in two contamination cases
handled  for the New Jersey State Department of Environmental
Protection.  (B.F. Dudenbostel,  FTS 201-548-3419, Coml.  201-548-3347)

              STORAGE AND TRANSPORT OF OILS IN SOLVENTS
                   FOR QUANTITATIVE ANALYSIS BY IR	

Dispersed oil in water samples is susceptible to evaporative and
microbial degradation losses.   Prolonged transport and storage is
therefore not recommended.  Prompt solvent extraction, and sub-
sequent  transport  and storage  in the extraction solvent is a
promising alternate approach.   A preliminary evaluation of the sta-
bility of oils  in  some common  solvents was therefore performed
A  No. 2  fuel oil and a South Louisiana Crude Oil,  which are non-
viscous, moderately volatile oils,  were used.  Ten  mg/100 ml solu-
tions were  prepared in carbon  tetrachloride (CC14)  and Freon 113,
while 2 mg/100  ml  solutions  were prepared in a 2%  CC14 - 98%
Freon 113 solvent  mixture.   The solvents were  selected to correlate
this work with  oil quantitation procedures in  the  Analytical
Quality Control  Laboratory Newsletters,  Nos.  12 and 15.   All solu-
tions were  stored  in stoppered 100  ml  volumetric flasks  for eight
days, at normal  room temperature and lighting  conditions.   Solution
stabilities were monitored by  measuring the 2930 cm'1  absorbance
band of oil.  All  the  solutions  proved to be  stable;  less  than 1%
change in absorbance occurred  during the test  period.  This  pre-
liminary study suggests  that dispersed oils should  be  promptly
extracted from water, prior  to  prolonged transport  or  storage.

      (M. Gruenfeld/J. PugUs, FTS 201-548-3543,  Coml.  201-548-3347)

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 CALCULATION OF ABSORBANCE  FROM  IR ORDINATE  EXPANSION MEASUREMENTS

An equation was developed in  order to  transpose into absorbance
units the 5X ordinate expansion  readings  obtained with a Perkin-
Elmer Model 457A  (now sold  as Model 467)  infrared spectrophoto-
meter.  This instrument  expands  any 20% portion of the transmit-
tance range to cover the entire  chart  ordinate.  The equation can
also be applied to  similar  data  output of other IR instruments
with ordinate expansion  features that  operate in the same manner.
Transposition of expansion  factors other  than 5XP into absorbance
units, can be accomplished  by substituting this new expansion
factor for 5 in the numerator and denominator of the equation.


                 Absorbance = log      5B.
                                  10   5B+D-C

  B = % T of the unexpanded  baseline
  C = % T of the expanded  baseline

  D = % T of the expanded  peak maximum.

         (M. Gruenfeld,  FfS 201-548-3542 - Coml. 201-548-2347)

   GLASSWARE CLEANING FOR  THE QUANTITATION OF OIL IN WATER

 Carbon tetrachloride is frequently used for cleaning glassware to
 remove trace  organic impurities.  This solvent is expensive and
 its excessive handling is hazardous.  A comparison of the ability
 to remove oil from glassware was  therefore made between carbon
 tetrachloride and a powdered detergent (Tide Laundry Detergent).
 A small quantity of detergent and warm water was added to a
 separatory funnel coated with oil.   Light and heavy crude and
 processed oils were used.   The funnel was thoroughly shaken, and
 then rinsed repeatedly with water.   After the elimination of suds
 no interference was encountered at 2930 cm"-'-, which is the in-
 frared region used for quantitating  oil.   The detergent appears
 essentially equal to carbon tetrachloride for removing the oils.
 It is believed that other detergents will yield similar results.

     (J. Puglish/M.  Gruenfeld, FTS 201-548-3542, Coml. 201-548-3347)
                 SOLVENT EXTRACTION OF OIL FROM WATER

Difficult  emulsion problems were encountered while analyzing 200
water  samples for oil,  by the IR method described in the AQCL
Newsletter #15,  October 1972.  These samples originated from the
overboard  discharges of a refined cargo tanker during its ballast
run.   It was  determined by visual inspection that many of the
samples contained more  than 2 ml of oil  as a surface layer.  This
exceeded the  upper concentration limit of the method and often
yielded difficult-to-break emulsions.  Use of the IR method was
possible only after additions of 100 ml portions of concentrated
hydrochloric  acid to the sample - carbon tetrachloride mixtures
in the separatory funnels.   This caused the emulsions to separate
readily into  two distinct layers.

              (U.  Frank, FTS 201-548-3510, Coml. 201-548-334?)

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        Analytical Quality Control Newsletter #20 January 1974
                SULFUR INTERFERENCE IN U.V. ANALYSIS

The U.V. absorption spectrum of elemental  sulfur  has  been  found  to
resemble the absorption spectra of some petroleum oils.  As  a
consequence, leaching of elemental sulfur  from  the  environment
(sediments, et al), by spilled oils, can result in  erroneous
analyses.  This possibility should be considered  whenever  quan-
titating oil in water by a U.V. absorption technique  (Harva, 0.,
Somersalo, A., Suomen Kern, 3_1  (B) :  384-7  (1958)),  and whenever
using a U.V. method for "passive  tagging"  oils  (Levy, E. M.,
Water Research, 6_, 57-69  (1972);  also - Shields,  D. C.,  "Forensic
Aspects of Oil Pollution", presented at the 1973  CIC-CCIW
Symposium on Water Quality Parameters, Author's address  -  R.C.M.P.
Chemistry Section, Crime Detection Laboratory,  Vancouver,  B.C.,
Canada).  These conclusions derive from our limited measurements
of South Louisiana and Bachaquero Crude, and No.  2  and No.  6 Fuel
Oils.  These are viscous and non-viscous crude  and  processed oils
that are considered to be representative of many  petroleum oils.
Solutions of the oils and sulfur, in cyclohexane, were prepared
for U-V. measurement.  The spectrum of sulfur was found  to exhibit
absorption maxima at approximately 225 mp  and 260 my.  The oil
spectra also exhibit absorption maxima or  shoulders at these
approximate wavelengths.  The  spectral profiles of  South Louisiana
Crude and No. 6 Fuel Oils most closely resemble that  of  elemental
sulfur, but the spectral profile  of No. 2  Fuel  Oil  differs from
it significantly.  (J. Lafornara/M, Gruenfeld,  FTS  202-548, 3543,
                                                Coml.  201-548-3347)
            REMOVAL OF CHARRED OIL DEPOSITS FROM GLASSWARE

During our laboratory's recent participation in an ASTM collabora-
tive study of methods for "passive tagging" oils, difficulties
arose in cleaning charred oil deposits from distillation equipment
and from GC injector sleeves.  These items we.re constantly reused
and, therefore, required frequent cleaning.  Scrubbing with steel
wool and detergent, and soaking in cold Chromerge solution
(sulfuric acid and sodium dichromate) proved ineffective.  Hot
Chromerge solution was effective, but is considered too dangerous
for routine use.  Complete removal of all visible traces of oil
deposits was achieved by heating the glassware for 10 minutes at
500C, in a muffle furnace.  It is thought that this technique
can be used to remove other carbonaceous deposits from glassware
that can withstand this temperature.

  (H.  JeZeniewski/U.  Frank, FTS 201-548-3510.. Coml. 201-548-3347)

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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  tr1chlorotrlf1uorethane,  a  solvent  that
Is often recommended  for  the  quantitative  analysis  of  water
dispersed  oils.  While this  term  describes  two  isomers,  the
Isomer  that  Is  actually the  recommended   solvent   Is   1,1,2-
trIchloro-1,2,2-trlfluoroethane.   It  is  available  from E.  I.
DuPont  De   Nemours and Company as a  refrigerant  (Freon-113)
and as  a cleaning agent (Freon  TF).   It   is   also   available
from  a  number  of   chemical   supply  companies,  in  several
grades  of  purity.  (M. Gruenfeld., FTS  201-548-3543, Coml.  201-548
                                                            3347)

       EVALUATION OF A PORTABLE IR SPECTROPHOTOMETER


A preliminary  evaluation   of   a  compact  portable   infrared
spectrophotometer  that   Is   suitable  for  the  quantitative
analysis of  oMs  In the field,  Is  described.   This   unit,   a
MIran   I   Fixed  Filter   Infrared  Analyzer (Wllks  Scientific
Corporation, South Norwalk,  Connecticut) is   a  single  beam
device  with  a  fixed 3.4 micron  filter.   It accommodates  a  10
mm  path length  sample  cell,  Incorporates  ordlnate  expansion
capability 5x  and 20x,  and can  operate off a   12-volt  auto-
mobile  battery.   Battery operation was used during  most  of
this study,  but AC operation  was found  to  yield   identical
readings.    Solutions   of  four  oils  (South  Louisiana  and
Bachaquero Crude and  No.  2 and  No. 6  Fuel  Oils)   with  known
concentrations   In  carbon tetrachloride were measured in  10
mm path length  cells  without  ordlnate  scale   expansion,   and
by  using  expansion  settings 5x and  20x.   Absorbance  versus
concentration  curves  were  generated,  and these prove   to   be
nonllnean.   Mlran  "absorbances"  do  not  match  absorbance
readings of  a  double  beam  IR  Instrument.  In  accordance  with
a recommendation by Wllks,   appropriate   correction  factors
were  determined as the difference between linearity  and  the
curve of one oil.  These  correction  factors,  when  applied  to
the curves of the oils, translated them   into,  linear  plots
that  passed  through  the  origin.    This  demonstrates  the
utility of the  Instrument  for single point analysis of oils.
Similar curves were also derived  by   using   ordinate  scale
expansions,  but  no  attempt  was  marie to  correct these  to
linear  plots.  At ordlnate expansion 20x,  points were  quite
scattered  and  did  not   yield a  smooth curve.  The  concen-
tration range 0.1 - 150  mg/100  ml  oil   in   carbon   tetra-
chlorlde  was  examined in this study, and the lower  concen-
tration figures Is considered to be the  reasonable  detection
limit of the Instrument at 20x expansion.    This   represents
an  oil In water concentration of  0.1  ppn  when 1  liter water
samples are extracted with 100 ml carbon tetroch1 oridp.

    (M.  Gruenfeld/U. Frank, FTS 201-548-3543,  Coml.  201-548-334?)

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          Analytical Quality Control Newsletter #24 January 1975
   IDENTIFICATION OF MILLIGRAM QUANTITIES OF  PETROLEUM  OILS

Several  new  methods  for  fingerprinting petroleum  oils  have
been published in the 19714 Annual Book of ASTM Standards,  Part
31, Water.  They include a procedure  for  sample   preparation
and    laboratory   weathering    (D332G-71+T),   and    for   gas
chromatographic correlation (D3328-74T).  Fairly  large  volumes
of oil (50 ml) are required by D332G, and this amount   may   be
difficult to  collect at the scene of a spill.  This is  also  an
.excessive  amount  of oil to extract from the water column for
those  analyses requiring  fingerprinting  of  water   dispersed
oils.  A procedure has therefore been developed for laboratory
weathering    and  gas  chromatographic  correlation   of  small
amounts  of   oil,  i.e.,   0.5-30  mg.   Our   procedure  yields
chromatograms  that closely resemble those of D3328,  Method  A,
(packed column GO; work with SCOT   columns   (Method  B)  will
soon be undertaken.  Carbon tetrachloride or  1,1,2  -  trichloro
- 1,2,2,  - trif1uorethane (Freon 113, et al.) solutions of the
spilled  oils  are  quantitatively   analyzed  by  IR.  Solution
volumes of 100 ml are then prepared,  which contain  either  0.5
or  30  mg  oil.    A steam table and a filtered air stream are
used to strip the solutions to a final 1-2 ml volume  in 150  ml
beakers,  and  these concentrates are  transferred to  10 x 30   nm
vials.   The  vials  that  contain   the 30 mg oil portions are
suspended in  a kQC water bath,  and  a filtered air  stream   is
used   to remove the final trace of solvent.    This condition  is
then maintained for an additional 10 minutes.  The  vials  that
contain  0.5  mg portions of oil are held at  room temperature,
the airflow is stopped just before total solvent  removal,  and
final  solvent  evaporation  is  achieved spontaneously.  This
condition is  then maintained for  an  additional   10  minutes.
Distilled   solvent   is  used  for  the  lower  concent ratior
determinations.  Small  amounts of CC1 4 (10-20 VI ) are then added
to each vial  prior to GC injection.   Laboratory weathering   of
the  reference oils Is achieved by suspending vials containing
70 mg of each reference oil  in a  kQ C  water  bath,   for   15
minutes,   in  the presence of a filtered air  stream.  Portions
of these  reference  oils  are  then  injected  onto  the  GC.
(Michael   Gruenfeld/R.  Frederick, FTS 201-548-35U3  Coml. 201-
5U8-33i*7)

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         Analytical Quality Control Newsletter #26 July 1975
         Separation of Petroleum and  Non-Petroleum  Oils

The  direct  addition of silica gel  to  freon  has  been  recommended
(Newsletter #22, p 7) for separating  petroleum  oils  from   animal
oils and vegetable oils.  Silica gel  removes  the  non-hydrocarbons
from  the  solution  while   leaving   the  hydrocarbons  unaffected.
This  technique  avoids  column    chromatographic   and    solvent
stripping  steps  and   is   therefore  very  straightforward.    We
examined this method as part of a  project  to  develop a  procedure
for  quantitatlng  1  ppb levels of  petroleum oils  in  water.   The
Interaction of silica gel with petroleum  and  non-petroleum  oils
In carbon tetrachloride was monitored.  The consequence  of  silica
gel  deactivatlon  was  also examined.  This  information was  then
plotted versus the degree of oil removal,  and  the   Influence  of
different  stirring  times.  The resulting  graph  suggests  optimum
operating  conditions,  and  Is  available    on   request.     (M.
Gruenfeld, FTS 201-5^8-351*3, Coml.  201-51+8-3347).

-------
        Analytical Quality Control Newsletter #29 April 1976
                     Replicate Oil Chromatograms

Replicate high resolution gas chromatograms of  four weathered
and unweathered crude oils were obtained in order  to  evaluate
a pattern recognition computer program for fingerprinting  oils.
This program was previously described in AQC Newsletter No.  25
April 1975, Page 8.  Ten replicate runs of each oil  (total of
80 chromatograms) were prepared, using a 50 ft  OV-101  SCOT
column.  The injection and quality assurance techniques used
were in  accordance with  Technical  Report  72-55  of  the Woods
Hole Oceanographic Institution  (1972) by  Zafiriou, 0., Blumer,
M., and  Myers, J.  Photocopies  of  the chromatograms are avail-
able on  loan,  for  duplication,  together with  information re-
garding  oil  identity  and weathering  history.    (M.  Gruenfeld,
FTS 342-7543,  Coml.  201-548-3347,  M. Urban, FTS 342-7517)

-------
         Analytical Quality Control Newsletter #30 July 1976
            Determination of  Oil  in Sediment by NMR

Nuclear magnetic  resonance  (NMR)  spectrometry was  found to be a
rapid and  useful  technique  for determining  the presence of
petroleum  contamination  in  aquatic sediment samples.   Deter-
minations  were  performed in  support of  a  study conducted by
Region II  to  establish whether a  major  oil  spill,  that occurred
in 1973, caused long  term irreversible  damage and  plant mor-
talities in a mangrove swamp in Puerto  Rico.   Column  chroma-
tographic  "clean-up11  procedures and gas chromatography (GLC-FID)
were initially  used to analyze carbon tetrachloride extracts of
the sediments,  but failed to provide conclusive results because
of interferences  from possible biogenic organics.  Extracts of
sediments  from  both the  spill site and  a  similar uncontaminated
location  (control) were  therefore examined  with our 60MHz  NMR
spectrometer.   The extracts  from  the spill  site were  found to
exhibit absorptions in the range  6.5 -  8.0  ppm (ring  aromatic
protons)  and  0.5  - 2.0 ppm  (paraffinic  protons)  relative to
tetramethylsilane  (TMS);  the control extracts exhibited no
aromatic absorptions.  The low-field aromatic absorptions  found
only in sediments from the spill  site were  ascribed to the pre-
sence of petroleum oil suspected  of originating from  the past
spill incident.   (U.  Frank^  FTS 342-7510, Coml.  201-548-3347,
M. Gruenfeld, FTS 342-7543,  Coml.  201-548-3347)

-------
 Reprinted  from:  Proceedings 1973  Conference  on Prevention  and Control  of  Oil
 Spills,  March  13-15,  1973, Washington, B.C., API,  Wash.,  DC
                 IDENTIFICATION OF OIL  POLLUTANTS:
                A  REVIEW  OF  SOME  RECENT METHODS
                                               Michael Gruenfeld
                                       U.S. Environmental Protection Agency
                                     Edison Water Quality Research Laboratory
                                National Environmental Research Center (Cincinnati)
                                               Edison, New Jersey
 ABSTRACT
    Some recent studies of methods for identifying weathered
oils through chemical fingerprints (passive tagging) are re-
ported. These studies were performed by Esso Research and
Engineering Company, Phillips Scientific Corporation, Woods
Hole Oceanographic Institution, and Baird Atomic Corpora-
tion under U.S.  Environmental  Protection  Agency (EPA)
sponsored grants and contracts. A broad range of analytical
instruments  and  techniques  were  used,  e.g., adsorption
chromalography,  molecular emission and absorption  spec-
trophotometry, atomic  absorption spectrophotometry, gas
chromalography,  computerized mass  spectrometry, el. al.
Some of the oil parameters evaluated as potential finger-
print indices are  vanadium,  nickel, sulfur and nitrogen con-
tent, gas chromatographic profile appearance,  carbon and
sulfur isotope ratios, API gravity, and pour point.  Several
promising methods for passive  tagging  oils are suggested
by these studies.

 INTRODUCTION
    Discharges and spills of crude,  residual, and  distillate
oils are  occurring with ever  increasing frequency in coastal
waters. They cause extensive damage to marine life, coastal
life, recreational beaches, and to their dependent industries,
and are therefore of special concern to  the U.S. Environ-
mental  Protection Agency  (EPA).   Establishing  the true
source of the pollutant, where  several suspect sources  exist,
and proving identity between the weathered  residue and an
unweathered authentic  often  requires extensive analytical
instrumental procedures. Many methods  are now used, but
there is  a  lack of agreement among laboratories about the
advantage or necessity  of any  single technique or combina-
tion of techniques. This variety of methods is illustrated in a
recent publication1 that reviews some current procedures for
correlating weathered and unweathered oils (passive  tagging).
Some techniques  such as infrared spectrophotometry require
only simple inexpensive instrumentation that is readily avail-
able and usable by most laboratory personnel. But other tech-
niques, such  as  neutron activation analysis,  require such
expensive instruments and highly specialized personnel  that
they are available in very few laboratories.
   The Edison  Water Quality Research  Laboratory,  a
Laboratory  of  EPA's National Environmental  Research
Center   in  Cincinnati  (NERC),  investigated  numerous
methods for passive tagging of oils  that occur  as slicks  and
shoreline  residues. Several analytical systems that integrate
simple  techniques  such as gas chromatography with more
sophisticated ones such as mass spectrometry were developed
through grants and contracts.  These  techniques were eval-
uated  singly and  in  combination  as a basis for  recom-
mending reasonable analytical  approaches to oil identifica-
tion problems.  Funding was provided to the Esso Research
and Engineering Company2, the Phillips Scientific Corpora-
tion3, the Woods Hole Oceanographic Institution4, and the
Baird  Atomic  Corporation^.  Resulting analytical methods
and oil parameters measured are summarized (Table 1).

The Esso  System
   Esso  examines  a  variety of crude and processed  oils
(Table  2) by mass spectrometry and gas chromatography to
determine  high  molecular  weight paraffins, naphthenes
(cyclic  paraffins),  and polynuclear  aromatics.  Emission
spectroscopy is used  to  determine  bulk  vanadium-nickel
content; X-ray spectroscopy  and Kjeldahl analysis are used
to  determine  bulk  sulfur-nitrogen   content.  Adsorption
chromatography with several different columns  is used to
isolate  paraffins, naphthenes,  and  aromatics  from  the oil
matrix  and to separate the  aromatics from the aliphatics.
A schematic  presentation of  these analysis  steps  is  pro-
vided (Figure 1).
    Portions of the  authentic  and  weathered, samples are
first separated  for  determination of bulk  nickel-vanadium
and sulfur-nitrogen content.  Separate  portions are distilled

'The Baird Atomic study was funded through the EPA Analytical
Quality Control  Laboratory, NERC, Cincinnati.
'Weathering of oils is simulated by using a continuously recirculat-
ing salt water system with controlled temperature,  agitation,  light
and wind conditions.
                                                     179

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180
IDENTIFICATION OF OIL
                         Table 1: Summary of Techniques, Fingerprint Indices, and Investigators
            Oil Parameter
     Vanadium & Nickel Content

     Nitrogen Content

     Sulfur Content

     Total Profile


     N-Paraffins

         Isotopic Composition
     Carbon & Sulfur in:
     Total High Boiling Residue
     Carbon in:
     Saturate Fraction
     Aromatic Fraction
     Asphaltic Fraction
     Polynuclear Aromatics
      Naphthenes
      Saturates
      Polynuclear Aromatics
      Asphaltics
      API Gravity
      Pour Point
                                           Method of Determination
                          Emission Spectrometry
                          Atomic Absorption Spectrophotometry
                          Kjeldahl Method
                          Dumas Method & Gas Chromatography
                          X-Ray Spectroscopy
                          ASTM Method3
                          Gas Chromatography (Flame lonization):
                             Packed Column
                             Open Tubular  Column
                          Gas Chromatography/Adsorption Chromatography
                          Gas Chromatography/Urea Adduction
                          Isotope Mass Spectrometry

                          Isotope Mass Spectrometry
                          Isotope Mass Spectrometry
                          Isotope Mass Spectrometry

                          Gas Chromatography
                          Mass Spectrometry (Low Voltage)
                          Ultraviolet Absorption Spectrophotometry
                          Fluorescence and Phophorescence Spectrophotometry (77K)
                          Mass Spectrometry (High Voltage)
                          Gravimetric/Adsorption  Chromatography
                          Gravimetric/Adsorption  Chromatography
                          Gravimetric/Adsorption  Chromatography

                          ASTM Method3
                          ASTM Method3
Investigator

     1
     2
     1
     2
     1
     2

     2
     3
     1
     2
                   1-Esso Research and Engineering Company
                   2-Phillips Scientific Corporation
                   3-Woods Hole Oceanographic Institute
                   4-Baird Atomic Corporation
      3ASTM Method cited in text.
to remove components boiling  below 400F; these volatiles
are  discarded.  Accurately  weighed portions  of  the  higher
boiling residues are dispersed in 50 cc n-pentane, insolubles
are  separated by centrifugation and discarded.  Each solu-
tion is then fed to a clay column and successively  eluted with
n-pentane, benzene-acetone,  and  acetone.  The  n-pentane
eluate preferentially contains  the paraffins,  naphthenes, and
aromatics, and  the acetone-benzene and  acetone eluates
contain the more polar sample fractions. These polar ma-
terials are thought to be more  water soluble and  therefore
unreliable indices; they are  not  used in the  analysis scheme.
                                                   The n-pentane eluate is stripped of solvent, and yields a con-
                                                   centrate rich in paraffins, naphthenes and aromatics which is
                                                   fed  to a  silica gel column. The  aliphatic  components are
                                                   selectively eluted with n-pentane, and  the  aromatic  com-
                                                   ponents with acetone. The two solvents are then stripped and
                                                   the  residues weighed. These material  balance data  may be
                                                   useful  for oil characterization (Table 3). The saturate frac-
                                                   tion is subsequently analyzed by gas Chromatography for n-
                                                   paraffins  and  by computerized  high  voltage  mass  spec-
                                                   trometry for various naphthene  types. The aromatic  fraction
                                                   is analyzed by low voltage  computerized mass  Spectrometry
                                       Table 2:  Oil Samples Examined by Esso

               Sample No.                   Sample Type                   Oil Field          Location
                    1             Crude oil                               Tia Juana         Venezuela
                    2             Crude oil                               Lago             Venezuela
                    3             Crude oil                               Grande Isle       Indonesia
                    4             Crude oil                               Nigeria           Nigeria
                    5             Crude oil                               Zuitina           Libya
                    6             No. 2 Heating oil
                    7             No. 4 Fuel oil     Refined and
                    8             No. 5 Fuel oil     formulated from
                                                  Venezuelan stock

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                                                                              POLLUTANTS
                                                                 181
Sulfur Analysis

     X-Ray
 Spectroscopy
          Pentane 1
Benzene + Acetone 2
          Acetone 3
          Pentane 1
 to
400F

ition
F

\

Nickel/ Vanadium
Analysis
Emission
Spectroscopy

\
!
Nitrogen
Analysis
Kjeldahl
                                    Residue
                           Centrifugation
                           Clay Separation
                                                Insolubles .
                                               Acetone  + Polars
                                               Benzene
                                    Pentane
                                    Paraffins + Naphthenes
                                    Aromatics
                                           Aromatics,
                                            Acetone
                     n-Paraffin      Aromatic Analysis - Low Voltage
                      Analysis       Naphthene Analysis (P + N fraction) - High Voltage
                             Figure 1: Analysis Schematic of the Esso System.

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182
IDENTIFICATION OF OIL
                       Table 3:  Typical Material Balance Data from Esso Sample Separation Processing3
               Oil Type
          Tia Juana Medium
            Crude Oil
          Lago Crude Oil
          Grande Isle Mix
            Crude Oil
          Nigerian Crude Oil
          Zuitina Crude Oil
          No. 2 Heating Oil
          No. 4 Heating Oil
          No. 5 Heating Oil

          Replicate  Tia Juana
          Medium Crude Oils
                          Paraffins
                              +
                         Naphthenes
                            46.0

                            39.8
                            73.0

                            61.2
                            66.1
                            49.7
                            63.2
                            64.2

                            48.2
                            47.7
                            47.4
Aromatics
  21.6

  21.8
  16.1

  20.7
  12.1
  46.6
  26.2
  20.4

  19.5
  20.8
  18.6
Weight

Polars
19.3
24.7
12.3
10.6
15.2
0.0
8.6
12.1
18.4
20.4
21.5
% of Sample
Pentane
Insolubles
9.0
15.5
1.2
1.0
3.1
0.2
1.0
5.8
10.9
9.4
10.9
2 Fractions
    95.9

   101.8
   102.6

    93.5
    96.5
    96.5
    99.0
   102.5

    97.0
    97.9
    98.4
          aData based on unweathered samples.
for various polynuclear aromatic types. Twenty-six promising
fingerprint indices derived by these techniques (Table 4) are
subjected to  Discriminant Function Analysis to identify the
best discriminators for the test oils (Table 5). This statistical
treatment is  needed to isolate five indices having the lowest
probability of mismatching  two  oils (Table 5).  Estimation
of  confidence  levels for  oil classification could  also  have
been performed with the  Bonferroni "t"  Statistics.
    In this  study,  Esso  uses only four fingerprint indices
and,  with high statistical confidence,  distinguishes among
any possible pairs of the  oils, even after the oils have ex-
perienced extensive  laboratory  weathering. These  indices
are generally  unaffected  by simulated  weathering, but  their
applicability  to other  oils has not been established. Other
indices may be required for individual situations.

Another Esso  Approach
    In a supplemental study Esso evaluates a combined gas
chromatographic-ultraviolet  spectrophotometric   procedure
for passive  tagging oils  (Figure  2). Individual polynuclear
aromatics (PNA's) separated by gas chromatography are col-
lected  and   measured by  ultraviolet  absorption  spectro-
photometry.  After 1-gram  portions of the weathered  and un-
weathered oils are extracted with caustic  and dissolved in
cyclohexane,    triphenyl   benzene  is   added  as   internal
standard. Each solution  is  fed  to a  deactivated alumina
column (2%H O)  that is successively eluted with cyclohexane,
cyclohexane/benzene, benzene, and  benzene/methanol. The
cyclohexane/benzene fraction,  which is enriched in three-
ring and heavier PNA's, is stripped of solvent and the residue
dispersed in  toluene. A gas  chromatogram  of  the PNA's is
then obtained (Figure 3). Selected components appearing as
gas chromatographic peaks are trapped and their ultraviolet
absorption spectra  obtained. These  spectra together  with
appropriate calibration coefficients are used to quantitate the
individual polynuclear aromatics  in the oils (Table 6).  Esso
uses these quantitative results and visual examination of the
chromatograms for passive tagging oils. Preliminary  data in-
dicate that this approach  is promising.

The Phillips Study
    Phillips surveys  a multitude of parameters that  may  be
useful for passive tagging oils. Main emphasis is on iden-
                                                    tifying  promising "fingerprint"  indices; extensive controlled
                                                    weathering experiments are  not  performed. Seventy-seven
                                                    crude oils representing world-wide production (Appendix A)
                                                    are stripped of components having boiling points below 600F
                                                    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 214F at  0.15 mm Hg.  This is equivalent to  ap-
                                                    proximately  600F atmospheric. The 600 F + residuals  are
                                                    analyzed for total nitrogen (Dumas method followed by  gas
                                                    chromatography); sulfur content (ASTM method D 1552-
                                                    64); API gravity and pour point (ASTM  methods D287-67
                                                    and  D97-66,  respectively);  total nickel and vanadium (in-
                                                    cineration in the presence  of  sulfur and subsequent analysis
                                                    by atomic absorption spectrophotometry); carbon and sulfur
                                                    isotope ratios (mass spectrometry);  and gas chromatographic
                                                    profile  appearance. A novel classification scheme is used for
                                                    coding  gas chromatographic information in a form amenable
                                                    to  computer input: a straight line is drawn between  the  20
                                                    and Cso peak tips, and the locations of the intermediate  nine
                                                    n-paraffin peaks are noted as "above" or "under" the line
                                                    (Figure  5). Designating the gas  chromatographic profile as
                                                    Type A  (above) or Type U (under) is based on the location of
                                                    the majority of peaks. The number  of peaks in the minority
                                                    is designated by a digit following the type letter. For example,
                                                    Type U-2 indicates that seven peaks are  under the line and
                                                    two above (Figure 5). A Type B profile  has a broad envelope
                                                    with n-paraffin peaks poorly defined, which prevents the con-
                                                    struction of the characterization line between peaks  (Figure
                                                    6). A summary of all these "fingerprint" indices  is presented
                                                    (Table 7).
                                                        Phillips  examines   still  more   potential  "fingerprint"
                                                    indices.  The 600 F + residuals are separated by adsorption
                                                    chromatography over silica into three fractions:  saturates,
                                                    aromatics,  and  asphaltics (Figure  4).  Pentane,  dichloro-
                                                    methane,  and methanol-dichloromethane, respectively,  are

-------
                                                                                        POLLUTANTS
                                                                                               183
                                    Table 4: Some Promising Esso Finger
                 V
                 Ni
                                                       23 Ring Naphthenes
                                                       2    (P + N)
                 N
                                                       24 Ring Naphthenes
                                                       2    (P + N)
               -20
            UnParaffin
          c = 20
          c = 40
5 Ring Naphthenes
2(P + N)
               C
                21
            EnParaffin
          c = 20
          c = 40
  CnH2n-6
  CnH2n-i8
               C24
            2nParaffin
           c = 20
           c = 40
                                                                   2 CnH2n -14
            2nParaffin
           c = 20
           c = 40
2-1 Ring + 2 Ring Naphthenes
25 Ring + 6 Ring Naphthenes
                -26
            2nParaffLn
           c=20
           c = 40
2CnH2n_6   (Benzenes)
2 Aromatics
               C
                27
            2nParaffin
           c = 20
           c = 40
2 CnH2n_6   c- 20
2 Aromatics
            2nParaffin
               C
                31
            2nParaffin
            2C
               20
2 C30
           c = 20
           c = 40
           c = 20
           c = 40
                    -1\
                          -22
                          C32
                                                                   2 CnH2n_i0  (Indenes)
                                                                   2 Aromatics
2CnH2n_10  c = 20
2 Aromatics
                                                       2CnH2n_14  (Acenopthenes)
                                                       2 Aromatics
            2 C20 + C21 + C22 + C30 + C31  + C32
2  C
                24
                      25
                               4- C27
                                        28
                                                       2CnH2n_16  (Acenopthalenes)
                                                       2 Aromatics
            2nParaffins
            2 (P+N)
                                                       2 Aromatics
                                                                    (Phenanthrenes)
used for elution. Percent composition of each fraction in the
600 F + residuals, and  the  carbon  isotope ratios in  these
fractions are measured as additional  indices. The n-paraffins
in the saturates fraction are further separated by urea adduc-
tion and measured by gas chromatography for relative abun-
dance  according  to  carbon  number  distribution.   Odd-
numbered  to even-numbered n-paraffins are calculated  as a
function of carbon number.  A summary of these additional
"fingerprint" indices  is  presented  (Table 8).  A  limited
                                               assessment is  also made of measurement repeatability and
                                               weathering effects.

                                               The Woods Hole Oceanographic
                                               Institution System
                                                  Woods Hole Oceanographic Institution (WHOI) uses gas
                                               chromatography with support-coated  open tubular (SCOT)
                                               columns for passive  tagging oils.  The oils are merely dis-

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184      IDENTIFICATION OF OIL
    Table 5:  Most Promising Esso Fingerprint Indices

                           V_
                           Ni
                      SnParaffins
                  S-5 Ring Naphthenes
                  2 (P + N)
                        _   _c=
                     HnParaffinc=4o
              S-l Ring+2 Ring Naphthenes
              2-5 Ring+6 Ring Naphthenes
solved  in  carbon disulfide (ca 5-10% W/V)  and  injected;
exact concentrations need not be known. The gas  chroma-
tograph injector is  equipped with a  glass liner that is re-
movable  for  cleaning.  Column  specifications are  50  feet
x  0.02  inch  packed  with  nonpolar liquid silicone  OV-101
and rated at  25,000 effective plates.  Oil  chromatograms
are  compared  visually,  and certain features  are  abstracted
and tabulated  when analyzing many samples  and  for  sta-
tistical  purposes.  These features  are measured as  vertical
distances between superimposed baselines  and appropriate
peak heights (Figure 7, Table 9).
    The WHOI method requires frequent evaluation of sys-
tem performance by injecting  a standard oi!3 that  yields a
repeatable known chromatogram. The extent of weathering
undergone  by oils  is estimated from  the onset of the gas
chromatographic  signal:  if below n-C]4 then the tabulated
indices  are considered unaffected by  weathering, but  if at
or  above n-C]5  then weathering  alterations  are  implied.
Tabulated indices of oils  that  exhibit considerable weath-
ering  should  not be compared directly  with the  indices of
unweathered  oils.  Instead,  each  unweathered  suspect  is
artificially weathered in a rotary  evaporator  by dropping
pressure slowly to a high vacuum while heating to  60 C or
warmer. Small  portions are  withdrawn periodically for GC
analysis and the procedure continued until the oil resembles
the environmental sample.  Similarity is judged by the shape
of low-boiling   range signals, or by comparing background
ratios at Q5 to any signal parameter at CI7 or Qg. The re-
maining indices (Table 9) are then tabulated and  compared
to those of the weathered sample.  SCOT column  lifetimes
generally exceed 200 oil  analyses (injections), and tabulated

3A Number 2 Fuel Oil "stabilized" by passage through  an alumina
and silica gel column.
                                                   Add Cyclohexane and

                                                   Internal Standard

                                                   to Sample
                                                 Separate on A1203 ( + 27. H20)

                                                 Elute by Cyclohexane,

                                                  Cyclohexane/Benzene, Benzene

                                                  Benzene/Mechanol
Cut 1A
125 ml
eye lohexane

Colorless
Fractions

Front Cue Point
a c Appearance
o Color

Evaporate, Add
Toluene


                                                     157.
                                   Figure 2:  Analysis Schematic of Another Esso Approach.

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                                                                                         POLLUTANTS      185
Figure 3: Gas Chromatogram by Esso of Polynuclear Aromatics in a Crude Oil. Marked Peaks Are Identified in Table 6. Triphenyl Benzene
(TPB) Is Used as Internal Standard.
                             Table 6: GC Components Trapped by Esso for UV Analysis
             Peak Aa          fluorene
             Peak B    methyl fluorene
             Peak C           phenanthrene
             Peak D  2-methyl phenanthrene
             Peak E  1-methyl phenanthrene
             Peak F  dimethyl phenanthrene
              aGC peaks are identified in Figure 3

-------
186
IDENTIFICATION OF OIL
                                  CRUDE   OIL
                                Isothermal
                                       Distillation
                                       0,15 nm
         RESIDUE, WT.  %
       (600 F+ Fraction)
        Liquid-Solid
       Chromatography
       Silica
                   Measxrra

                     API  Gravity
                     Pour Point
                     Sulfur  Content
                     Nitrogen  Content
                     Nickel  +  Vanadium Content
                     Carbon  Isotopic Composition
                     Sulfur  Isotopic Composition
                     GLC  Profile
                           DISTILLATE
   SATURATES
                                       AROMATICS


ASPHALTICS
           Measure

         %(w/w) In 600F+ Fraction
         Carbon Isotopic Composition
    Urea Adduction
                               Measure
                                  Measure
                                           600 F+
%(w/w) In
Fraction
Carbon Isotopic
Composition
600F+
                                                                 Fraction
                                                                 Carbon Isotopic
                                                                 Composition
  n-Paraffins
       Measure
         GLC Carbon Nimber Distribution-
         Calculate OEP Ratio
                            Figure 4: Analysis Schematic of the Phillips Study.

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                               POLLUTANTS
                                                                                                                  187
                         Typo U-2 Profile
                         Figure 5:  Coding of Gas Chromatogram Profile by Phillips  Type U-2 System.
indices of individual  oils remain quite  stable from  column
to column.
    WHOI reports on a preliminary evaluation of their system
with oils exposed by the Edison EPA Laboratory to various
weathering  conditions for different  time intervals. Common
spill-control  chemicals were also added  to some of the oils.
Unweathered portions of  16 out of 17 oils used for this test
accompanied 35  simulated  spill  samples.  All the samples
were number coded and identified only as  potential sources
or  simulated spills.  Test results are reported  as  "definite
correlation",  "probable  correlation",  or  "no  definite or
probable  correlation"   Correct  "definite  correlation"  is
achieved in 74% of the cases, and only one "probable correla-
tion"  is incorrect (Table 10).  An evaluation of the system's
ability to distinguish among similar unweathered oils and oil
products is also reported;  in  the majority of cases  the  oils
were uniquely identifiable (Table  11).  It is thought that since
short  term weathering does not significantly affect the cor-
relation ability of the method,  these  results  obtained  with
unweathered oils also apply to many weathered oils.


The  Baird Atomic Study
    Baird-Atomic  provides a preliminary  assessment  of
molecular emission  (fluorescence  and  phosphorescence) at
low temperatures as a method for identifying oils. Their ef-

-------
188      IDENTIFICATION OF OIL
                                C29
                                                                             18
                            Figure 6: Coding of Gas Chromatogram Profile by Phillips  Type B System.
 fort  is a probe  to evaluate the utility  of the technique for
 discriminating among oils and to determine  whether an ad-
 vantage  derives from using  cryogenic temperatures.  This
 study  deals  mostly  with unweathered  heavy  crude  and
 processed oils  (Table 12);  only two  weathered oils  were
 tested and no substantial effort was made to correlate  them
 with the appropriate unweathered oils. Emphasis is on asses-
 sing  the utility  of low temperature molecular emission for
 distinguishing among different oils and not  to  demonstrate
 the  utility  of  this  technique  for  correlating  appropriate
 weathered and unweathered oils; i.e., passive tagging.
    The oils are dissolved in methylcyclohexane and cooled to
 77 K. Methylcyclohexane is a  good solvent that forms a clear
 glass at the cryogenic temperature.  The use of other solvents
 and  different oil concentrations is  described; 10 ppm oil in
 methylcyclohexane  yields the  most  satisfactory  results.
 290  mju and 340  mji  are  the diagnostic excitation wave-
 lengths  used  to  compare the  oils. In every case oil emission
 spectra  resulting from excitation at  340 m^t are more intense
 than those resulting from excitation at 290 m/j., but excitation
 at  this latter  wavelength yields broader emission spectra; all
the oils  yield characteristic  emissions  in  the  380-400 m^t
region when excited at 340 mju. Dramatically improved spec-
tral resolution is achieved by cooling to 77K (Figures  8a
and  8b).  These  emission  spectra at cryogenic temperature
are generally a mixture of fluorescence and phosphorescence,
the  latter  occurring  at  the  longer  wavelengths.   Phos-
phorescence and fluorescence spectra of several oils are com-
pared; they  differ sufficiently  to distinguish the oils. This
study also attempts to identify major luminescing compound
types in the oils. Porphyrins,  compounds similar to 4-methyl-
pyrene, et.  al.,  are  identified.  Several  solvents  in addition
to methylcyclohexane  are  also  evaluated; they include pen-
tane,  hexane, heptane, and  octane.  Initial difficulties with
spectral resolution were traced to oxygen-quenching that was
more evident in these normal paraffin solvents.  The difficulty
was resolved by degassing the solutions with dry nitrogen.
    The  Baird  Atomic  study  demonstrates  the  potential
utility of low temperature molecular emission  for  passive
tagging oils. Cooling to cryogenic temperature enhances spec-
tral  resolution  and  the  technique can  differentiate  among
very similar oils; e.g., several number 6 f"l oils (Figure  9).
         Table 7:  Initial Phillips Fingerprint Indices

             API Gravity
             Pour Point
             Carbon Isotopic Composition
             Sulfur Isotopic Composition
             Nickel/Vanadium Content (Ratio)
             Sulfur/Nitrogen Content (Ratio)
             GC Profile
      Table 8: Additional Phillips Fingerprint Indices

         % (w/w) Saturates
         % (w/w) Aromatics
         % (w/w) Asphaltics
         Odd-Even N-Paraffin Predominance
         Carbon Isotopic Composition (Saturates)
         Carbon Isotopic Composition (Aromatics)
         Carbon Isotopic Composition (Asphaltics)

-------
                                                                                     POLLUTANTS
                                                                                         189
                                     DEFINITION  OF TERMS
                                                                 CHROM'ATOGRAM SERIES NO:
                                                                       216
                                                                       -^ ^ ^~r
                                                                   COLUMN
                                                                       NO.
                                                                        INJECTION
                                                                             NO.
                                                            CHART  SPEED	
                                                           I/2"=IMIN= 6C  !
                                                                  i             '
                                                             -PROGRAM  START!
                                                             CS2 SOLVENT PEAK
                                                                BASE LINE
                               -INCREASING TEMPERATURE
                                                                                             INJECTIONJ
                   Figure 7: Gas Chromatogram by Woods Hole Oceanographic Institution with Terms Defined.
      Table 9: Woods Hole Oceanographic Institute
                 Fingerprint Indices3
     Pristane/Phytane
     C17/ Pristane
     C18/Phytane
           C17/Background
           C17/C17-Pristane Valley
     a - Ratios measured as in Figure 7.


This  study does not evaluate  the  susceptibility of low
temperature fluorescence spectra  to  changes  that  result
from weathering.

Discussion
   These EPA sponsored  studies are an effort to develop
passive  tagging methods  having broad scope of application,
and to appraise the usefulness of many analytical techniques
for this purpose. Methods are sought that permit identifying
   weathered oils by  comparing  them with libraries of unn-
   weathered oil indices, but that  can also be readily simplified
   for comparing a weathered oil  with only  a few unweathered
   oils. Although comparison with  a few unweathered oils is now
   the common procedure, libraries of standard oil indices may
   come into use around  harbors and other  high  density oil
   storage and handling locations.
      Of these  studies,  only  those by Esso and WHOI yield
   ready-to-use  passive  tagging methods.  The  others mostly
   evaluate  promising oil parameters and analytical  techniques,
   and determine their  utility for pairing  and  discriminating
   among unweathered oils. Phillips and Baird Atomic do not
   perform extensive weathering tests and, therefore, do not con-
   firm the reliability of their parameters and techniques for
   passive  tagging oils.  A considerable difference  also  exists
   in  the  usability  of the instrumental  techniques  in  these
   studies by "average" analytical laboratories.
      The  Esso System yields a ready-to-use  passive tagging
   method that can be applied, in  whole or in part, according to
   analysis needs and available instrumentation. For example, if
                  Table 10: Woods Hole Oceanographic Institute Test Results of EPA Weathered Samples
                        Prepared by EPA
              17 Source Oils:   35 Simulated Spills:
               8 Crudes       Exposures:
5 Fuels

4 Re-refined
                             1,3, 30 Days

                             Freshwater, Saltwater,
                             Beach Sand
                             With or Without 1 of 4
                             Spill Control Chemicals
                             Added.
Correlated by Woods Hole
26 Unique Correlations
 6 "Probable" Correlations
   (A Second Source Possible)

 3 No Definite or Probable
Correlations Found
                                                                         Result
                                                                      All Correct
                                                                      1 Error2
No Correlation
Existsb
          aError attributable to presence of oil-based spill control chemical.
          bThe source of these samples was not supplied to Woods Hole by EPA.

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190
IDENTIFICATION OF OIL
                                Table 11: Ability by Woods Hole Oceanographic Institute
                                      to Distinguish Among 30 Oils and Oil Products
                                             from Greater New York Harbora
                    Product Type
                    Gasoline
                    #2 Fuel Oil
                    #4 Fuel Oil
                    #6 Fuel Oil
                    Miscellaneous:
                    Kerosene
                    Marine Diesel
                    Nigerian Crude
                    Asphalt
                    Overall:
                                   No. of Samples
                                         12
                                         14
         Assignment
Not Analyzed
7 Unique
3 Pairs of Indistinguishable Oils
1 Trio of Indistinguishable Oils
All Unique
All Unique


Unique
Unique
Unique
Unique
77% Unique
20% Indistinguishable Pairs
 3% Indistinguishable Triplets
                    aEach oil was analyzed and compared with the 29 others to determine distinguishability.
 pairing a weathered oil to one of only a few unweathered oils
 is required,  then  nickel-vanadium, sulfur-nitrogen, and GC
 analyses may suffice; but, where comparison with many simi-
 lar unweathered oils is to be made, the entire Esso Method
 should be used. This assumes, of course, that a computerized
 mass  spectrometer  is  available.  Therefore,  the extent of
 method utilization depends on problem complexity and avail-
 able instrumentation.
    The  Esso  Method  is  somewhat  inconvenient because
 Adsorption chromatography is  used repeatedly and because
 new selections among the  26 fingerprint indices (Table 4)
 may have to be  made  for different analyses. Additionally,
 computerized mass spectrometry is  not available in  most
 laboratories,  thereby limiting  the usability  of  the  Esso
 Method.  The method should permit unique correlations with
 extensive libraries of oil indices, however. It also benefits
 from the  extensive weathering studies that were performed in
 its development by Esso.
    Another Esso approach evaluates  a combined GC-UV
 procedure that  may provide additional  fingerprint  indices
 for passive tagging oils. Weathered and unweathered portions
 of the  same oils are used in order to establish method integrity
 in the  presence of weathering.  If successful,  this technique
 should find wide acceptance because gas chromatographs and
 ultraviolet  spectrophotometers  are generally  available in
 analytical laboratories.  It could also be  combined  to ad-
 vantage with those portions of the Esso Method that  require
 more commonly available instruments. The chromatograms of
 polynuclear aromatics differ considerably from the aliphatic
         Table 12:  Oil Types3 Tested by Baird-Atomic

                  Five Crude Oils
                  Two Weathered Crudes
                  Seven Number 6 Fuel Oils
                  Two Asphalts

   a - Individual oils not identified.
                                                    GC profiles of the Esso Method. These aromatic profiles are
                                                    easily obtained and may facilitate  passive tagging  oils, even
                                                    without further UV analysis.
                                                        Use of adsorption chromatography and GC fraction col-
                                                    lection make this procedure somewhat inconvenient. Losses
                                                    of oil components can  also occur during these steps, thereby
                                                    yielding erroneous results.  But, Esso's work at the time of
                                                    this writing is still too preliminary for meaningful conclusions.
                                                    Usable chromatograms  of  polynuclear aromatics could per-
                                                    haps also have been obtained in the previous Esso study if
                                                    the aromatic fraction, following silica gel separation (Figure
                                                    1), had been analyzed by GC.
                                                        The  Phillips  Study evaluates  the  suitability  of  many
                                                    parameters and  techniques for distinguishing and  correctly
                                                    pairing  unweathered  crude oils  that  represent  worldwide
                                                    production.  Emphasis  is directed  toward  identifying  those
                                                    parameters that distinguish oils and  that  may retain their
                                                    integrity  in the presence of weathering.  No in-depth evalua-
                                                    tion of weathering effects  is  done, however, and  therefore,
                                                    no ready-to-use passive tagging method results.
                                                        The potential fingerprint  indices examined in this  study
                                                    include  those that require only minimal  equipment  (API
                                                    gravity  and pour point) and those that require  unusually
                                                    expensive instruments  (carbon and sulfur isotope ratios).
                                                    The suitability  of  some commonly  used  passive tagging
                                                    indices  (sulfur,  nitrogen,  nickel,   and   vanadium  content)
                                                    for distinguishing among  a large  number  of crude  oils is
                                                    also determined.  Phillips  aims to  evaluate  a sufficiently
                                                    broad range of parameters  to yield a comprehensive method,
                                                    such as the Esso Method.
                                                        Phillips' general scheme (Figure 4)  suggests  a compre-
                                                    hensive  passive tagging  method. Identification of parameters
                                                    that are unaffected by weathering and extension of the study
                                                    to processed oils (distillates and residuals)  must  necessarily
                                                    precede acceptance of  this  outline as a method. Use  of ad-
                                                    sorption  chromatography and  urea adduction are thought to
                                                    be somewhat inconvenient.
                                                        The  WHOl System is a ready-to-use passive tagging
                                                    method requiring minimum sample pretreatment and  using
                                                    only one instrumental  technique.   WHOI  provides explicit

-------
                                                                                             POLLUTANTS       191
                                                                             CONCENTRATION   Hi l'i"i
                                                                             SLITS 22
                                                                             TIME CONSTANT   ".3
                                                                             GAIN 30/0  u 0.01/MAX
                                                                             TEMPERATURE
                                                                             EXCITATION WAVELEr-GTH  2''UMU
                                                                            EXCITATION WAVELENGTH  310HU
                                                                             GAIN  30/0
                                                                            FLUORESCENCE WAVELENGTH 3UOMU
                                                                            SLITS
                                                                            GAIN  3'./"  K 0.01/HAX
                                                                            TEMPERATURE  ROUM
                      EX   SOMU  FI.   E*
         FL. 3<40MU
200
                                                                             CONCENTRATION  IOPPM
                                                                             SLITS  22/11
                                                                             TIME CONSTANT  0.3
                                                                             GAIN  30/6  R 0.1 MAX
                                                                             TEMPERATURE    77K
                                                                             EXCITATION WAVELENGTH  290
                                                                            EXCITATION WAVELENGTH   3M
                                                                            GAIN  30/0
                                                                            FLUORESCENCE WAVELENGTH
                                                                            SLITS   11/22
                                                                            GAIN  30/6  R 0.01 MAX
                                                                            TEMPERATURE  77K
                     300
                                                                507T
                                          WAVELENGTH (nanometers)
                                                                                     600
Figure 8: Fluorescence  Excitation and Emission Spectra by Baird Atomic  of a Crude Oil (a) at  Room Temperature,
and (b) at 77K. Concentrations Are 10 ppm in Methylcyclohexane.

-------
192
IDENTIFICATION OF OIL
                                                                                  CONCENTRATION  10 PPM
                                                                                  SLITS  22/12   R  0.01 MAX
                                                                                  TIME CONSTANT 0.3
                                                                                  GAIN   30/10
                                                                                  TEMPERATURE   " K
                                                                                  EXCITATION  WAVELENGTH  290MU
                                                                                  PHOSPHOROSCOPE   16.5 VOLTS
                                                OIL (A)
 200
                        300
                                    400                    500
                                      WAVELENGTH (ncmomuluis)
                                                                                           600
                                                                                                                   700
 Figure 9: Phosphorescence Spectra  of Three  Different Number 6 Fuel  Oils at 77"K by  Baird Atomic.  Concentrations Are  1U  ppm in
 Methylcyclohexane.
 instructions for separating oils from water, sand, and animal
 tissue matrices. The oils are then injected directly into  the
 GC instrument in  carbon disulfide solution.  Unweathered
 suspect  oils  are also merely  dissolved in carbon disulfide
 and injected.  No preweathering,  high  speed centrifugation,
 adsorption  chromatographic,  or  multi-instrumental  tech-
 niques, such  as  those used by Esso and Phillips, are used by
 WHOI.  The method requires some modification of most  GC
 instruments,   however.  These  are  now  generally  available
 instruments in most analytical  laboratories.
    The WHOI Method was  evaluated by using  weathered
 and unweathered  portions of oils supplied by EPA's Edison
 Laboratory.  Most,  but  not  all of the  weathered oils were
 uniquely correlated; several correlations  were only "prob-
 able"  The WHOI Method, therefore, seems usable for com-
 parisons to small numbers of  standard oils and possibly for
 correlations with small libraries of oil indices. But,  it may not
 achieve  the  "definite"  correct correlations  with  extensive
 libraries of oil indices that should be achievable by the Esso
 Method, and  by a method that may derive from the Phillips
 study. Use of additional fingerprint indices,  such as nickel,
 vanadium, sulfur, and nitrogen content, in conjunction with
 the WHOI Method, should yield further improvement.
    The  requirement for support-coated open tubular (SCOT)
 columns, capillary injectors, glass  liners, etc., may diminish
                                                     the acceptability of this method. SCOT columns are unusually
                                                     expensive  GC  columns (ca.  $160)  and have useful lifetimes
                                                     that may not greatly exceed  200  injections. Excellent resolu-
                                                     tion and reproducibility of fingerprint indices are achieved.
                                                     however; the usual packed GC columns yield inferior results.
                                                     The use of capillary  injectors and glass  liners  will require
                                                     modification of many  GC instruments.
                                                        The Baird Atomic Study is an evaluation of low tempera-
                                                     ture molecular  emission  as  a technique  for  distinguishing
                                                     among  oils. Efforts  are also made  to  identify  individual
                                                     fluorescing and phosphorescing  components in  oil and  to
                                                     maximize  spectral  differences among  oils.  An enhanced
                                                     ability to distinguish between oils and to yield more detailed
                                                     spectra, by cooling to  77K, 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

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                                                                                           POLLUTANTS
                                                                                     193
approach in their entirety.  But, the effect of weathering on
the low  temperature molecular emission of oils has not yet
been determined.

 CONCLUSION
    These EPA-sponsored studies were performed to evaluate
many existing and  suggested techniques for passive tagging
oils. The gas  chromatographic Method of WHOI and  the
fluorescence spectroscopic  approach of  Baird  Atomic  are
readily  usable in many "normal"  laboratory  facilities;  the
Esso  Method  and Phillips study encompass techniques that
range beyond the capabilities of most laboratories. Considera-
tion is given in these studies  to "simple" methods that permit
correct  pairing of  weathered and  unweathered oils  when
dealing with a small selection of oils, and to more complex
methods that  may  permit identification of  totally unknown
weathered  oils by  using extensive  libraries of appropriate
"fingerprint" indices. Evaluation  of so many analytical tech-
niques by these studies should facilitate the selection by other
personnel of the most appropriate passive tagging procedures
that suit individual situations and available facilities. These
descriptions of the Esso and Phillips projects are preliminary
and subject to change; final reports are not yet available to
EPA at  this  time.  Most tables  and figures presented were
extracted in whole or in part from investigators' reports.

 REFERENCES
1. E. R.  Adlard, J.  Inst. Petrol. 58, 63 (1972).
2. Esso  Research and Engineering Company  (EPA Project
Number  68-01-0058).
3. Phillips Scientific  Corporation  (EPA  Project  Number
68-01-0059).
4. Woods  Hole  Oceanographic Institution (EPA  Project
Number  15080 HEC).
5. Baird Atomic Corporation (EPA  Project Number  16020
GBW).
APPENDIX A

Crude Oils Tested By Phillips
                       Crude Oil
                        Sample
       Crude Oil         Number      County j Region
Abu Dhabi
Abu Dhabi
Abu Dhabi
Alaska
Alaska
Alaska
Alaska
Alaska
Alaska
Algeria
Algeria
Algeria
Algeria
Argentina
California
California
California
20
59
60
 1
 2
 3
 4
 5
 6
21
22
23
24
70
 7
Offshore
Cook Inlet
Cook Inlet
Cook Inlet
Gulf of Alaska Shore
North Slope
North Slope
Off Shore St. Barbara
Los Angeles Co.
St. Barbara Co.
Crude Oil


California
California
Canada
Colombia
Cuba
Cuba
Cuba
Florida
Florida
Florida
Florida
Florida
Gabon
Gabon
Indonesia
Indonesia
Iran
Iran
Iran
Iran
Israel
Israel
Kuwait
Kuwait Neu
Kuwait Neu
Libya
Libya
Libya
Louisiana
Louisiana
Louisiana
Louisiana
Louisiana
Louisiana
Louisiana
Mississippi
Mississippi
Mississippi
Mississippi
Mississippi
Nigeria
Nigeria
Norway
Norway
Norway
Qatar
Qatar
Saudi Arabia
Saudi Arabia
Saudi Arabia
Texas
Texas
Texas
United Arab Republic
United Arab Republic
United Arab Republic
Venezuela
Venezuela
Venezuela
Venezuela
Crude Oil
Sample
Number
75
76
47
71
72
73
74
51
52
53
54
55
57
58
25
61
26
28
29
30
62
63
64
31
27
32
33
34
13
14
77
78
79
80
81
15
16
17
18
19
35
36
37
56
82
65
66
67
68
69
10
11
12
41
42
43
44
45
46
50
County 1 Region


St. Barbara
St. Barbara
Alberta




Collier
Collier
Hendry
Lee
Hendry-Lee


Brunei


Offshore
Offshore
Offshore




Khasji

Cyrenaica

Timbalier Off Shore
Jackson Parish
Claiborne Parish
Offshore
Offshore
Offshore
Offshore
Wayne
Wayne
Clark
Jones
Jasper


North Sea
North Sea






Yoakum & Gaines Co.
Nueces Co.
Brazoria Co.



Lake Maracaibo

Lake Maracaibo
Anzoatequi

-------
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 Receiued for review November 8, 1972. Accepted March 19, 1973.
 Work supported by the Connecticut Research Commission, Grant
 RSA-71-20. Supplementary Material Available. Tablet, 2, 3, and
 4, in complete form, will appear following these pages in the mi-
 crofilm edition of this volume of the journal. Photocopies of the
 supplementary material from this paper only or microfiche (105 x
 148 mm, 20X reduction,  negatives) containing all of the supple-
 mentary  material for the papers in this issue  mav be obtained
 from the Journals Department, American Chemical Society, 1155
 16th St., N.W.,  Washington,  B.C. 20036. Remit checks or money
 order for $3.00 for photocopy or $2.00 for microfiche, referring to
 code number ES&T-73-631.
 NOTES

 Extraction of Dispersed Oils from  Water for Quantitative
 Analysis by Infrared Spectrophotometry

 MICHAEL  GRUENFELD
 Environmental Protection Agency, Edison Water Quality Research Laboratory, National Environmental
 Research Center (Cincinnati), Edison, N.J. 08817
  Some parameters that optimize  the extraction  of dis-
 persed oils  from  water  for quantitative  analysis  by  in-
 frared Spectrophotometry (ir) are examined,  and an im-
 proved extraction procedure is  recommended. Trichloro-
 trifluoroethane,  an essentially nonpoisonous solvent (1000
 ppm TLV),  is compared with carbon tetrachloride, which
 is highly poisonous  to  operating laboratory personnel (10
 ppm TLV).  Both solvents are  usable  for  extracting  dis-
 persed oils from water, and  for their quantitative analysis
 by ir,  but trichlorotrifluoroethane is preferred. Changes in
 extraction efficiency following small additions of sulfuric
 acid and sodium  chloride are examined.  Great improve-
 ment  results in  extraction efficiency,  but  no  further  im-
provement derives from addition of more salt.  Absorbance
 measurements are at 2930  cm"1 in 10-mm path  length
cells.
  Many biological processes necessary for the survival  of
aquatic organisms may be adversely affected  by the pres-
ence of extremely low levels0.1 mg/1.of petroleum oils
in water. Jacobson (1972), for example,  has shown that
kerosine extracts in water, in the  part-per-billion  range,
upset the chemotactic response of Nansarius  obsoletus  to
oyster and  scallop tissue.  Identification of the particular
oil  and its  quantitative determination  in the  water col-
umn are essential properly to  monitor and assess potential
biological damage resulting from oil spill incidents.
  The development of a method that can be  used to rap-
idly and completely extract dispersed oils from water for
quantitative analysis  by infrared Spectrophotometry, and
one that can be safely used in a mobile  laboratory operat-
ing at the  scene of a spill incident  is described in this
paper. Methods currently available  for the  quantitative
analysis  of petroleums  dispersed  in  water  are broadly
identified as  gravimetric  and spectroscopic  procedures.
636   Environmental Science & Technology

-------
Table I. Fraction of Oil Removed by Individual Extracts from Synthetic Dispersions Containing 5 Ml of 50% H2S04
        and 5 Grams of NaCI
                                                        Percent recovered0







a
No. of
25-ml
extrac-
tions
1
2
3
4
South Louisiana
Crude Oil
Freon 113
92.6
99.3
99.8
100
Determined as A/ A, X 100 + 8: XI.
tracts (2/1); B,

CCU
94 4
99.7
100

No 2
Fuel Oil
Freon 113
97.2
99.5
100

infrared absorbance at 2930 cm
total percent oil recovered by the
previous extracts.
Bachaquero

ecu
97.8
100


-' due to


Crude Oil
Freon 1 13
90.0
98.7
99.7
100
the extract of





ecu
95.4
99.5
99.9

Freon
91.
98.
100
No. 6
Fuel Oi
113
1
7


I
CCU
92.2
98.6
100
100
nterest, XI;

, sum of absoi

bances at 2930


cm ' of all the ex

The gravimetric methods produce losses of the more vola-
tile petroleum fractions making their use questionable for
measuring light oils and distillates. Spectroscopic meth-
ods are inherently more sensitive and accurate, as indicat-
ed by Harva and Somersalo (1958). Infrared and ultravio-
let procedures therefore seem  to hold greater promise for
yielding sensitive and accurate techniques.
  The extraction of petroleum pollutants  from water is  a
necessary part of quantitative analysis by either gravimet-
ric or spectroscopic  methods. Parameters  that influence
these extractions are evaluated in this study, and an opti-
mum  extraction  scheme is  presented. These parameters
include the degree  to which addition's of acid  and salt in-
fluence extraction efficiency and the utility of trichlorotri-
fluoroethane (Freon 113) and carbon tetrachloride  (CC1J
for such  extractions;  the  latter solvent  is  highly toxic
when  inhaled  (10 ppm TLV)  or when  absorbed through
the skin (Sax, 1968).
  The literature contains reports on the extraction of oils
from  water with  carbon tetrachloride and trichlorotrifluo-
roethane, and  acid and salt have previously been used to
increase extraction  efficiency. The  American Petroleum
Institute's spectroscopic procedure  (1958) utilizes  carbon
tetrachloride  as the  extracting solvent, after addition  of
sulfuric acid and salt. Carbon tetrachloride is  also used in
a spectroscopic procedure  developed by the Beckman In-
strument Co.  (1968)  without,  however, the  addition of
acid or salt. Freon  113  is used as the extracting solvent in
a gravimetric procedure reported by the American  Public
Health Association  (1971), following addition of sulfuric
acid,  but not salt.  These methods suggest that there is  a
lack of uniformity  and general agreement about the ad-
vantages of using acid and salt. They also do not examine
the extent to which additions of these materials influence
extraction efficiency, or the  possibility of using Freon 113
for spectroscopic  analyses.  Freon  is safer than  carbon
tetrachloride from the analyst's viewpoint.
  In the present study  four oils were used to compare the
efficiencies  of the two  solvents and  the influence of acid
and salt: No. 2 Fuel Oil, which is a  low-viscosity distillate
oil (2.4 cSt at  100F); No. 6 Fuel Oil, which is a high-vis-
cosity residual oil  (2300 cSt at  100F); South Louisiana
Crude Oil which has a low viscosity (4.8 cSt at 100F);
and Bachaquero Crude  Oil which has a moderately high
viscosity (1070 cSt at  100F).  Consecutive extractions of
each oil from synthetic dispersions  in water  were carried
out with each solvent; the quantity of oil in the individual
extracts was monitored by measuring the oil absorbance
band  intensity at 2930 cm-1 in the ir spectral  region (Fig-
ure 1). This band is not unique  to  oils, but derives from
the CH2 group that is  common  to  many organics.  Freon
113 and carbon tetrachloride yield minimal absorbance in
the 2930 cm-1 region and are amenable for such analyses.
                0.70 -

                0.80

                090-
                1.0
                      3200   3000   2BOO
                        WAVENUME CM'1
Figure 1. Infrared absorbance band of No. 2 Fuel Oil dissolved in
Freon 113 (0.034% W/V), using 10-mm path length silica cells;
Freon is in the reference beam

Absorbance at 2930 cm"1  is determined as the  difference  between
points A and 8
The changes  in  extraction  efficiency that  accompanied
additions of sulfuric acid and sodium chloride were estab-
lished  by monitoring resulting changes in the quantity of
oil  separated  by the  individual  extracts.  The  degree to
which  the dispersed oils  were separated by  each extract,
following addition  of  acid  and  salt, is also  estimated
(Table 1). Since the tabulated results are  derived  solely
from  our synthetic oil-water dispersions  however,  they
should not be extrapolated  to other types  of dispersions
without further study.

Experimental
  Apparatus.  Perkin  Elmer Model 457A  and  Beckman
IR-33 infrared grating spectrophotometers were used for
the determinations. Absorbance of the solutions was mea-
sured in  10-mm  path  length glass-stoppered rectangular
silica  cells (Beckman  Instruments, Inc.,   Catalog  No.
580015).
  Reagents.  Extractions were performed with  Freon TF
(Freon 113) solvent (E. I. Du Pont De Nemours  and Coin-
                                                                                   Volume 7, Number 7, July 1973  637

-------
                           A FREON 113
                            C Cl,
                                     10   11   12
 Figure  2. Number  2  Fuel  Oil extracted  from  1-liter duplicate
 synthetic oil-water samples  containing no added acid or salt
                                 LEGEND
                            NO ACID OR SALT
                            5
-------
ing the ratio of its absorbance to the sum of the absorban-
ces of all the extracts (Table I).

Results and Discussion
  Freon  113 and  carbon tetrachloride  were found  to be
about equally  effective for extracting  the  dispersed  oils
from  water.  Virtually  the  same  number of  extractions
with  each  solvent effected  removal of the oils (Figures 2
and  4). Additions of sulfuric acid  and sodium  chloride
dramatically improved  extraction efficiency. In  the  ab-
sence of these materials, the complete separation of No. 2
Fuel Oil was not possible even after  15  separate 25-ml ex-
tractions with  carbon tetrachloride and Freon 113.  How-
ever,  complete separation  of oil  was achieved  with only
four extractions when  5  grams of sodium  chloride  and 5
ml of 50%  sulfuric acid were added to the 1-liter synthetic
samples. The addition  of more than  5 grams of salt  yield-
ed no further improvement  (Figures  2 and 3). Four  25-ml
extractions with either solvent achieved complete separa-
tion of all  the  test oils when these quantities of acid and
salt  were added  to  the synthetic 1-liter samples (Figure
4). In these latter determinations, more than 90% of each
emulsified  oil was removed by the first extract  (Table I).
  Freon  113  is recommended as  the solvent of choice for
extracting  dispersed oils from water, because it is virtual-
ly as efficient for these extractions and as usable for the
infrared determination of oil as  carbon tetrachloride,  but
is much less poisonous to laboratory personnel. It is espe-
cially  preferable  to carbon tetrachloride   in  situations
where adequate  ventilation  may  be lacking,  such  as in
some mobile laboratory and field use.
  The recommended  procedure  for extracting dispersed
oils from water is the addition of 5 ml of 50% sulfuric acid
and 5 grams of sodium chloride to 1-liter samples. Extrac-
tion  should  be  carried out with  four  25-ml  portions  of
Freon 113 in 2-liter separatory funnels.  Checks for acidity
(below  pH  3) and  completeness of extraction  should be
performed. Initial dilution is to 100 ml. Seawater samples
are an  exception because they already contain  adequate
salt and can therefore probably be analyzed without addi-
tion of sodium chloride. Such samples were not examined
in the present study, however. A "blank" determination of
the reagents and water should be performed to prevent in-
terference with the oil  measurement at 2930 cm"1 by ex-
traneous solvent extractable organics.
Acknowledgment
  Special thanks are given to Joseph Lafornara for provid-
ing valuable background information, and to Henry Jelen-
iewski,  Midhael Killeen, Susan Rattner, and  Peter Furth
for their assistance.
Literature Cited

American Petroleum Institute, "Manual on  Disposal of Refinery
  Wastes," Vol. IV, Method 733-58, 1958.
American Public Health Association, "Standard Methods  for the
  Examination of Water  and  Wastewater,"  13th  ed.,  APhA,
  AWWA, and WPCF, New York, N.Y., 254-6, 1971.
Beckman Instruments, Inc., Mountainside, N.J., Infrared Appli-
  cation Note 68-2, 1968.
Harva, O., Somersalo, A., Suomen Kern., 3l(b), 384-7 (1958).
Jacobson, S., Woods Hole Oceanographic Institution,  Woods
  Hole, Mass., personal communication, June 7, 1972.
Sax, I.  N., "Dangerous Properties  of Industrial  Materials," 3rd
  ed., pp 535, 1192, Reinhold, New York, N.Y., 1968.

Received for review August 10, 1972. Accepted March 26, 1973.
                                                                                    Volume 7, Number 7, July 1973   639

-------
Reprinted from:   ASTM Special Technical Publication 573
Nov.  1973, ASTM, Phil.,  PA
                            Michael Gruenfeld*


                            Quantitative  Analysis   of   Petroleum
                            Oil   Pollutants   by   Infrared
                            Spectrophotometry
                             REFERENCE: Gruenfeld, Michael, "Quantitative Analysis of Petroleum Oil Pollutants
                             by Infrared Spectrophotometry," Water Quality Parameters,  ASTM STP 573, American
                             Society for Testing and Materials, 1975, pp. 290-308.

                             ABSTRACT: The accuracy and sensitivity of infrared Spectrophotometry are evaluated
                             for the quantitative analysis of water dispersed oils, by single point analysis. Absorbance
                             versus concentration (Beer-Bouguer Law) plots are prepared for viscous and nonviscous
                             crude and processed oils in Freon 113, carbon tetrachloride,  and in a mixture of these
                             solvents. Absorbances at 2930/cm are measured  in  10 and  100-mm path length cells,
                             with and without ordinate scale expansion. Solution concentrations in the range 0.5 to
                             40 mg/100 ml oil in solvent yield linear plots that pass through the origin. The concen-
                             tration 0.05 mg/100 ml oil in solvent  yields a recognizable absorption band at approx-
                             imately 2930/cm when measured in  100-mm path length  cells with ordinate scale
                             expansion X5. This is  considered  the practical  detection  limit of these  oils by the
                             infrared (1R)  technique. Stability of oil absorptivities following solution storage, and use
                             of IR absorptivities for oil identification are also examined briefly.

                             KEY WORDS: water  quality, oils,  infrared spectrophotometers, Spectrophotometry,
                             environmental tests, water pollution


                              Improved methods  for  the  quantitative  analysis  of  water dispersed
                            petroleum oils are  under investigation  by  the Industrial Waste Treatment
                            Research  Laboratory  of the  National Environmental  Research  Center
                            (NERC),  Cincinnati.  Such methods  are  needed  to  evaluate  the  per-
                            formance of oil-water separator  devices, to  measure the dispersed oil con-
                            centration in  water   below surface floating oil  slicks,  to  monitor  oil
                            concentrations in effluent waters of various industrial plants and  water-
                            craft, and to support  studies of the effect  of water dispersed petroleum
                            oils on the  biosphere.  That low concentrations of water dispersed  oils
                            adversely  affect living organisms  is well known: for  example, Jacobson [I]2
                            has shown that kerosene extracts in water,  in the parts-per-billion  range,

                             'Supervisory chemist, U.S. Environmental Protection Agency,  Industrial Waste Treatment
                            Research Laboratory, National Environmental Research Center, Edison, NJ. 08817.
                             'The italic numbers in brackets refer to the list of references appended to this paper.

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     GRUENFELD  ON ANALYSIS  OF PETROLEUM OIL POLLUTANTS    291

 upset  the  chemotactic response  of Nassarius Obsoletus  to oyster and
 scallop tissue.
   Most methods that are currently used  for the quantitative analysis  of
 water dispersed oils can be classified as gravimetric or spectroscopic pro-
 cedures. The determinative step of gravimetric methods  is simple  weigh-
 ing, while the determinative step of spectroscopic methods is the measure-
 ment  of  radiation  absorption  by  dissolved oils. .Solvent  extraction  is
 commonly used by gravimetric  and spectroscopic methods to isolate and
 concentrate  water  dispersed  oils for  quantitative measurement.  Solvent
 evaporation  (stripping), which precedes  weighing  in gravimetric methods,
 has a drawback because the more volatile petroleum fractions are lost.  A
 gravimetric  method  by  the American  Public  Health   Association  [2]
 illustrates yet another drawback: questionable sensitivity and  accuracy are
 achieved by  weighing minute oil residues in "large"  (approximately 125
 ml) distilling flasks.  Harva and  Somersalo [3] conclude that the spectro-
 scopic methods are more accurate and sensitivea justifiable conclusion.
   Infrared spectrophotometry (IR)  is the most commonly used spectro-
 scopic technique for the quantitative analysis of water dispersed oils.  In an
 American  Petroleum Institute procedure [4]  dispersed oils are  extracted
 from water  with  carbon  tetrachloride;  a  mechanical shaking apparatus
 and a 5-liter extraction  flask are  used. The absorption  band maxima of oil
 at  2850 and 2930/cm  are measured,  using  10-mm path length quartz
 cells. Oil concentrations are determined by comparing the sum of these
 absorbances  to the sum of absorbances derived from a prepared carbon
 tetrachloride solution containing an accurately known concentration of the
 oil. The latter solution  is  the standard solution, and the  determination  is
 performed by  single  point analysis. These terms  are  discussed  later. In
 this method  a blend  of hydrocarbons (37.5 percent  isooctane,  37.5
 percent cetane, and 25 percent benzene) that is thought  to  approximate
 the IR  absorptivity of  an  average petroleum oil  is used  to  prepare the
 standard solution when  a portion of the dispersed oil is not available.
   In  an  IR method by  Beckman Instruments,  Inc.   [5],  100-ml of
 dispersed oil in  water  samples  are extracted  with 2-ml carbon  tetra-
 chloride, using hand-held  separatory funnels. The absorption band  max-
 imum  of oil  at 2930/cm  is  measured as  in the previous method,  using
 10-mm  path  length  near IR silica cells. The  2850/cm  band  is not  used.
 Reference solutions are  prepared  as oil in water dispersions having known
 oil  content, and each of these dispersions is  extracted with  2-ml carbon
tetrachloride. The extracts are then measured at 2930/cm, and a concen-
tration versus absorbance plot is derived.  This plot is  used  to quantitate
dispersed oil  in water samples.
  The two preceding methods utilize the same extraction solvent, but they
differ in most  other  respects. The method  by the American  Petroleum
Institute uses a large oil  extraction flask  and a mechanical  shaker; salt

-------
292   WATER QUALITY PARAMETERS

and  acid are  added;  extraction  is  performed  with  successive  100-ml
portions of carbon  tetrachloride; two IR absorption  band  maxima are
measured; and comparison is made to only a single standard solution. The
Beckman Instrument  method  uses a  small hand-held  separatory  funnel;
one  2-ml portion of  carbon  tetrachloride is  used to extract a  100-ml
sample volume;  acid  or salt  are not added;  one IR absorption  band
maximum is measured; and  a concentration versus absorbance plot
derived from several  oil-in-water reference solutions  is used for  sample
determination. These  methods thus differ in the  type of apparatus used,
sample and solvent volumes, addition of salt and acid,  and the method of
IR measurement and  data  handling.  Gruenfeld  [6]  recently investigated
some of these parameters in detail. He examined the  influence of salt and
acid, and compared the  extraction efficiencies of carbon tetrachloride and
Freon 113 (l,l,2-trichloro-l,2,2-trifluoroethane).  Freon 113, like  carbon
tetrachloride, can  be used  for  IR  measurement of  oil  at  2930/cm.
Gruenfeld [6]  extracts  1-liter  dispersed  oil  in  water samples with four
consecutive  25-ml portions  of  solvent,  using 2-liter hand-held separatory
funnels. Use of  Freon  113 is  of  special interest because  it is  far less
poisonous to exposed  personnel than  carbon tetrachloride.  According to
Sax  [7], carbon tetrachloride is highly toxic (10 ppm TLV)  when inhaled
or absorbed  through the skin,  while Freon 113 is  much safer (1000 ppm
TLV).
   The present paper describes  a  study of the  accuracy and sensitivity of
the  IR  technique,  when used for  the quantitative determination  of
petroleum oils by single point analysis.  Carbon tetrachloride, Freon 113,
and  a mixture of these solvents are used.  The detection  limit of oils by IR,
the  stability  of oil  absorptivities during  prolonged solution storage, and
the  utility of these absorptivities for oil identification are  also examined.
Four oils were used: No. 2 Fuel Oil,  which is a low viscosity distillate  oil
(2.4 cSt at 100F); Number 6 Fuel Oil,  which  is a high viscosity residual
oil (2300 cSt at  100F); South Louisiana Crude Oil, which has  a low
viscosity (4.8 cSt at 100F);  and Bachaquero Crude  Oil,  which  has  a
moderately  high  viscosity (1070 cSt at  100F).  IR  measurements were
made  in 10 and  100-mm  path  length silica cells,  at  approximately
2930/cm. The oils,  solvents, final solution volumes, and cells were selected
in order to  correlate this work  with the  previously reported  investigation
by Gruenfeld [6].
   Atwood et  al  [8] reported  a  somewhat  similar, though  more  limited
study of IR for the quantitative analysis of oils. A blend of hydrocarbons
(isooctane, hexadecane, and benzene) was used to represent "typical" oils,
and  carbon  tetrachloride solutions of this blend were measured in  10 and
100-mm path length quartz cells.  No actual oils,  or solvents other than
carbon  tetrachloride were used.  An  effort  was  not made  to  establish
whether concentration  versus absorbance  (Beer-Bouguer Law) plots  of oils

-------
     GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS   293

are linear and pass through the origin; that is, whether oils can be quanti-
tatively determined by single point analysis.

Experimental

Apparatus
  A Perkin Elmer Model  457A infrared  grating spectrophotometer3 was
used  for  the determinations.  Solution  absorbances  were measured  in
10-mm (Beckman Instruments,  Inc., Catalog No.  580015) and 100-mm
(Fisher Scientific Co., Catalog No. 14-385-930F) path length  cells. These
are  rectangular silica and cylindrical Supracil cells, respectively.  Cell
holders obtained from the Perkin  Elmer Corporation (Catalog No.  186-
0091) were used for  the  10-mm  path length  cells, while holders obtained
from International Crystal Laboratories (Catalog No. R 100-22 with Teflon
gaskets as spacers) were used for the 100-mm path length cells.

Reagents
  The oil solutions were prepared in spectroanalyzed carbon tetrachloride
(Fisher Scientific  Co.,  Catalog  No. C-199), Freon  113  solvent  (E.  I.
DuPont De Nemours and Company,  Inc.),  and in  a 98 percent Freon
113/2 percent carbon tetrachloride  (by volume) solvent mixture.  Freon 113
is a  DuPont  refrigerant. It is  l,l,2-trichloro-l,2,2-trifluoroethane,  of
specified purity. This isomer of trichlorotrifluoroethane is available from
several manufacturers, under a variety of trade names.

Procedure

  Oil solution concentrations were adjusted to yield  absorbances that were
within the ordinate scale range of the IR chart paper. Measurements were
made  with matched  10 and  100-mm path length cells, without ordinate
scale expansion, and  with ordinate scale expansion x5. Measurements
without scale expansion required oil concentrations of 2 to 40 mg/100  ml
and 0.5 to 4.0 mg/100 ml for measurements in  the  10 and 100-mm path
length cells, respectively. Measurements with  ordinate scale expansion  x5
required concentrations of 0.5 to 4.0 mg/100 ml and 0.05 to 0.4 mg/100
ml for measurements in the 10 and 100-mm path length cells,  respectively.
All  the  solutions contained accurately known  oil  concentrations.  The
reference cell was filled with solvent from the same reagent bottle that was
used to prepare the  oil solution. Absorbances derived from  measurements
without scale  expansion  were read directly from a  nonlinear absorbance
type chart paper. These  absorbances were measured as vertical distances
between the  2930/cm absorption  band maximum  of oil and  a baseline
  'Mention of trade names or commercial  products does nut constitute endorsement hy the
U.S. Government.

-------
294   WATER QUALITY  PARAMETERS


drawn tangent to absorption  minima adjoining the band  maximum  (Fig.
1). Absorbances that were determined by using ordinate  scale expansion
x5 required use of a linear transmittance type chart paper, and calculation
by special equation  (Fig. 2).  The solvents used were Freon 113, carbon
tetrachloride, and 98  percent Freon  113/2  percent carbon  tetrachloride
(by volume).  Absorbance versus concentration  (Beer-Bouguer Law)  plots
of the oils in the solvents were derived.
                        0.70

                        O.SO

                        0.90
                        1.0
                               3200    3000    2800
                                 WAVENUMBER CM'1

  FIG.  IIn/rared absorption band of No. 2 Fuel Oil dissolved in Freon 113 (0.034% w/v),
using I0-mni path length  silica cells: Freon 113 is in  the reference beam.  Absorbance  at
2^30/cm is determined as the difference between Points A and B.
  The utility of IR absorptivities as a parameter for identifying weathered
oils was also  briefly investigated. Diverging  slopes of Beer-Bouguer Law
plots  illustrate differences  in  oil absorptivities.  Eiomm1''0   values (absorb-
ances  measured  in  10-mm path length  cells,  normalized  to  1  percent
weight/ volume dissolved oil) of the oils were calculated  and evaluated for
stability,  following  loss of volatile  oil components  as  a  consequence  of

-------
      GRUENFELD ON ANALYSIS OF PETROLEUM  OIL POLLUTANTS    295
       100-
        90-
     Z
     <
        60-
        50-
                   2930cm'1

                       Absorbance = Iog10
    2930cm"1
5B
                                       5B 4- D  C
                       B = % T of the unexpanded baseline
                       C = % T of the expanded baseline
                       D = % T of the expanded peak maximum
  FIG. 2Infrared absorption band of No. 2 Fuel Oil dissolved in Freon  113:  (/) without
nrdinate scale expansion, and (2) with ordinate scale expansion x5.

solvent distillation  (stripping). An oil solution was stripped  free of solvent,
following IR measurement in 10-mm  path length cells.  The solvent  was
discarded and  the  oil residue weighed. The  residue was  then redissolved,
diluted to the  previous volume  (100-ml), and the solution measured  again
by IR. The new  Eiomm1"7'  absorptivity was compared to the original value.
The solvent stripping procedure  of the American  Public Health Associa-
tion [2] was used.
  A brief investigation was also made of the stability of oil absorptivities
following prolonged  solution storage  under  normal room  light and tem-
perature conditions.  Oil  solutions were prepared  in carbon tetrachloride,
Freon 113, and  in  the 98  percent/2  percent solvent   mixture.  South
Louisiana Crude and No. 2 Fuel Oils were  used; solution concentrations
were  2 and  10 mg/100  ml. Following  the initial absorbance measure-
ments, the solutions  were stored  for eight  days on an exposed laboratory
shelf,  in clear-glass  tightly sealed  100-ml volumetric flasks.  The absor-
bance measurements were then  repeated and compared  with  the  original
values.

Results and Discussions

  The major purpose of this study was to  assess the utility of IR for  the

-------
296   WATER  QUALITY  PARAMETERS

quantitative  determination of petroleum  oils, by  single point  analysis.
Solutions of four representative oils  in two solvents, and in  a mixture of
these solvents,  were used  to  prepare the appropriate absorbance versus
concentration (Beer-Bouguer  Law)  plots.  Quantitative determination  by
single point analysis requires linear plots that pass through the origin. Use
of single point analysis  usually offers a considerable time saving  because
only one standard  solution  is used for each  analysis, rather  than  a
Beer-Bouguer Law plot derived from several solutions.  The term standard
solution  designates  a  solution containing an accurately known  concen-
tration  of oil with  the  same  identity  as  the dispersed  oil in the  water
sample. The following equation is used for single point  analysis
where
          Cx = the unknown oil concentration of the sample extract used
                 for IR measurement;
  Ax and As = the absorbances of  the  sample  extract  and  standard
                 solution,  respectively; and
          Cy = the standard solution concentration used for IR measure-
                 ment.
The measurements were made in  cells with identical path  lengths,  and
using identical  ordinate scale expansion settings. The four oils selected for
study were thought to be  fairly  representative  of viscous and nonviscous
crude and processed petroleum oils. The choice of solvents was determined
by the previously cited publications, and by  consideration of oil solubility,
IR absorptivity, and solvent toxicity.
  Carbon tetrachloride is used in the previously described IR methods by
the American Petroleum Institute [4], Beckman Instruments,  Inc. [5], and
Atwood et al  [8].  As previously indicated, this solvent is highly toxic.
Freon  113 is used in a gravimetric  procedure by the American  Public
Health  Association  [2], and is  much  safer than  carbon  tetrachloride.
Freon  113 is evaluated in  the present  study for IR measurement  of  oils.
Gruenfeld  [6] compared the ability of these solvents  to extract  dispersed
oils from  water. He found  them  equally effective  for  extracting small
quantities (less  than 200 ppm) of water dispersed oils. Although Freon 113
is adequate for extracting low concentrations of water dispersed  oils, it
does not readily dissolve (cut) undispersed viscous oils such as No.  6 Fuel
and Bachaquero Crude oils.  Freon 113 is  therefore not recommended for
preparing IR standard solutions of viscous oils. This problem is solved by
first dissolving  a sufficient  amount of viscous oil in carbon tetrachloride to
yield an accurately known concentration  approximating 20  mg/ml.  This
solution (2.0-ml) is then diluted to  100-ml with  Freon 113,  to yield  a final
98 percent Freon 113/2 percent carbon tetrachloride  mixture that contains

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      GRUENFELD  ON  ANALYSIS OF PETROLEUM  OIL POLLUTANTS    297
approximately 40-mg  oil. A fresh  portion  of  the  98  percent/2percent
solvent mixture is used for further dilutions.  These standard  solutions will
be discussed further.
  The oils used in the present study were dissolved directly in the solvents,
to yield solutions having accurately known  concentrations. These solutions
simulate  solvent extracts of  actual dispersed oil  in water  samples. Beer-
Bouguer Law plots  were prepared  and evaluated  for compliance with the
criteria for single point  analysis; that is,  plots  that are linear and  pass
through the origin.  Solutions of the four oils in the two solvents and in the
solvent mixture  were measured by  IR.  A range of concentrations  was
examined in 10 and 100-mm  path length cells, with and without ordinate
scale expansion. Linear  plots that pass through  the  origin were obtained
for  the four oils in  carbon tetrachloride (Fig.  3)  and in 98 percent Freon
                                            NO 1 FUEL OIL
                                            NO 6 FUEL OIL
                                            BACHAQUERO CRUDE OIL
                                            SOUTH LOUISIANA CRUDE OIL
                        10      20     30      40     50
                       CONCENTRATION |mg/100 ml) OIL IN SOLVENT

  FIG. .1Oil solutions in carbon tetrachloriJe measured in 10-mtn path length cr//i with-
out ordinale scale expansion.

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298   WATER QUALITY  PARAMETERS

113/2 percent carbon tetrachloride (Fig. 4),  and for the two less viscous
oils  in  Freon  113 (Fig.  5).  Number  6 Fuel  and  Bachaquero  Crude  oils
were not readily soluble,, in  Freon  113. Therefore,  they  were dissolved
initially  in carbon tetrachloride, and  then diluted further with Freon 113,
by the  previously described-procedure, to yield  a final  98 percent  Freon
113/2 percent carbon tetrachloride solvent mixture. The potential  absorp-
tivities of viscous oils in Freon  113 are thought to match their absorptivi-
ties  in  the  solvent   mixture,  because Beer-Bouguer Law  plots  of  the
nonviscous  oils in Freon 113 yield slopes that  match the slopes of these
oils  in 98  percent Freon 113/2  percent carbon  tetrachloride  (Figs.  4, 5,
and  6). Preparation of standard  solutions  in  98 percent Freon  113/2
                                                  KEY

                                           NO  2 FUEL OIL
                                           NO  6 FUEL OIL
                                           BACHAQUERO CRUDE OIL
                                           SOUTH LOUISIANA CRUDE OIL
                     10       20      30      40     50
                      CONCENTRATION (mg/100 ml) OIL IN SOLVENT
  FIG. 4Oil solutions in 98 percent Freon 113/2 percent carbon tetrachloride measured in
10-mm path length cells without ordinate scale expansion.

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      GRUENFELD ON ANALYSIS OF PETROLEUM  OIL POLLUTANTS   299
                                                KEY

                                         NO 1 FUEL OIL

                                         SOUTH LOUISANA CRUDE OIL
                     10      20      30     40      50
                     CONCENTRATION (mg/100 ml) OIL IN SOLVENT
  FIG. 5Oil solutions in Frcon 113 measured in W-mm path length cells without ordinate
scale expansion.
percent carbon  tetrachloride  is recommended whenever Freon 113  is used
as the extraction solvent for quantitating dispersed oil in water samples.
   Use of IR for the quantitation of water dispersed  oils at  concentrations
below 1  ppm, by single  point analysis, was  also  examined.  Appropriate
Beer-Bouguer Law plots were derived from measurements in 100-mm path
length cells without ordinate scale expansion, using solutions of the four
oils in carbon tetrachloride (Fig.  7), in 98 percent  Freon  113/2 percent
carbon tetrachloride (Fig. 8), and solutions of the two less  viscous oils  in
Freon 113  (Fig.  9).  While  some  of the plots  exhibit  deviations  from
linearity  above  0.7 absorbance, all  the plots are  linear below  this value
and pass  through the origin. In  some  cases,  portions of an  oil used for

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300   WATER  QUALITY  PARAMETERS
                                         9B% FREON-113 -
                                         2% CARBON IETRACHLORIDE
                    1.0     2.0     3.0     4.0      5.0
                      CONCENTRATION (mg/100 ml] OIL IN SOLVENT
  FIG.  6Solutions oj No. 2 Fuel Oil measured  in 100-mm path length  cells without
animate scale expansion.
measurements  in 10  and  100-mm  path length cells  were  inadvertently
taken from different drums. This caused the slopes of some plots deriving
from  measurements in  10-mm path length cells (Figs.  3,  4,  and  5)  to
differ from slopes of plots deriving from measurement  of the same oils  in
100-mm path length cells  (Figs.  7, 8,  and  9). Use  of Freon  113 or  98
percent Freon 113/2  percent carbon tetrachloride in  100-mm path length
cells results  in a sluggish  response of the IR instrument  recorder  pen;
Freon  113 has a  higher absorptivity at 2930/cm than  carbon tetrachloride.
The IR instrument gain  setting should be increased  sufficiently for  these
measurements  to yield  a properly shaped  absorption band at  approxi-
mately  2930/cm  (Fig.   1).   Linear  Beer-Bouguer Law  plots  that  pass

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      GRUENFELD ON  ANALYSIS OF PETROLEUM OIL POLLUTANTS   301
         z
         2 05-
         oc
         O
      KEY
NO 2 FUEL OIL
NO. 6 FUEL OIL
BACHAQUERO CRUDE OIL
SOUTH LOUISANA CRUDE OIL
                    1.0     20      30      4.0     5.0
                     CONCENTRATION (mg/100 ml) OIL IN SOLVENT
  FIG. 1Oil Mtliitinns in carbon Ictrachlnride measured in 100-mm path length cc//.v with-
 nl  nntiiitili' .sra/r expansion.
through the origin were also obtained for measurements of the four oils  in
carbon tetrachloride, using 10-mm  path  length  cells with ordinate  scale
expansion x5  (Fig. 10).  But, considerable  deviation from linearity resulted
from the measurement  of Bachaquero Crude oil  in  100-mm  path length
cells with ordinate scale expansion  *5 (Fig. 11).
  Accurate quantitative determination  of water  dispersed oils by  single
point  analysis can be  accomplished in  the concentration  range  2  to 40
mg/liter (ppm) oil  in  water,  by using  10-mm path length cells without
ordinate scale expansion (Figs. 3 to  5).  This concentration range  assumes
use of the  extraction procedure by  Gruenfeld [6|,  whereby  1-litcr  oil  in
water samples are extracted with four 25-ml portions of solvent. Improved

-------
 302    WATER QUALITY PARAMETERS
                                                          KEY
                                                  NO 1 FUEL OIL
                                                  NO 6 FUEL OIL
                                                  BACHAOUERO CRUDE OIL
                                                  SOUTH LOUISIANA CRUDE OIL
                       10       20       30      4.0       5.0
                        CONCENTRATION (mg/100 ml) OIL IN  SOLVENT
  FIG. 8Oil solutions in 98 percent Freon 113/2 percent carbon tetrachloride measured in
100-mm path length  cells without ordinate scale expansion. A higher than normal IR  instru-
ment gain setting was used.

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     GRUENFELD ON ANALYSIS OF  PETROLEUM  OIL POLLUTANTS    303
                                                KEY

                                         NO 2 FUEL OIL

                                         SOUTH LOUISIANA CRUDE OIL
                   1.0     2.0
                    CONCENTRATION I
  3.0      40     50
ng/100 ml) OIL IN SOLVENT
  FIG. 9Oil solutions in  Freon 113 measured in 100-mm path length cells without or-
dinal? scale expansion. A higher than normal 1R instrument gain setting was used.
sensitivity can be achieved by using less  solvent. Accurate  quantitation  of
oils  in the concentration range 0.5 to 3 ppm oil in water can be achieved
by  single  point  analysis when  using 100-mm  path  length  cells without
ordinate scale expansion (Figs.  7 to 9),  of 10-mm path length cells with
ordinate  scale  expansion  x5 (Fig.  10).  The  measured  absorbances  in
100-mm path length  cells should  not exceed"  0.7.  Use of 100-mm  path
length  cells  with  ordinate  scale  expansion   x5 yields nonlinear  Beer-
Bouguer Law plots (Fig.  11). While accurate quantitation  of oils by single
point analysis is not possible under these conditions,  useful  information is
gained about the detection  limit of the 1R technique.  These  measurements

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 304     WATER QUALITY  PARAMETERS
               0 10 T	
                                                              KEY

                                                     NO  1 FUEL Oil
                                                     NO.  6 FUEL OIL
                                                     BACHAQUERO  CRUDE Oil
                                                     SOUTH LOUISIANA CRUDE OIL
10       20
 CONCENTRATION (
                                             3.0
                                            g/100
   4.0       5.0
I OIL IN SOLVENT
  FIG. 10Oil solutions in  carbon tetruchloride measured in I0-mni path length cells with
tmliiititt' scale expansion*5.

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     GRUENFELD  ON  ANALYSIS OF  PETROLEUM OIL POLLUTANTS   305
                      0.)     02    03     04     05    06
                        CONCENTRATION (m9/IOO ml) OIL IN SOLVENT
  FIG. 1 \Carbon tetrachloride solutions of Bachaquero Crude Oil measured in 100-mm
path length cells with ordinate scale expansion X5.
yield  a recognizable absorption  band at approximately 2930/cm  (Fig. 1)
for an  oil solution containing  0.05  mg/100  ml oil  in solvent.  This is
equivalent to  0.05 ppm dispersed  oil  in  water  when  the extraction
procedure by  Gruenfeld  [6]  is  used.  This  detection  limit  of  the  IR
technique can  be improved  further by  reducing the volume  of extraction
solvent.
  Consideration  of the Beer-Bouguer  Law  plots  demonstrates  that the
lines  diverge and, therefore,  that  the  oils have different absorptivities.
These absorptivities appear to remain reasonably stable, despite loss of the
more  volatile  oil  components  (Table  1).  They are   solvent  dependent,
however,  as demonstrated by the diverging Beer-Bouguer  Law plots  that

-------
 306    WATER  QUALITY  PARAMETERS
                                                  IN CARBON TETRACHLORIDE
                                                  IN FREON-II3
                        10       20       30       40       50
                        CONCENTRATION (mg/100 ml) OIL IN SOLVENT
  FIG.  12Solutions oj No. 2  Fuel Oil measured in  10-mm path  length  cells  without
ontinule scale expansion.

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     GRUENFELD  ON  ANALYSIS OF  PETROLEUM  OIL  POLLUTANTS    307
                                         IN CARBON TETRACHLORIDE

                                         IN FREON-113
                    10      20      30      40      50
                     CONCENTRATION (ma/100 ml) OIL IN SOLVENT
  FIG.  1.1Solutions of South Louisiana Crude Oil measured in 10-mm path  length cells
without  nnlintilf scale expansion.
are obtained from the same oil in different solvents  (Figs. 12 and  13): No.
2 Fuel and South Louisiana  Crude  oils yield  absorptivities in Freon 113
that  differ  from  their  absorptivities  in   carbon  tetrachloride.  These
differences are also  apparent when comparing Figs. 3 and 4, and others.
IR absorptivities are thus a promising  parameter for "passive tagging" oils
because they differ from oil to oil, and yet remain reasonably stable. Their
solvent dependence  may  also be useful for  distinguishing among similar
oils.  The  term  "passive  tagging"  describes  a   procedure  whereby  a
weathered  oil residue, that is, an  environmental pollutant, is  correlated
(matched)  with an unweathered portion of the  same oil.
  The stability  of solution absorbances, and  therefore  oil  absorptivities,

-------
308    WATER  QUALITY  PARAMETERS
 TABLE 1Effect uj so/vent distillation on ad absorptivity. (No. 2 Fuel and South Louisiana
  Crude Oils are in Freon 1/3 solution: No.  t> Fuel and Bachauiiero Crude Oils are in 98%
                     Freon  113/2% carbon  tetrachloride solutum).

                    Before Distillation                  After Distillation"
               Oil in Solvent  Absorptivity"     % Loss     Absorptivity       %
    Oil         (nig/100 ml)   (EiOmm1%>   (by weight)   (H1omml%)     Change
No. 2 Fuel

South Louisiana
Crude
No. 6 Fuel
Bachaquero
Crude
104
14.5
101
10.5
10. fa
103
21.4

22.4

23.8
16.1
11.5
23.0
3.0
14.3
0.0
4.4
21.1
19.6
21.6
21.7
23.8
15.3
1.4
8.4
3.6
3.1
0.0
5.0
  "Solvent distilled  (slripped) from 100-ml  solutions having known oil content, using the
procedure of the  APHA |2|. The weighed residues are redilmed to 100-ml and the solution
absorbances measured at 2930/cm.
  ^E|0 mm  " values are absorbances at 2930/cm, measured in  10-mm path length cells,
normalized to 1% (weight/volume) dissolved oil.


following prolonged solution storage was  also examined.  Oil  solutions in
carbon tetrachloride,  Freon  113,  and  98  percent  Freon  113/2  percent
carbon tetrachloride were stored  under normal  room  light and  tempera-
ture  conditions,  in  clear  glass  flasks  for  eight  days. The   absorbances
following storage matched those prior to storage,  within 1  percent. Prompt
solvent extraction  of dispersed oil  in water  samples  is therefore  recom-
mended.  Oil  transport and storage  in carbon tetrachloride or Freon  113
solution should prevent biological degradation and evaporative losses that
can occur in the  water  matrix.

References

|/]  Jacobson,  S.,  personal communication,  Woods  Hole Oceanographic Institution, Woods
    Hole, Mass.,  7 June 1972.
[2|  Standard Methods for the Examination of Water and Wastewater,  13th ed., American
    Public Health Association,  New  York, 1971. pp. 254-256.
|.?|  Harva, O. and Somersalo, A., Suomen Kemistilehti, Vol. 31 (b), 1958, pp. 384-387.
[4\  Manual on Disposal of  Refinery Wastes. Vol. IV, Method 733-58,  American Petroleum
    Institute, 1958.
151  Infrared Application Note 68-2, Beckman  Instruments, Inc., Mountainside, N.J., 1968.
|6|  Gruenfeld,  M., Environmental Science and Technology.  Vol. 7, 1973, pp. 636-639.
|7|  Sax. I.  N., Dangerous Properties of Industrial Materials. 3rd ed., Reinhold, New York,
    1968, pp. 535. 1192.
|,V|  Atwood, M.  R., Hannah,  R. W., and Zeller,  M.  V.,  Infrared Bulletin  No.  24,  The
    Perkin-Klmer Corp.. Norwalk, Conn. 1972.

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    Reprinted  from:  Proceedings 1975  Conference on Prevention  and Control
    of  Oil  Pollution,  March 25  -  27,  1975,  San Francisco, API,  Wash., DC
         PRELIMINARY  OBSERVATIONS ON THE  MODE OF
                    ACCUMULATION OF  #2  FUEL  OIL BY
                THE SOFT SHELL  CLAM,  MYA ARENARIA
                                               Dennis M. Stainken
                                               Rutgers University
                                       Department of Zoology and Physiology
                                              Newark, New Jersey
ABSTRACT

   Chemical analysis has shown that various components of oils can
accumulate within marine invertebrates.  Several mechanisms by
which  this may occur have been conjectured. This paper offers
experimental verification of a mechanism by which a commercially
important bivalve, Mya arenaria,  can accumulate oil within its tis-
sues.  The paper also documents the behavioral response of Mya
arenenaria and deleterious ecological side effects resulting from oil
accumulation.
   Young Mya (25-35 mm) were exposed to #2 fuel oil and an oil
soluble dye (Oil Red 0) which  were ultrasonically emulsified in
water.  The concentrations tested were 50 ppm, 100 ppm and 150
ppm. Exposures were done in both natural and artificial seawater at
4C and 22 C. Exposure periods ranged from 3 hours to 4 days.
   Macroscopic observations were performed to determine the
effects of the dyed oil contacting the gill surfaces and the means by
which  the oil was either ingested or ejected. Definite patterns of
response  to the dyed oil were  established. Essentially, the clams
treat oil  micelles and globules as food or detritus  particles.  The
smallest oil micelles are passed by ciliary currents directly  to the
stomach.  Larger globules are bound by mucus secreted by the gill
ctenidia.  Gas chromatography and mass spectrometry confirmed the
binding of oil-mucus. The oil-mucus is ingested or rejected by means
of the clam ciliary pathways. Implications of the oil-mucus mecha-
nism and the ejection of this mucus into the environment are dis-
cussed.

INTRODUCTION

   Oil spillage  continues to be a serious problem in coastal and
estuarine waters.  Many  spills reported from barges, tankers, and
industrial installations involved fuel oils [8,2,32,22,20] . Spilled oil
may be altered  by many factors including evaporation and  photo-
oxidation. The water soluble portions of the oil, mostly aromatics
[5,21], will dissolve and  be dispersed.  Many of the petroleum
hydrocarbons may be dispersed throughout the water  column by
formation of emulsions, either by turbulent wave action [13,12] or
chemical  dispersion. Morris [23] documented the occurrence of oil
emulsions in the  eastern Mediterranean. Some of the  oil may be
adsorbed to particulate matter within the water column or mixed in
the sediment. Boehm and Quinn [4], experimenting with #2 Fuel
Oil, have demonstrated the existence of accomodated (particulate or
droplet),  solubilized (colloidal micelles), and soluble hydrocarbon
components in fuel oil-seawater mixtures.
   The solubilized petroleum hydrocarbons dispersed  throughout
the water column and sediments may be ingested by benthic marine
filter feeders. These animals filter the water to obtajn planktonic
food, maintain respiration, and  excrete wastes. Chemical analyses
have shown that various components of oils can accumulate within
marine bivalves [11,6,7,33,10].
 Figure 1.  Gas Chromatograms:  (a) 8 ppm #2 fuel oil in hexane,
 attenuation 1 X 128Mb) Carbon tetrachloride extract of clam mucus,
 attenuation 10 X 1024

   Number two fuel oil was chosen for study because it is com-
monly shipped in coastal waters, used in coastal industrial installa-
tions, and  has already been involved in a well-documented  spill [2].
The #2 fuel oil was supplied by  the United States Environmental
Protection Agency,  Industrial Waste Treatment Research Labora-
tory in Edison, New Jersey. Two temperatures, 4C and 22C, were
used to represent the winter and summer temperature regime found
in the New York region. The bulk of the study was performed at
4C assuming that  the winter with its accompanying wind and
                                                     463

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464
CONFERENCE ON  PREVENTION AND CONTROL OF OIL  POLLUTION
storms would be the most likely time for  spills to occur. Oil was
added as an emulsion assuming that initially much of the spilled oil
is  naturally  emulsified through turbulent wave action. Subsequent
treatment by oil dispersants would increase emulsion formation.
   Mya arenaria were chosen to study because they are a. commer-
cially valuable bivalve commonly found in the littoral and sublitto-
ral zones on the American eastern Atlantic coast from the coast of
Labrador to the region of Cape Hatteras, North Carolina, and on the
Pacific coast from Monterey, California,  to Alaska. Mya occur on
the European coast from Norway to the Bay of Biscay, France, and
along the western Pacific coast from  the Kamchatka Peninsula to
the southern regions of the Japanese Islands [16]. Young Mya with
a  mean length  of  25 mm  [25,27]  were utilized  because  young
bivalves tend  to have higher  respiratory and filtration rates than
older bivalves [28,29,19,9,35]. Young clams would therefore prob-
ably accumulate oil present in the water column faster than older
clams. They would also be prone to recurrent exposure to oil during
their maturation process with the concurrent possibility of continu-
ous low-level accumulation of petroleum hydrocarbons within their
tissues. A continuing re-exposure to oil can occur in cases where the
oil is mixed into the sediments and slowly released again. Farrington
and Quinn [ 11 ], Zafiriou [36], Blumer et al.  [2], and Scarratt and
Zitko [31]  have reported  the  occurrence of  petroleum hydrocar-
bons  from marine sediments. Blumer et al. [2]  demonstrated that
oil-contaminated sediments can continue to release  relatively unde-
graded oil for extended periods of time.
   Numerous studies have reported  the occurrence of petroleum-
derived  hydrocarbons in bivalved molluscs, and several mechanisms
by which this may occur have  been conjectured. This paper offers
experimental verification of a mechanism by which a commercially
important bivalve, Mya arenaria, can  accumulate oil within its tis-
sues. The  paper also shows the  behavioral response  of Mya arenaria
and  the possible deleterious ecological side effects that result from
oil accumulation.
Methods and Materials

   The mode  of accumulation  of #2 fuel oil  and behavioral
response of Mya was followed by using an oil soluble dye, Oil Red
0. Adapting a procedure developed by Gruenfeld and Behm [14],
#2 fuel  oil and Oil Red  0 (5  mg) were emulsified in 100 ml  of
chilled tap water by ultrasonification to produce a stable 20,000
ppm dyed oil in water  emulsion. This was diluted to the desired
concentrations for each test. After  exposure  to the dyed oil, each
clam was removed from the water  and  its left valve was removed.
Dyed oil accumulation was then  followed by observations using a
binocular dissecting scope. A color  photographic record was main-
tained  of selected specimens.
   A preliminary series of four experiments  was run at  different
exposure periods (3-24 h)  and concentrations  at 22C to determine
procedures and  patterns.  In the  first experiments, lab acclimated
clams were placed in specimen dishes with natural filtered seawater
(26%)  or artificial Rila seawater mix (26%). The dyed oil emulsion
was  added either by syringe when  the  clam  was siphoning or by
adding the desired concentration to the water.  In subsequent experi-
ments, clams were placed in 1,600 ml of Rila artificial seawater mix
(26%),  and enough dyed oil emulsion was added to achieve the test
concentration. The salinity chosen for the tests was the salinity  of
the water at the time the clams were collected. In those experiments
in which clams were kept  in 1,600 ml seawater, aeration was pro-
vided through a 1 ml pipet  with a moderate airflow.
   In the experiments performed  at 4C, clams were  kept in 1,600
ml of artificial seawater  (26%) and exposed to concentrations of 50
ppm, 100 ppm, and 150 ppm dyed oil emulsion for periods of 1, 2,
3, and 4 days.
   The dye was determined to be totally insoluble  in mucus by
taking  human mucus and mixing the raw dye in. The dye and mucus
remained insoluble. A drop of #2  fuel oil instantly solubilized both.
After mixing  and standing  for several minutes in seawater, the
mucus-oil-dye combination had  the same appearance as seen in the
clams in the experiments.
                                                        Results

                                                           The dyed oil emulsions were very stable in the water column and
                                                        did not crack readily.  Whenever the dyed oil  emulsion cracked,
                                                        small red oil globules would appear on the water surface. Even when
                                                        some of the dyed oil emulsion  accumulated on the surface, there
                                                        was still a visible red tint in the  water column. This may have been
                                                        due to the solubilized (colloidal micelles) and soluble hydrocarbon
                                                        components in  the  dyed fuel oil emulsion-seawater mixture [4].
                                                        Because some of the dyed emulsion did crack with time, actual
                                                        exposure concentrations are relative only to time 0 when the emul-
                                                        sion was added. The Oil Red 0 (C.I. 26125) is a neutral dis-azo dye
                                                        which does not contain water solubilizing groups and hence is insol-
                                                        uble in aqueous media but is soluble in oils, fats, waxes, etc. [15].
                                                        The Oil Red 0 dye was  totally insoluble in  aerated and nonaerated
                                                        control  mixtures of dye and  artificial  seawater  and  dye and
                                                        natural seawater.  Earlier toxicity  and ancillary experiments had
                                                        confirmed  that  the  patterns  of mucus  secretion  and  behavior
                                                        response were elicited by the emulsified fuel oil. The dye had no
                                                        effect on the clam.
                                                           In  preliminary experiment I  at 22C, seven clams (25-35 mm)
                                                        were used. Tests were run for 3  hours. All clams behaved similarly.
                                                        The dyed  oil emulsion  present  in  the  water while  the clam was
                                                        filtering resulted in  periodic  "coughing" and periodic rejection of
                                                        dye-oil in a mucus binding. This periodic coughing is a rapid con-
                                                        traction of the adductor muscles which forces water out the inhal-
                                                        ant siphon. It is a mechanism  commonly used by bivalves to rid
                                                        themselves of accumulated detritus and pseudo feces.
                                                           Within 2 to 3 hours, two clams had dyed oil-mucus visible in the
                                                        stomach.  One of these plus two  others now had  dyed oil-mucus in
                                                        the digestive diverticula  and around the style. Most commonly, the
                                                        dyed oil was found bound in mucus near the palps. The behavior of
                                                        the dyed oil on the gills and palps was closely followed. It was noted
                                                        that the oil droplets and globules were  passed  to the edge of the
                                                        gills,  bound  in  mucus as a food or  detritus particle, and  passed
                                                        towards the palps. Smaller oil droplets passed directly to the palps.
                                                        The oil usually followed the ciliary pathways outlined by  Kellog
                                                        [18].
                                                           The results from  preliminary  experiments II and III are listed in
                                                        tables  1 and 2 respectively. In preliminary experiment IV at 22C,
                                                        clams were  exposed  to  50 ppm  and 100 ppm of dyed #2 fuel oil
                                                        emulsion for 24 hours. Large clams were used (40-50 mm), four per
                                                        concentration. At 50 ppm exposure, one clam accumulated dyed oil
                                                        and oil-dye-mucus in the stomach  and  diverticula.  All had much
                                                        oil-dye-mucus on the mantle, gills, and some on the palps. At 100
                                                        ppm exposure, three died. One accumulated oil  in the stomach and
                                                        diverticula, with oil-dye-mucus on gills and mantle.
                                                           The first  accumulation experiment at 4C was a 24-hour expo-
                                                        sure of clams (20-25mm) to the dyed-oil emulsion at concentrations
                                                        of  50 ppm and 100 ppm. Fourteen clams were used in the test at 50
                                                        ppm exposure. Four  clams accumulated dyed oil in the stomach and
                                                        diverticula. Two  clams had no  oil visible and ten had dyed oil in
                                                        mucus  on the gills, palps, mantle, and foot. Some dyed oil-mucus
                                                        exited through the pedal aperture. Seven clams were exposed to the
                                                        100 ppm concentration.  Five clams accumulated dyed  oil in the
                                                        stomach and digestive diverticula. All clams had oil-dye-mucus on
                                                        gills, mantle, and palps.
                                                           The results from  accumulation experiment II at 4C are listed in
                                                        table 3. In  these experiments, clams were exposed to the dyed oil
                                                        emulsion  for  1, 2, 3, and 4 days. Figure 2 illustrates the results of
                                                        accumulation experiment II  at  4C.  Experimental problems pre-
                                                        cluded  the measurement of the incorporation of the  150 ppm con-
                                                        centration of the dyed oil emulsion on day one.
                                                           Chemical  confirmation of the oil  being  bound or adsorbed to
                                                        mucus  is shown in figure 1. The data was obtained from a different
                                                        series of experiments in which Mya were exposed to sublethal con-
                                                        centrations of #2 fuel oil. Complete results from these experiments
                                                        will be published at  a later date. The mucus extracted was secreted
                                                        by clams exposed to an initial concentration of 100 ppm emulsified
                                                        #2 fuel oil.  Four weeks later some of the  mucus was collected. It
                                                        had a  gray-green flocculent appearance  and  formed  flocculent
                                                        clumps on the surface and in the water  column. A sample of water
                                                        and mucus was  taken from the water  column (there was no surface

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                                                                                                           EFFECTS
                                                          465
     Table 1. Preliminary experiment //-22C, natural filtered
     seawater, clams (20-25 mm) were placed in specimen dishes
                      and dyed oil added

 Group 1 66 ppm, exposure 5 hours (3 clams)
          Much  of  the dyed  oil  was visibly bundled in  mucus.
          appearing as  semisolid viscous globs  on the surface. No oil
          was readily visible  in the gut. Most oil  was bundled in
          mucus near pedal and palp area or following general cili-
          ary rejection  pathways.
 Group 2  83 ppm, exposure 5 hours (5 clams)
          All clams had  packaged th(  ill-dye within  mucus. This
          oil-dye-mucus was always found on  and around the palps.
          A small amount was sometimes found on the  gills.
 Group 3  83 ppm,  exposure 5 hours (kept  anaerobic l'/i hours
          before test by placing on  wet towel to  induce  filtering
          when placed in oil and water) (7 clams)
          Oil-dye-mucus strings were near, on, and around the palps
          in 5 clams. None had oil visible in the visceral mass; some
          oil-dye-mucus was being shunted  out the pedal aperture
          and siphon in several clams.
 Group 4  83 ppm, exposure 8 hours (kept anaerobic 6  hours before
          test to induce filtering when placed in oil and water) (8
          clams)
          Three  clams  showed no trace of oil. One clam's digestive
          diverticula were tinged red. Five clams had oil-dye-mucus
          on, near,  and around  palps and  siphon and very small
          amounts in mantle rejection tracts.
 Group 5-83 ppm, exposed 9  hours (kept anaerobic 6  hours before
          test) (8 clams)
          Two clams showed no trace of oil. The other 6 clams had
          oil-dye bound in mucus  near the  palps and/or siphon. A
          few had some in the mantle rejection tracts.
 Group 6Kept anaerobic l'/2 hours prior to test, placed in seawater,
          and fed 2,000  ppm oil-dye emulsion from syringe when-
          ever pumping for 8 hours (5 clams)
          Four  clams had accumulated oil in stomach. No  oil was
          visible in the digestive diverticula. Several clams had some
          oil-dye bound in mucus near the palps or siphon.
 Groups 7 & 8-Kept anaerobic 6 hours prior to test, then placed in
          seawater and fed dye-oil  emulsion by syringe for 9 hours.
          Four  clams were fed  a  100 ppm  emulsion.  All accumu-
          lated oil in the stomach and in mucus around  palps and on
          mantle. Four clams were fed a 2,500 ppm  emulsion. Three
          accumulated  oil  in stomach; all accumulated  oil in mucus
          on palps, near siphon, and some on gills and mantle.
slick) and filtered through no.  1 Whatman paper. Infrared analysis
of the water at  the  time  of sampling indicated a concentration of
approximately 0.466 ppm  oil  in the water column. Solvents were
glass  distilled  and checked for impurities.  All glassware was pre-
washed with solvents. Approximately 40 ml of mucus-water sample
was filtered and  then washed with artificial seawater. The filter was
then extracted in an erlenmeyer flask with 50 ml of carbon tetra-
chloride  with  constant  shaking for ten minutes. The sample was
then filtered through prewashed sodium sulfate and concentrated to
a volume of 0.15 ml  under  nitrogen. During  this  procedure,  all
carbon tetrachloride  evaporated. Gas chromatographic analysis was
done using a Perkin Elmer Model 900 gas chromatograph equipped
with a flame ionization detector and a six-foot stainless steel column
packed with 8% Dexsil on 80/100 mesh of Chromosorb W. Standard
operating conditions were as  follows: carrier gas N2,  2 cc/min;
detector  H2, 20  cc/min, air 40 cc/min; injector temperature 200C,
detector  temperature 300C, manifold temperature 300C; column
temperature 70C with two-minute hold, programmed  at an increase
of 8C per minute to 300C; injection sample  0.1 micro liter. The
mucus sample gas chromatographic tracing was  quantitated gravi-
metrically by  compaiison to  the peak aiea of 16 micrograms  of
octacosane dissolved in hexane. The  calculated quantity of oil
derived from the mucus was 833 micrograms. The hydrocarbon con-
tent of the water was 4.66 ppm or  0.466  micrograms per milliliter.
Therefore, the most  the  hydrocarbon background of  the water
   Table 2. Preliminary experiment I1J-22C, clams (25-34 mm)
   placed in 1,600 ml Rila seawater mix (26%), 12-houi exposure

      cone.: 25 ppm (6 clams), 50 ppm (6 clams),  100 ppm
             (5 clams)-all in #2 fuel + dye
             50 ppm (5 clams) in So. Lo. crude oil
25 ppm (#2 Fuel):
50 ppm (#2 Fuel):
100 ppm (#2 Fuel):
50 ppm (So. Lo. Crude):
Five clams had oil-dye and or dye-mucus
accumulated  in the stomach and divertic-
ula.  All  had  oil-dye wrapped in  mucus
near and  on palps, and  some on mantle
and gills.
All clams  had  oil-dye  and  or  oil-dye-
mucus accumulated  in  the  stomach and
diverticula.  In some the  style tip was
slightly  tinted. All had oil-dye-mucus on,
near,  and around  palps, some near foot
and on mantle, gills, and pedal aperature.
Two clams accumulated  oil-dye in  stom-
ach and diverticula, but no oil was visible
in three.  All had large amounts of oil-dye-
mucus on gills, palps, mantle, siphon, and
pedal aperture.
All 5 clams  accumulated  dyed  oil and
dyed oil-mucus in the stomach and  diges-
tive diverticula.  Only 2  clams had small
amounts  of oil-dye-mucus visible on gills,
mantle, and pedal aperture.
could have contributed by adhering to the filter was i 8.64 micro-
grams, and the remainder was from the oil bound in mucus. Mass
spectrometric analysis of  the extracted sample was provided by the
U.S. Environmental Protection Agency, Industrial Waste Treatment
Research Laboratory. Edison.  New lersey. Analysis demonstrated
that the  sample  was predominantly dimethylnaphthalenes and tri-
methylnaphthalenes with  paraffins,  mainly in the C-14 and C-15
regions.
Discussion

   When dyed oil initially contacts the gill surfaces, the beating of
the gill frontal cilia often disintegrates the film  or globules. The
resultant small oil globules and micelles then pass along ciliary tracts
to the palps directly, or enwrap in mucus secreted by  the gills. The
clams treat the oil globules and micelles as food or detritus particles.
The  mucus  covered particles  will then  either  fall onto rejection
paths of the mantle or enter the esophagus and stomach. When the
dyed  oil is bound in mucus it follows the ciliary  pathways as out-
lined  by Kellog  [18].  The movement is similar to the general pat-
terns  described for bivalves by Morton [24] and J^rgensen [17], If
the mucus strings accumulate on the gills and palps and fall onto the
mantle, they may be ejected out the siphon or pedal aperture. As oil
concentration and exposure  time increases, production of mucus
binding the  oil  increases. This tends to  accumulate in  the anterior
portion of  the clam around the palps causing the  clams to increas-
ingly shunt  the bulk of the rejecta  out the pedal aperture.
   Bernard [ 11 described particle  sorting and labial palp function in
the Pacific oyster. Suspended  particles, both organic and inorganic.
which  impinge on  the  gill  ctenidium  are entrapped m mucus and
transported by the action of the frontal cilia. Stimulation of two or
more  adjacent ctenidial filaments  results in a copius secretion. The
ctenidial surfaces are normally covered with a thin watery fluid not
subject to ciliary movements. A definite band of mucus overlays the
frontal cilia. Tactile stimulation of the ctenidia results in the abun-
dant  production of "rejection" mucus  forming a sheet over the
ctenidium. The mucus masses are then carried to the free margins of
the ctenidia. Overstimulation of the filaments by particles wili cause
the release of rejectory mucus which is removed from the ctenidium
by muscular action  or its inability to enter the food grooves. Much
of the mucus is  carried to  the palps. In  the normal state the inner
surfaces of the  palpi are always pressed  together.  The complicated
ciliation of the palps brings  any large mucus masses to the free edges

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 466
CONFERENCE ON  PREVENTION AND CONTROL OF OIL  POLLUTION
   Table 3. Accumulation experiment 11-4C, clams (20-25 mm)
       exposed 1, 2, 3, and 4 days to #2 fuel oil-dye emulsion.

 Cone.: 50 ppm, 100 ppm, and 150 ppm in  1600 ml Rila s.w. (26%)

           Day 1
 100 ppm:  (6 c!ams)-Four clams accumulated dyed oil in stomach
           and  digestive diver ticula.  One  had no oil visible.  All
           others  had varying degrees of  oil-dye-mucus on palps,
           gills, mantle, and  in mantle rejection tracts near siphon
           and pedal aperture.

           Day 2
 50 ppm:   (6 clams)-Three  clams accumulated  dyed  oil in  the
           stomach and digestive diverticula.  All clams had varying
           amounts of oil-dye-mucus on  gills, palps, mantle, and
           foot, and near siphon  and pedal aperture, and in mantle
           rejection tracts. There were many  visual observations of
           oil following ciliary pathways.
 100 ppm:  (6 clams)-Two clams  had dyed  oil and oil-dye-mucus in
           stomach and diverticula. All had varying amounts of oil-
           dye-mucus on gills, palps, mantle,  and  foot. Oil globules
           in all clams followed standard ciliary pathways, [18].

           Day 3
 50 ppm:   (6 clams)Four clams accumulated dyed oil in the stom-
           ach and diverticula. All had varying amounts of oil-dye-
           mucus  on gills,  palps, mantle, and foot, and out siphon
           and pedal aperture. There were many visual observations
           of oil  being bound in mucus and passing along ciliary
           paths.
 100 ppm:  (6 clams)-Four clams accumulated dyed oil in stomach
           and  diverticula.  Oil-dye-mucus observations  were  the
           same as for 50 ppm, except more was present.
 150 ppm:  (6 clams)-Two clams  accumulated dyed oil and oil-dye-
           mucus  in  stomach and  diverticula.  All  had  varying
           amounts of oil-dye-mucus on gills, palps, etc., as did 100
           ppm and 50 ppm, except much more was present for 150
           ppm.  Many observations  were  made of oil-dye being
           passed  along ciliary tracts, wrapped in mucus, and pass-
           ing to palps and into stomach or  along rejection tracts.

           Day 4
 50 ppm:   (6 clams)-All accumulated dyed oil and dye-oil-mucus in
           either the stomach, digestive diverticula, or both. All had
           varying amounts of oil-dye-mucus  on gills, palps, mantle,
           etc.,  as outlined. There were  many visual observations of
           oil  following ciliary  tracts  and  being enwrapped in
           mucus.
 100 ppm:  (6 clams)-Four clams accumulated dyed oil and dye-oil-
           mucus  in stomach. All had much  oil-dye-mucus passing
           out the pedal aperture, near palps  and  siphon, and some
           was on gills, mantle, and foot. There were many observa-
           tions of oil being  ensnared in mucus and passing along
           ciliary paths.
 150 ppm:  (6 clams)-Four clams accumulated some dyed oil and
           oil-dye-mucus in stomach, digestive diverticula, or both.
           All were relatively unresponsive  and slow. Much oil-dye-
           mucus was in each clam on gills, mantle, and near siphon.
           It was also passing out the pedal aperture, and it was near
           the siphon  and on visceral mass and foot. There were
           many observations  of  oil  following ciliary paths and
           being ensnared in mucus.
of the palps for rejection. The mechanism is presumably the cause of
,oi]-dye-mucus commonly  occurring in large masses on, near, and
around the palps of My a. When sufficient rejecta accumulate in the
palp region,  the clam contracts the  tip  of the siphons  and  then
rapidly  contracts the anterior adductor muscle forcing water and
rejecta out the pedal aperture. There also is a slow ciliary beat  on
(he mantle expelling rejecta out the pedal aperture. This pattern of
rejection may not be a primary pathway in the field. When the clam
is burrowed in the  sand mud  substrate, it is extremely difficult to
                                                        Figure  2.   Accumulation experiment
                                                        #2 fuel oil emulsion at4C
Clams exposed to dyed
                                                        expel  rejecta  out  the pedal aperture. The bulk of the rejecta accu-
                                                        mulates within the mantle cavity. If much rejecta accumulates, the
                                                        clam has to do more work to expel it out the inhalant siphon. In the
                                                        interim, the oil-dye-mucus may  possibly release some  of the  oil
                                                        again. In  essence,  this would  constitute an internal re-exposure to
                                                        oil. Other detrimental effects are the increased energy demand  on
                                                        the clam for mucus secretion, ciliary beating, and muscular contrac-
                                                        tions  and  the synergistic  effects of narcotization by the oil and
                                                        clogging of gills, palps and digestive tract. Concentrations of #2 fuel
                                                        oil  above  100 ppm over  a 24-hour or longer exposure cause  an
                                                        increasingly greater degree of general narcotization with increasing
                                                        oil concentration.
                                                           Figure  3 illustrates the general ciliary pathways which dyed #2
                                                        fuel oil globules and micelles follow in Mya, as they are bound in
                                                        mucus. The oil enters through the inhalant siphon into the pallial
                                                        cavity.  Here the oil  follows pathways  depicted where  the  mi-
                                                        celles  pass to the edge  of gill  ctenidia  to  the  marginal groove.
                                                        Ciliary currents then sweep the material along the marginal groove
                                                        towards the palps. Some  of the rejecta passes slowly along mantle
                                                        rejection tracts towards the inhalant siphon and accumulates at the
                                                        base of the inhalant siphon. Occasionally,  the clam contracts expel-
                                                        ling the dye-oil-mucus out the inhalant siphon.
                                                        Figure 3.  General ciliary pathways which dyed #2 fuel oil droplets
                                                        follow as they are bound in mucus

                                                           Whether a clam will accumulate the dyed oil as dyed oil-mucus
                                                        in the stomach and diverticula seems to be related to prior feeding,
                                                        oil concentrations,  length of exposure, and temperature. Figure 2
                                                        illustrates  the results of accumulation experiment II at 4C. 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

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                                                                                                           EFFECTS
                                                          467
only to time zero at the beginning of the test. The higher tempera-
ture, 22C, seemed to accelerate the accumulation of dye-oil-mucus,
general  narcotization,  and death. The lower  temperature, 4C,
tended to enhance the stability of the oil emulsion and slow  the
narcotization of the clam.
   If the clam's gut is full of detritus  and silt there is little visual
accumulation. The most rapid accumulation of oil seems to be cor-
related with a partially empty gut.  When  the dyed  oil is in  the
stomach, it may  be in mucus fragments  or as minute droplets. Occa-
sionally, the style tip may be tinted red. Next, the digestive divertic-
ula gradually become tinted from  the stomach outward toward the
limit of the digestive diverticula where the tissue becomes undiffer-
endated gonad.
   The ability of the  clam to accumulate  oil, concentrate it in
mucus, and release it, may have deleterious ecological side effects.
While the clam is accumulating oil  within its own tissues, it may also
bind and concentrate any oil entering its pallial cavity.  Some of this
is  accumulated  and some ejected from the clam as  concentrated
oil-mucus. This may occur at high-oil concentrations even while the
clam is dying. At low-oil concentrations, the effect would be more
insidious. Filter-feeding  bivalves must filter large volumes of water
to sustain their nourishment and respiration. Even very low concen-
trations of  oil in the water column can be concentrated by the clam
in its tissues and mucus. Essentially, the  clams serve as bioconcentra-
tors, accumulating oil in their tissues and rejecting some in the form
of concentrated oil-mucus. This situation is greatly aggravated in the
case  of coastal spills when dispersants and sinking agents are used.
The effect  of sinking  oil or dispersing  it is primarily cosmetic and
increases the potential  for oil  accumulation  by filter  and detritus
feeders.
   The ability of the  clam to accumulate and concentrate oil  has
other side effects. Eventually, the effects of the accumulated oil  can
cause death. The oil in the decaying clam flesh is deposited into the
sediments  at depths of  2-14 centimeters. Several studies including
Blumer et al. [2], Blumer and Sass [3], and Farrington and Quinn
[11] have shown that oil may exist in sediments in an undegraded
state for extended periods of time  with the potential for release into
the environment again. The release of oil-mucus rejecta by the clam
also has the potential for further contamination. The mucus strings
may be worked into the sediment or utilized as food by a variety of
detritus feeders and benthic scavengers, resulting in their contamina-
tion. Scavengers  such as green  crabs and brown shrimp  are common
prey for fish. The clams themselves are a common food for many
food  and  game  fish  such as  striped bass [26], black drum, and
flounder.  Trevallion et  al. [34] reported that the siphons of clam
were a major source of food for plaice.  Many crabs, including those
used for human consumption and those utilized by marine predators
such as striped bass, also feed on clams [30]. By these means, low
concentrations of oil may rapidly be disseminated  through  the
marine sediments and food chains.
ACKNOWLEDGMENT

   The analyses for this investigation were performed at the analyti-
cal  facility of the  U.S. Environmental Protection  Agency (EPA),
Industrial  Waste Treatment  Research  Laboratory in Edison,  New
Jersey. Use of these facilities was provided by an EPA program that
supports graduate level research of the environment.
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2. Blumer, M.;  Sass,  J.; Souza,  G.; Sanders, H.; Grassle, F.; and
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3. Blumer, M.,  and  Sass,  J.  1972. Oil pollution:  persistence and
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10. Ehrhardt,  M.  1972. Petroleum hydrocarbons in oysters from
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12. Forrester, W.D.  1971.  Distribution  of suspended oil particles
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My subject is - The Ultrasonic Dispersion, Source Identification, and
                Quantitative Analysis of Petroleum Oils In Water*


     I would like to discuss three projects that are now underway in our

laboratory.  We are developing methods for identifying and quantitating

small amounts of petroleum oils in water, and a procedure for preparing

stable oil in water dispersions containing known amounts of oil.  The

identification method will correlate milligram and sub-milligram amounts

of water dispersed oils with undispersed oils, for the purpose of source

identification; the quantisation method will  estimate part-per-billion

amounts of petroleum oils in water, in the presence of non-hydrocarbons;

and the oil in water dispersion method is needed by biologists for oil

toxicity evaluations and by chemists for evaluation of oil quantitation

methodology.  Our work on these projects is still  in progress and our

conclusions are therefore tentative.  In fact, the present discussion

and paper deal only with the rather limited segments of each project that

have actually been completed.

     Source identification of waterborne oils is accomplished in three

steps.  The oils are first separated from water.  The weathered and  neat

oils to be compared are then submitted to distillation or to some other

vaporization procedure, which induces comparable volatility losses in

the oils.  The non-volatiles are then compared by techniques such as gas

chromatography, infrared, and fluorescence spectroscopy.  We used a  gas

chromatographic procedure of the American Society for Testing and Materials

(ASTM) to compare the oils.   But, we developed a procedure for inducing

volatility losses in milligram and sub-milligram amounts of oils, that

simulated volatility losses  resulting from an ASTM distillation method

that requires approximately  40 gm quantities  of oil.  My present discus-

sion of oil identification is restricted to our vaporization procedure.
^Presented by Michael  Gruenfeld, September 8, 1975, at the ICES Workshop
 on Petroleum Hydrocarbons, in Aberdeen, Scotland.   Mr.  Gruenfeld is a
 Supervisory Chemist with the U.S. Environmental  Protection Agency,
 Industrial  Environmental  Research Laboratory-Ci,  Edison, NO  08817
                              -l-

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                               - 2




     Oil  quantitation is the second subject of my discussion.   We are de-



veloping an IR spectroscopic method for estimating part-per-billion amounts



of petroleum oils in water, even in the presence of non-hydrocarbons.  We



are evaluating as part of this method a rapid silica gel adsorption tech-



nique for separating petroleums from non-hydrocarbons, directly in carbon



tetrachloride solution.  My present discussion of oil quantitation deals



only with this adsorption procedure.



     Preparation of stable oil in water dispersions having a known oil con-



tent is my third subject for discussion.  A commercial ultrasonification



device is used to prepare oil in water concentrates that readily mix fur-



ther with water.  We also checked whether ultrasonification causes spectral



or chromatographic changes in oils.



     My discussion therefore emphasizes the procedures and results of the



oil vaporization, silica gel adsorption, and ultrasonic dispersion techni-



ques.



     We evaluated vaporization techniques by preparing oil in water disper-



sions and then extracting them with Freon 113.  Dilutions were then made



to yield solutions containing 0.5 or 30 mg oil.   (Slide 1) These were trans



ferred to 150 ml beakers and stripped to 1-2 ml, with a steam table and a



filtered air stream.   (Slide 2)  The concentrates were then transferred to



small glass vials (10  x 30 mm).  Vials containing 30 mg oil were sus-



pended in water at 40  C.  The filtered air stream was used to remove



final solvent traces,  and this condition was maintained for 10 additional



minutes.  Vials containing 0.5 mg oil were maintained at room tempera-



ture, and the air was  turned off just before total solvent removal.   Final



evaporation then occurred spontaneously.  These vials remained open at

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                              -  3




room temperature for 10  additional  minutes.   Finally,  small  amounts  of car-


bon tetrachloride (10 -  20  ill) were added  to  each  vial  for gas  chromato-


graphic injection.


     The neat reference  oils  were  treated  somewhat differently.   Seventy


milligram oil was added  directly to the  vials,  which  were then  suspended


In water for 15 minutes, at 40C,  while  exposed to the air stream.   The


oils were then injected  onto  the gas chromatographic  instrument without


further dilution.

     ,       .     vaporization  ,  .          T         n     ,.     ,
     (Slide 3) Our             techniques, as I previously mentioned,  in-


duced losses in small amounts of oil that  approximate  losses induced by an


ASTM distillation procedure for  large  amounts of oil.  This slide illustrates


the results of the ASTM  procedure.  The  upper chromatogram is South  Louisiana


Crude oil before distillation;  the lower chromatogram results from use of


the ASTM distillation.  The numbers refer  to  n-alkanes, while pristane and


phytane are abbreviated  as  Pr and Ph.   The ASTM procedure causes major loss


of components below the  C-,-, n-alkane.   (Slide 4) Our  procedure  for inducing


vaporization losses in 70 mg neat oils yields similar results.   The  upper


chromatogram is South Louisiana  oil after  ASTM distillation; the lower


chromatogram is the neat oil  after our procedure.   (Slide 5) The lower chro-


matogram here results from  our treatment of 30  mg oil that was extracted


from water.  The upper chromatogram is the 70 mg portion of neat oil.


(Slide 6) The upper chromatogram here is again the 70 mg portion of neat


oil; the lower chromatogram results from our  treatment of 0.5 mg oil that


was extracted from water.  The lower chromatogram  shows evidence of gas


chromatographic column degeneration, but adequate resolution of pristane


and phytane from the normal alkanes was  achieved with a new column.   I


should emphasize that although oils that actually weathered in the water

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                               - 4 -
column have not yet been evaluated, our techniques  are usable  for identi-
fying small amounts of oil  that occur as slicks and shoreline  residues.
     Next I will discuss the silica gel adsorption  technique that was
evaluated in the oil quantitation project.  Silica  gel was  tested for  sep-
arating petroleum oils from animal and vegetable oils directly in carbon
tetrachloride solution.  We contrasted the sorption capacity of silica gel
with its solution contact time, and degree of activation.   Carbon tetra-
chloride solutions of two petroleum oils at 20 mg/100 ml,  and  vegetable,
olive and cod liver oils at 100 mg/100 ml  were prepared in  100 ml volumet-
ric flasks.  3.0 gram portions of activated and partly deactivated silica
gel were added to the flasks.  (Slide 7) The solutions were vigorously
stirred for five and ten minute intervals  with a magnetic  stirrer.  (Slide
8) A plot was prepared contrasting oil removal, with silica gel  deactivation
and solution stirring time.  Maximum separation of petroleum from non-
petroleum oils resulted from fully activated silica gel and ten minute
stirring.  No loss of petroleum oils occurred.  Decreased  activation,  and
reduced stirring yielded adverse results.
     I will now briefly describe the preparation of oil in  water dispersions
[Slide 9) The probe of an ultrasonic instrument was inserted into a 50 ml
graduated cyclinder containing water and an accurately weighed amount  of
oil.  Maximum dispersion energy was applied for two minutes, while keeping
the cylinder in an ice bath.  Cooling prevented oil vapor losses during
ultrasonification, which causes substantial heating.  Dispersion stability
diminished with increasing oil viscosity.   A non-viscous crude oil yielded
stable 1%  (10,000 PPM) dispersions that were stable for days; but a more
viscous oil yielded less stable and less concentrated dispersions.  This
technique permitted oil recoveries in the range 96 -  100%.   A check for

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                              - 5 -
ultrasonically induced changes  showed no substansive chromatographic or



spectral  alterations  of the oils.



     In closing,  I  would like to  repeat that this  discussion  and paper



deal with only limited portions  of three projects.   Our work  is  still



underway, and we  hope to provide  final  results  in  the near future.







                               THANK YOU -

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Slide 1

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Slide 2

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Slide 3

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Slide 4

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ETENTION TIME (min.
           Slide  5

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30 "^RETENTION TIM
                 Slide 6

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Slide 7

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   110 r-
   100$
                               A
                               A
i   90
              Key
A  South Louisiana Crude Oil
A  No.2 Fuel Oil
O  Vegetable  Oil 10 min. stirring
  Vegetable  Oil 5 min. stirring
  Cod Liver Oil 10 min. stirring
O  Olive  Oil 10 min.
                    3         5
                    PERCENT DEACTIVATION
                           Slide 8

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Slide 9

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           The Effect of a No. 2 Fuel Oil and a South
        Louisiana Crude Oil on the Behavior of the Soft
                   Shell Clam, My a arenaria L.
                          Dennis M. Stainken
                           Rutgers University
                         Dept. Zoo/. & Physiology
                             Newark, N.J.
             Present Address: U.S. Environmental Protection Agency
                Industrial Environmental Research Laboratory-Ci
                   Oil & Hazardous Materials Spills Branch
                           Edison, N.J. 08317

                            Introduction

     The toxic effects other than lethality  of oils has  often
been treated as a  secondary problem  in bioassays.   According to
STIRLING (1975), there is  a need  to  enlarge  the  concept  of the
routine bioassay test to  include  quantitative  measurements of the
effects of pollutants on  behavior, physiology  and  metabolism.
The inherent interspecific  and  intraspecific physiological dif-
ferences of test species  and various  test conditions make direct
comparison of bioassay results  difficult.  VAUGHAN (1973) and
BEAN, et_ a]^. (1974) discussed  these  problems in  detail.

     Few studies have characterized  the  behavioral effects of
oils on bivalves or examined the  effects  of  temperature  simultan-
eously.  WILSON  (1974) noted that bivalve molluscs have  been
avoided for toxicity tests  because it is  difficult to establish
a simple criteria  of effect.   This study  was therefore performed
to determine the behavioral effects  of a  No. 2 fuel oil  and a
South Louisiana crude oil  in bioassay tests  conducted at winter
temperatures.  The winter  temperatures were  chosen because spills
are more likely to occur  during  the  inclement  winter weather.

     The oils were added  in an  emulsified form to  simulate a po-
tential naturally  occurring condition.   FORRESTER  (1971), FOSTER,
et_ al_.  (1971), GORDON, et_ a^.  (1973)  and  RANTER (1974) have re-
corded the formation of  oil emulsions in  sea water by various
mechanisms.  In the event  of an oil  spill during the colder
months, it is probable that much  of  the  oil  would  be dispersed
and emulsified  in  the water column through  turbulent wave action.
Many of these emulsions  tend to be relatively  stable and a mech-
ansim for the accumulation of  emulsified  oil by  bivalves has
been reported  (STAINKEN,  1975).

Materials and Method

     Behavioral observations were recorded  during  bioassay tests.
The tactile responses of  the clams were  examined by lightly tap-
ping the shell with a glass stirring rod.  Tests were conducted
according to the "Standard Dispersant Effectiveness and  Toxicity
Tests" published by the  U.S. Environmental  Protection Agency
(MCCARTHY, 1973).  An experimental concentration at which 50% of
the experimental animals  survived (LC5g)  was determined  during a
96 hr. exposure period.   The method  employed requires the use of
a standard toxicant.  Benzene was used initially but in  subse-
quent tests phenol was utilized  because  benzene emulsions were
not stable.
                                 724

Bulletin of Environmental Contamination & Toxicology,
Vol. 16, No. 6  1976 by Springer-Verlag New York Inc.

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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 4C and 14 C.
Benzene was tested at 14C.  The oils were added at concentra-
tions from 50-800 ppm.  The test concentrations of the oils
were later increased to 1,600 ppm.

     Clams for the experiments were collected from the Princes
Bay - Sequine Point Area of Staten Island, N.Y. from December,
1973 - February, 1974.  Clams were collected at water tempera-
tures and salinity closely approximating test conditions.  Young
clams with a mean shell length of 25 mm or less were utilized.
Prior to each experiment, clams were acclimated 24-48 hours in
1600 ml of artificial sea water  (salinity - 26%0).  Fifteen clams
(15-24 mm) were used at each oil concentration.  Five clams were
placed in each aerated container.  Five clams per container were
also used for standard toxicant testing (benzene or phenol) at
each concentration.

Results
     Behavioural observations were made in each LC5Q test.  A
noticeable reaction was not observed in clams exposed to benzene.
The clams merely contracted upon tactile stimulation.

     Clams exhibited an identical response pattern to oil exposure
in all tests.  Initially, at low oil concentrations  (50 ppm)
mucus was given off by the clam out the pedal opening and siphon.
Higher oil concentrations resulted in proportionally higher mucus
secretion.  As more mucus was secreted, the clams increasingly
shunted more out the pedal opening.  All effects appeared to be
both time and dose related, although crude oil effects were never
as severe as the effect of fuel oil.  Both oils depressed muscular
contraction.  A decrease in irritability and contractibility of
the siphon was noticeable at 50 ppm.  At concentrations greater
than 100 ppm, the pedal opening musculature rapidly became to-
tally relaxed and did not contract .  The adductor muscles simul-
taneously lost the ability to contract rapidly.  At concentra-
tions greater than 400 ppm, the adductors began to relax in less
than 15-20 sec and the animal consequently  'gaped'.  It appeared
that the anterior adductor was affected first, then the posterior.
                               725

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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 14C compared to responses at 4C.

     The response of clams to phenol was not completely similar to
that of oil.  Mucus secretions were never as heavy in response to
phenol, but phenol appeared to narcotize the musculature quicker.
Phenol exposed clams reacted differently than oil exposed clams.
The muscles remained at one length, turgid and lost their irrit-
ability.  Adductor muscles remained half or sometimes fully con-
tracted and the pedal aperture remained partially open.  The
muscles of the phenol exposed group rapidly became turgid at death.
The' tactile response of control clams remained normal during the
experiments and mucus secretion was not evident.

     The static bioassay data derived from the tests were inconclu-
sive.  The LCcjQ values were computed on semilog paper.  Results
of the tests are in Table I.  Tests numbers 1A and IB were run in
natural filtered sea water to determine whether testing in natural
sea water or artificial sea water would have an effect.  An ef-
fect was not found and all other tests were conducted in arti-
ficial sea water.  Scattered mortality was observed in the major-
ity of tests but in most cases there was insufficient mortality
after 96 hours to calculate a LC^Q.  Mortality was not found in
the controls.  Death was defined as a total lack of muscle re-
sponse.

     At 14C, two 96 hour LC^g values for No. 2 fuel were obtained.
These were 475 ppm (Test HA) and 535 ppm (Test IIIA).  Compari-
son by a t test revealed no significant difference and a mean
LCjg value of 505 ppm was calculated.  Some of the tests were
continued 3 days beyond the 96 hour period, to see if there was
a time effect.  At 14C, test IIIA and HIP were continued.  An
LC5Q (7 day) was found for No. 2 fuel oil to be less than 100
ppm, compared to test IIIA 1X59 (96 hour) of 535 ppm.  The LCjQ
(7 day) of phenol in test IIIB dropped to 535 ppm.

     At 4C, test IVA, IVP, VIA and VIP were also continued for
3 days (total 7 days exposure).  In test IVA, a LC$Q could not
be found.  In test IVP, the 7 day semilog plot had the shape of
a backwards  'S' and two values were derived, 80 and 225 ppm
phenol.  In test VIP, the LC5Q  (7 day) for phenol was 450 ppm.
It is probable that more mortality would have been encountered
if the tests were continued beyond seven days.
                                726

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TABLE I.  Calculated toxicity  (LC  ) during a 96 hour exposure
          period.
                              14C
(IA) So. Louisiana Crude
cone:  50,100,200,4'00,800 ppm
LC5Q = none
(IIA) i2 Fuel Oil
cone:  50,100,200,400,800 ppm
LCcjg = 475 ppm
(IIIA) //2 Fuel Oil
cone:  100,200,400,800,1600 ppm
LC5Q = 535 ppm
(IB) Benzene
10,20,30,40,50 ppm
LC__ = none
(IIB) Benzene
50,60,70,80,90,100 ppm
LC^Q = none
(HIP) Phenol
50,100,200,400,800 ppm
LC5Q = 565 ppm
                               4C
(IVA) So. Louisiana Crude
cone:  100,200,400,800,1600 ppm
LC;-,-, = none
(VA) //2 Fuel Oil
cone:  50,100,200,400,800 ppm
LC... = none
(VIA) //2 Fuel Oil
cone:  100,200,400,800,1600 ppm
LC_  = none
(IVP) Phenol
50,100,200,400,800 ppm
  cn
     = 365 ppm
(VP) Phenol
10,20,30,40,50 ppm
LC_. = none
(VIP) Phenol
50,100,200,400,800 ppm
LC5Q = 535 ppm
     The Southern Louisiana Crude oil appeared to have a few
acute toxic effects within the test parameters.
     In the tests with No. 2 fuel oil, there seemed to be a
temperature effect.  At 4C, a LC5Q was not found in any tests
(except VIA, 7 days), while at 14C a mean LC5Q of 505 ppm was
found in the two tests with No. 2 fuel oil.  A temperature
effect was not apparent in clams exposed to phenol.
                               727

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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, e_t al_. (1973) reported the
effect of crude oil and oil emulsions on bivalves and found a
similar decrease in tactile response.  The general behavior se-
quence they reported was:  increased activity; successively im-
ppaired activity; immobilization and death.  The observations of
this report are similar to their observations.

     The copious production of mucus by M. arenaria had several
effects.  It imposed a steady drain on the energy reserves of
the clams.  The continual production of mucus clogged the gills
and mantle cavity and would disrupt normal feeding mechanisms.
The clogging mucus must be expelled by the clam by increased con-
traction of the adductors and mantle musculature which puts an
additional strain on the metabolic rate.  The increased metabolic
demands for mucus production and excretion and the disruption of
normal physiological and biochemical processes occurred at much
lower concentrations of oil exposure than the LC^g indicates.

     A problem encountered in comparing the results of this study
was the lack of definition of death in bivalves in published re-
ports.  Terms such as "moribund" are found in the literature but
the criteria of death was not defined.  The definition of death
in this report was the total lack of muscular response, though
this was often difficult to determine.  Stimulation of the mus-
culature by pinching or light rapping with a stirring rod often
elicited muscular twitches though the clam appeared "dead".

     The bioassay LC.-Q values obtained for M. arenaria are
greater in concentration (ppm) than those reported for other
species exposed to No.  2 fuel oil (VAUGHAN, 1973; ANDERSON,
et_ ajL_. , 1974).  However, the values are lesser than those re-
ported for many species exposed to crude oils.  According to
the ranking system of SPRAGUE and CARSON (1970), the No. 2 fuel
tested in this report can be classified as moderately toxic to
M. arenaria.  An additional factor noted in the bioassay test
was the time of exposure.  Increasing the period of exposure from
96 hours to seven days decreased the LC5Q values obtained.
Similar increases in mortality of molluscs during longer exposure
periods were reported by SPRAGUE and CARSON (1970), KANTER et al.,
(1971) and KASYMOV and ALIEV (1973).

     Future bioassay work with molluscs, particularly bivalves,
should be determined over a 7 day exposure period.  However, be-
havioral observations appear to have advantages as a toxic
criterion over lethality, and shorter exposure periods can be
employed.
                               728

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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. REISER, and N.J.  PROUSE: J. Fish. Res.  Bd.
     Can. 3Q_, 1611  (1973).
GRUENFELD, M. and F. BEHM: Anal. Quality Contr. Newsl, U.S.E.P.A.
     16_, 6  (1973).
KANTER, R., D. STRAUGHAN, and W.N.  JESSEE: Proc. Joint Conf.
     Prevent. & Contr. Oil Spills,  Wash., D.C., A.P.I, 485  (1971).
KANTER, R.: U. So. Cal.,  Sea Grant  Prog. Publ. No. USC-SG-4-74
     (1974).
KASYMOV, A.G. and A.D. ALIEV: Water, Air & Soil Pollut. _2_,  235
     (1973).
MCCARTHY, L.T. jr., i. WILDER, and  j.s. DORRLER: U.S.E.P.A. Publ.
     No. EPA-R2-73-201 (1973).
SPRAGUE, J.B. and W.G. CARSON: Fish. Res. Bd. Can. Tech. Rept.
     201  (1970).
SWEDMARK, M. , A. GRANMO,  and S.KOLLBERG: Water Res. ]_, 1649 (1973)
STAINKEN, D.M.: Proc. Joint Conf. Prevent. &  Contr. Oil Spills,
     San Francisco, Calif., A.P.I.  (1975).
STIRLING, E.A.: Mar. Pollut. Bull.  6_:8, 122  (1975).
WILSON, K.W.: In: Ecological aspects of toxicity testing of oils
     and dispersants.  (ed. L.R. Beynon, E.B.  Cowell),  John  Wiley
     & Sons, New York. 11  (1974).
VAUGHAN, B.E.: A.P.I. Pub. No. 4191 (1973).
                                 729

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           A Descriptive Evaluation of the Effects
              of No. 2 Fuel  Oil on the Tissues of
             the Soft Shell Clam, Mya arenaria L.
                         Dennis M. Stainken
                  U.S. Environmental Protection Agency
              Industrial Environmental Research Laboratory-Ci
                  Oil & Hazardous Materials Spills Branch
                          Edison, N.J. 08817
                           Introduction

     Bivalve molluscs have been  found  to  accumulate petroleum and
petrochemical derivatives.  BLUMER,  e_t^ al_ (1970)  described hydro-
carbon uptake by oysters and scallops  following a fuel oil spill.
ZITKO (1971), LEE, t al.  (1972), STEGEMAN and TEAL (1973),
VAUGHAN (1973), and NEFF and ANDERSON  (1975)  have reported the
occurrence and uptake of petroleum hydrocarbons by bivalve mol-
luscs.  A mechanism by which soft shell clams may accumulate oil
was described by STAINKEN  (1975).

     The effects of petroleum oils and derivatives on bivalves
are varied.  Some of the effects reported have described altera-
tions in oxygen consumption, carbon  budgets,  larval development,
behavior, filtration rates, mortality  and biochemical effects.
The histological effects of petrochemicals are also varied.
Deleterious effects of oil on bivalve  tissue  structure have been
reported by LAROCHE (1972), CLARK, et_  ajL.  (1974)  and GARDNER,
e_t _al. (1975).  Histological aberrations  in bivalves have been
reported by BARRY, &t_ al.  (1971), JEFFRIES (1972), and BARRY and
YEVICH (1975).  The aberrations were believed to  be due to pollu-
tion effects.  In contrast, VAUGHAN  (1973) found  few effects of
oils on bivalves.

     Reports on the effects of petroleum  oils are often conflict-
ing.  Some of the studies were field studies  and  exposure concen-
trations were unknown.  The reported effects  of petroleum oil
exposure have ranged from  extensive  to relatively none.  Bivalves
in the environment are frequently exposed to  single spill or
chronic discharges.  This  study  was  therefore performed to exper-
imentally determine the effects  of subacute concentrations of
No. 2 fuel oil on the soft shell clam.

     A No. 2 fuel oil was  chosen for study because it is commonly
shipped in coastal waters, used  in coastal industrial installa-
tions, and has already been involved in a well documented spill
(BLUMER, e_t_ al. 1970) .  A winter temperature  (AC) was chosen
because spills are more likely to occur during the inclement
winter weather.  In the event of a spill  during  the colder months,
it is probable that much of the  oil  would be  dispersed and emulsi-
fied in the water column through turbulent wave  action.  The
clams were therefore exposed 28  days to oil initially added  in an
emulsified form to simulate a potential naturally occurring  condi-
tion of chronic exposure.
                                730
 Bulletin of Environmental Contamination & Toxicology,
 Vol. 16, No. 6  1976 by Springer-Verlag New York Inc.

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Materials and Method

     The No. 2 fuel oil was supplied by the U. S. Environmental
Protection Agency, Industrial Waste Treatment Laboratory,
Edison, N. J.  The specific gravity of the oil was 2.40 centi-
stokes.  The oil was composed of  14% aromatics and 86% nonaroma-
tics according to ASTM method No. D2549-68.  Oil-in-water emul-
sions were ultrasonically prepared according to a procedure
developed by GRUENFELD and BEHM  (1973).

     Clams for the experiments were collected from Sequine Point,
Staten Island, N. Y.  Young clams with a mean shell length of
25 mm were utilized because young bivalves tend to have greater
filtration rates than those of older bivalves.  The clams were
acclimated to the experimental conditions for a duration of 6
days before the emulsions were added.

     An exposure period of 28 days to No. 2 fuel oil emulsions
having concentrations of 10, 50 and 100 ppm was utilized.  Four
20 gallon aquaria containing 60 liters of filtered sea water/aquaria
(salinity = 20%0)were employed.  The sea water was collected from
Sandy Hook Bay and filtered through a coarse plankton net.  One
aquaria served as a control and each of the remaining aquaria
received either 10, 50 or 100 ppm of oil emulsion.  The water was
continuously aerated and the temperature was maintained at 4 C.
Sampling for hydrocarbon content of water and clams was performed
every 7 days.  The hydrocarbon content of the water was determined
by the method of GRUENFELD (1972).  Complete results from these
experiments will be published at a later date.

     At the beginning of the experiment, just before addition of
the emulsions (Time 0), five clams were fixed in Davidson's fixa-
tive (SHAW and BATTLE, 1957).  At the end of the 28 day exposure
period, ten clams from each concentration and the control tank
were removed for histological examination.  The clams sampled
represented 10% of the experimental population.  Five of the ten
clams randomly removed from each concentration were fixed in
Davidson's fixative and five were fixed in cold 10% acetate buf-
fered neutral formalin, pH 7.  The two fixatives were employed to
determine whether alterations in tissue structure were fixation
artifacts.  The results indicated that artifacts did not occur.

     All fixation was done in a refrigerator.  After 24 hours,
clams fixed in Davidson's were transferred to cold 70% ethanol.
All fixed material was stored in a refrigerator until further
processing.  Prior to embedding, the formalin fixed animals were
washed 14 hours in running tap water.  All tissues were dehydrated
and brought to paraffin utilizing an Autotechnicon.  Final infil-
tration was accomplished employing a vacuum infiltrator for 15
minutes at 13-15 inches Hg.   The paraffin embedded clams were
oriented to cut beginning at the anterior pedal opening.  Serial
cross sections were cut at 6 and 7 microns to the level of the
                                 731

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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*       Week 1 Week 2 Week 3 Week 4
Control
10
50
100
ppm
ppm
ppm
1
2
3
4
0
4.
43.
60.

5
72 
71
0
1.31
1.04
1.52
0
0.
0.
0.

56
71
78
0
0.
0.
0.

37
37
32
0
0
0.
0.


29
46
          * The Time 0 sample measurement was made two hours after
            addition of the emulsified oil.

Several factors were probably responsible for the gradual deple-
tion of oil from the water.  Much of the oil was apparently
removed from the water column by the mucociliary feeding and
ejection mechanisms of the clams.  Large masses of mucus were
                                732

-------
ejected from the clams and were accumulated on the cooling coils.
Subsequent chemical analysis revealed a large content of oil in
the mucus.  After 28 days, a sample of the mucus from the 100 ppm
aquaria was found to contain 833 micrograms of hydrocarbons.
Mass spectrometric analysis demonstrated that these hydrocarbons
were mostly dimethyl and trimethyl naphthalenes and paraffins in
the C-14 and C-15 regions  (STAINKEN, 1975).

     Harris's Hematoxylin was used to examine the general mor-
phology of clams.  Radical tissue aberrations were not observed.
A gradation of tissue effects was apparent.  The clams exposed to
100 ppm exhibited the largest number of anomalies from the con-
trols.  The pallial muscle appeared edematous in four clams,
similar to that described by PAULEY and SPARKS (1965, 1966).
There were more leukocytes in the pallial blood sinuses of 100 ppm
exposed clams than controls, with occasional leukocyte nests as
described by PAULEY and CHENG (1968) occurring in the pallial
blood sinuses.  The leukocytes often formed a band underlying the
mantle epithelium similar to that illustrated by DES VOIGNE and
SPARKS (1968).  In some clams, the anterior adductor muscle was
midly edematous and infiltrated with leukocytes.  The area between
the mantle membrane and the cell layer next to the shell was also
edematous and contained many leukocytes in some clams.  In one
clam, they formed a plug in the hemocoel of part of the foot.  In
seven clams,  the style sac, intestine and diverticula appeared
very vacuolar and the diverticula appeared much reduced in size.
There was a small loss of chromatophilic material at the top of
the gill filaments in several clams exposed to 100 ppm oil.

     At 50 ppm, the effects were less marked, except in the di-
verticula and intestine.  The diverticula of seven clams were
shrunken in size.  The diverticula epithelium appeared almost
cuboidal instead of the normal columnar epithelium.  Portions of
the intestinal mucosa appeared to be sloughing into the lumen
which was not prevalent in the controls.  The intestine, style sac
and diverticula were abnormally vacuolar in appearance.  In one
clam, a few diverticula near the gonad appeared necrotic and
undergoing resorbtion.  The pallial muscle was edematous in one
clam.  Several clams had more leukocytes and leukocyte nests in
the blood sinuses below the inner pallial epithelium than the
controls.  Only one clam had more than normal leukocytes and
leukocyte nests between the mantle epithelium and the cell layer
next to the shell.  In one clam, a portion of the gill had a
pavement of leukocytes along the lining of the blood sinuses next
to the central water tube.

     The 10 ppm clams showed fewer effects of oil exposure than the
100 or 50 ppm clams.  The diverticula were reduced in size and the
diverticula,  stomach and intestine were vacuolated in appearance.
In two clams, a moderate number of leukocytes were observable in
the pallial blood sinuses.
                                 733

-------
Cross sections of the clam visceral mass and pallium were stained
with azure A/eosin B to demonstrate necrotic tissues.  Major his-
tological differences were not found between controls and oil
exposed clams.  The general effect of holding the clams for four
weeks was an increase in vacuolization of the digestive diverticu-
lar and intestinal cells.  The vacuolization was present in all
groups.  The diverticular cells of the Time 0 clams were distinct
and contained few vacuoles.  After four weeks, the diverticular
cells of all clams appeared to contain many vacuoles and the cell
membranes were often indistinct.  However, this effect was exacer-
bated in the oil exposed clams compared to controls (Figure 1-3).

Figure 1.   Section of the digestive diverticula.   lOOx.   Stained
Azure A/eoson B.   Time 0.
                                734

-------
Figure 2.  Section of the digestive diverticula.   lOOx.   Stained
Azure A/eosin B.  Control.
Figure 3.  Section of the digestive diverticula.   lOOx.   Stained
Azure A/eosin B.  100 ppm exposed.
                               735

-------
     An aldehyde Fuchsin - Alcian Blue 8GX stain was used to stain
mucosubstances.  Histological differences were not found between
exposed and control groups along the intestines and the periphery
of the visceral mass.  There was a decrease in mucoid cells and
staining intensities from two of the 100 ,ppm clams.  The same
effect occurred in one 50 ppm clam.

     Clam sections were stained for glycogen according to the PAS
technique.  There was a gradation of effects of oil exposure in
the digestive diverticular and intestinal cells.  The 100 ppm clams
had the least amount of PAS positive material (glycogen).  Six of
the 100 ppm clams had observable differences from the control
clams.  Generally, the diverticular cells decreased in size and
contained much less PAS positive material.  The diverticular cells
of all 100 ppm clams appeared very vacuolar with few glycogen
deposits.  Several cells almost appeared amylase treated.  The
intestinal cells and basement membranes also contained less PAS
positive material.  Most of the diverticular cells appeared to be
devoid of cytoplasm.  The stomach mucosa of two of the 100 ppm
clams contained less PAS positive material than did the controls.
The gill filament tips and margins appeared to have a decrease in
PAS positive material.

     Generally, a pattern of cellular glycogen depletion was ob-
served in 50 ppm clams similar to that described for 100 ppm clams.
The diverticular cells were reduced in size, vacuolar in appearance
and contained less PAS positive material than did the controls.
The cells also appeared to be depleted of cytoplasm.  The intestin-
al mucosa appeared to be sloughed into the lumen.  The stomach
mucosa of the 50 ppm clams also showed a depletion of PAS positive
material as compared to controls.

     Most digestive cells in the 10 ppm clams appeared normal.
However, several clams diverticular and intestinal cells were
depleted of PAS positive material and were more vacuolar in appear-
ance than were the controls.

     In the control clams, the bulk of the diverticular cells were
full, round, and most of the cytoplasm was red.   In the 10 ppm,
50 ppm and 100 ppm clams, the diverticular cells were more vac-
uolar and contained less cytoplasm than did those of the control
group.

Discussion

     Petroleum hydrocarbons are generally assumed to be carcino-
genic, particularly the polycyclic aromatics.  Reviews of the
general carcinogenic effects of oil have been published by
HEIDELBERGER (1970) and ZOBELL (1971).  CLARK, et_ al. (1974)
reported alterations in tissue structure of oysters and mussels
exposed to outboard motor effluent.  LAROCHE (1972), BARRY and
YEVICH (1975) have reported a high incidence of gonadal tumors
in soft shell clams exposed to oils.  Hyperplastic germ cell
                                736

-------
tumors were also present in the gills.  BARRY, j2t_ al_.  (1971) re-
ported a high incidence of hyperplasia of the gills and kidneys in
soft shell clams from areas believed polluted.  In contrast,
VAUGHAN (1973) did not find evidence of histopathological change
in oysters exposed to No. 2 fuel oil.  There were some nonpatho-
logical changes evident in the epithelial layer of the inner
mantle lobe, and it was suggested that oil restricted feeding
activity.

     The results of this study with Mya arenaria revealed that
radical tissue changes did not occur after exposure to No. 2 fuel
oil.  It is possible, however, that either the very low concentra-
tion of oil present in the water column was not suffiecient to
alter tissue structure (i.e. neoplasms), or the exposure time was
not long enough.  The hydrocarbon concentrations in the water
column of each tank measured during the last 3 weeks of exposure
varied from 1.52 to 0.29 ppm.

     The general effects of subacute oil exposure can be charac-
terized as a depletion of glycogen and generalized leukocytosis
particularly evident in the blood sinuses of the pallium and
mantle membrane.  There was also an increase in vacuolization
of the diverticula, stomach and intestines.  The histological
effects in Mya arenaria appeared to be dose dependent.  The clams
exposed to the initial 100 ppm oil emulsion had more frequent
and noticeable histological differences from the controls.  The
depletion of glycogen and vacuolization may have been due to a
suppression of feeding and consequent use of body reserved coupled
with an altered respiratory rate.  The increased vacuolization of
oil-exposed clams may also represent inclusion and intracellular
compartmentalization of hydrocarbons.  The leukocytosis of the
mantle blood sinuses beneath the inner epithelium probably repre-
sents an inflammation reaction with a migration of leukocytes
into the affected areas.

Acknowledgement

     The analyses for this investigation were performed at the
analytical facility of the U. S. Environmental Protection Agency
(EPA), Industrial Waste Treatment Research Laboratory  in Edison,
N. J.  Use of these facilities was provided by an EPA  program that
supports graduate level research of the environment.
                                 737

-------
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BARRY, M.M., P.P. YEVICH, and N.H. THAYER:  J.  Invert.  Path.  17, 7
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GRUENFELD,. M. : Anal. Qual. Contr. Newsl. ,  U.S.E.P.A.,  _15_,  5  (1972).
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                                 738

-------
UNITED STATES ENVIRONMENIAL PRO'T ECTION AGENCY
INDUSTRIAL ENVIRONMENTAL RESEARCH LABORATORY-Ci
                   LDISON NLW JbRStY O8817

                      June 16, 1976
    This bibliography of petroleum oil analysis methods
    addresses major publications during 1974 and 1975.
    Its availability was announced in EPA's Analytical
    Quality Control Newsletter No. 29, April, 1976.
    The bibliography is rather incomplete, however,
    and information regarding obvious omissions would
    certainly be appreciated.
                   Michael Gruenfeld
                 Chief, Chemistry Staff

-------
Adams, C. E. (1974):  "A Method for the Separation of Oil from an Aqueous Oil-
     Detergent Solution Prior to IR Analysis", Government Reports Announcements,
     74(25): Technical Report NOLTR-74-102

Ahmed, S. M.,  et al. (1974):  "Sampling Errors in the Quantitation of Petroleum
     in Boston Harbor Water", Anal. Chem.. 46:1858

American Petroleum Institute (1974):  "A Second Oil Pollution Survey of the
     Southeast Florida Coast", API Publication No. 4231

Anbar, M., Scolnick, M.  E.,  and Scott, A. C.  (1974):  "Identification of Mineral
     Oils by Field lonization Mass Spectrometry", In the Proceedings of the
     Marine Pollution Monitoring Symposium, NBS Special Publication 409:229

Anonymous (1974):  "Analytical Techniques Seek to Fingerprint Oil Spills",
     Chem. & Eng. News,  52:30

Anonymous (1974):  "'Fingerprints' Trace Spill Culprits", Chemical Week, 114:40

Atanus, H. (1974):  "TLC Finds Hexane Solubles", Water and Wastes Engineering,
     J_l:26-28

Bean, R. M. (1974):  "Suspensions of Crude Oils in Sea Water: Rapid Methods of
     Characterizing Light Hydrocarbon Solutes", In the Proceedings of the Marine
     Pollution Monitoring Symposium, NBS Special Publication 409:127

Belcher, R. S. (1974):  "Determination of Mineral Oil in Water",  In:  Examination
     of Waters: Evaluation of Methods for Selected Characteristics; Australian
     Water Resources Council Technical Paper No. 8:79-83

Bieri, R. H.,  et al. (1974):  "Identification of Hydrocarbons in  an Extract From
     Estuarine Water Accommodated No. 2 Fuel Oil", NBS Special Publication
     409:149

Bird, C. W., and Lynch,  J.  M. (1974):  "Formation of Hydrocarbons by Micro-
     Organisms", Chem. Soc.  Rev., 3^39

Blaylock, J. W., Bean, R. M., and Wildung, R. E. (1974):  "Determination of
     Hydrocarbon Types in Lake and Coastal Sediments", NBS Special Publication
     409:217

Boehm, P. D.,  and Quinn, J.  G. (1974):  "The Solubility Behavior  of No. 2 Fuel
     Oil in Sea Water'1,  Marine Pollution Bulletin, _5:101-105

Bogatie, C. F. (1974):  "Rapid Identification of Oil and Grease Spills from Pulp
     and Paper Mills by Infrared Spectroscopy", Tappi, 57:130-134

Brown, C. W.,  Lynch, P.  F.,  Ahmadjian, M. (1974):  "Monitoring Narragansett Bay
     Oil Spills by Infrared Spectroscopy", Environmental Science  and Technology,
     18:669-670

Brown, C. W.,  Lynch, P.  F.,  and Ahmadjian, M. (1974):  "Novel Method for Sampling
     Oil Spills and for Measuring Infrared Spectra of Oil Samples", Anal. Chem..
     46:183-184

-------
Brown, R. A., Elliot, J. J., and Searl, T. D.  (1974):   "Measurement and Charac-
     terization of Nonvolatile Hydrocarbons in Ocean Water", NBS Special Publi-
     cation 409:131

Brown, R. A., et al.  (1974):  "Measurement and Interpretation of Nonvolatile
     Hydrocarbons in  the Ocean.  Part I.  Measurements  in Atlantic, Mediter-
     ranean, Gulf of Mexico, and Persian Gulf", U. S. Department of Commerce,
     Washington, D. C.

Bruce, H. E., and Cram, S. P. (1974):  "Sampling Marine Organisms and Sediments
     for High Precision Gas Chromatographic Analysis of Aromatic Hydrocarbons",
     NBS Special Publication 409:181

Budininkas, P., and Remus, G. A. (1974):  "Development  of Classification Scale
     for Characterizing Bilge Waters Used in Evaluating Oil Removal Techniques",
     USCG-D-75-74.  Contract DOT-CG-32521-A

Chernatskaya, A. N. (1974):  "Determination Using Modern Methods of Impurities
     Polluting the Waste Waters from Petroleum Refineries", Khimiya Tekhnolo-
     giya Topliv i Masel, 9^:24

Clark, R. C. Jr. (1974):  "Methods for Establishing Levels of Petroleum Contam-
     ination in Organisms and Sediment as Related to Marine Pollution Monitoring",
     NBS Special Publication 409:189

Clark, R. C. Jr., and Finley, J. S. (1974):  "Analytical Techniques for Isolating
     and Quantifying Petroleum Paraffin Hydrocarbons in Marine Organisms",  NBS
     Special Publication 409:209

Cretney, W. J., and Wong, C. S. (1974):  "Fluorescence Monitoring Study of  Ocean
     Weather Station "p"", NBS Special Publication 409:175

Domostroeva, N. G. (1974):  "Determination of the Content of Petroleum Products
     in Water by an Optical Acoustical Method", Izmeritel'naya Tekhnika, ^3:66

Ehrhardt, M., and Heineman, J. (1974):  "Hydrocarbons in Blue Mussels from the
     Kiel Bight", NBS Special Publication 409:221

Farrington, J. W. (1974):  "Some Problems Associated with the Collection of
     Marine Samples and Analysis of Hydrocarbons", Paper Presented at:  Confer-
     ence/Workshop on Marine Environmental Implications of Offshore Drilling in
     the Eastern Gulf of Mexico

Farrington, J. W., et al. (1974):  "Analysis of Hydrocarbons in Marine Organisms:
     Results of IDOE Intercalibration Exercises", NBS Special Publication 409:163

Feldman, M. H., and Cawlfield, D. E. (1974):  "Marine Environmental Monitoring:
     Trace Elements in Persistent Tar Ball Oil Residues", NBS Special Publication
     409:237

Garza, M. E., and Muth, J. (1974):   "Characterization of Crude, Semirefined and
     Refined Oils by Gas-Liquid Chromatography", Environmental jacience and Tech-
     nology, 8^:249-255

-------
Giger, W.,  and Blumer, M.  (1974):   "Polycyclic Aromatic Hydrocarbons in the
     Environment:   Isolation and Characterization by Chromatography, Visible,
     Ultraviolet,  and Mass Spectrometry",  Anal.  Chem.,  46 :1663

Giger, W.,  Reinhard, M.,  Schaffner, C., and Stuiran, W. (1974):   "Petroleum-
     Derived and Indigenous Hydrocarbons in Recent Sediments of Lake Zug,
     Switzerland", Environmental Science and Technology, :454-455

Gordon, D.  C., and Keizer, P. D. (1974):  "Estimation of Petroleum Hydrocarbons
     in Seawater by Fluorescence Spectroscopy:  Improved Sampling and Analytical
     Methods", Technical  Report No. 481 Marine Ecology Lab.  Bedford Institute
     of Oceanography, Dartmouth,Nova Scotia

Gordon, D.  C. Jr., and Keizer, P.  D. (1974):  "Hydrocarbon  Concentrations in
     Seawater Along the Halifax-Bermuda Section:  Lessons Learned Regarding
     Sampling and Some Results", In the Proceedings of the  Marine Pollution
     Monitoring Symposium, NBS Special Publication 409:113

Gordon, D.  C. Jr., Keizer, P- D.,  and Dale, J. (1974):   "Estimates Using
     Fluorescence Spectroscopy of  the Present State of Petroleum Hydrocarbon
     Contamination in the Water Column of  the Northwest Atlantic Ocean1',
     Marine Chemistry. 2^:251-261

Hellman, H. (1974) :  "Differentiation Between Hydrocarbons  of  Biogenous and
     Petrol Origin by Way of Fluorescence  Spectroscopy", Zeits Anal. Chem. (Ger),
     272:30

Hellmann,  H., and Zehle,  H. (1974):  "On Which Conditions are  Identifications of
     Mineral Oils on Water Surfaces Possible?",  Zeits Anal.  Chem. (Ger), 269:353

Hertz, H.  S., et al. (1974):  "Methods for Trace Organic Analysis in Sediments
     and Marine Organisms", NBS Special Publication 409:197

Hornig, A.  W. (1974):  "Identification, Estimation and Monitoring of Petroleum
     in Marine Waters by  Luminescence Methods", NBS Special Publication 409:135

Hunter, I., Guard, H. E., and DiSalvo, L.  H. (1974):  "Determination of Hydro-
     carbons in Marine Organisms and Sediments by Thin Layer Chromatography",
     NBS Special Publication 409:213

Ilordi, A.  M. (1974):  "Identification of  Crude Oil Leaks at Sea", La Rivista dei
     Combustibili, 28:367-371

Iliffe, T.  M., and Calder, J. A. (1974):  ''Dissolved Hydrocarbons in the Eastern
     Gulf of Mexico Loop  Current and the Caribbean Sea", Deep  Sea Res., 21:481

Jeffrey, L. M., et al. (1974):  "Pelagic Tar in the Gulf of Mexico and Caribbean
     Sea",  NBS Special Publication 409:233

Jeltes, R.  (1974):  "Fingerprinting Techniques as Aides in  the Analysis of
     Composite Chemical Pollutants in the Environment", Jour.  Chromatog. Sci.,
     12:599

-------
 Jeltes,  R.  (1974):   "Prompt  Detection and Tracing of Oils  and Other Detrimental
      Chemicals  in the Environment",  Water Res.  (G.B.),  8_:977

 Johnson,  J.  D.,  and  Gram,  H.  R.  (1974):   "Discrimination of Waste  Oils  by  Micro
      Emission Spectro-Chemical Analysis", Available from the  National Technical
      Information Service,  Springfield,  Virginia:   Report No.  CG-D-21-75

 Kawahara, F. K.  (1974):   "Recent  Developments  in  the Identification of  Asphalts
      and  Other  Petroleum Products",  NBS  Special Publication 409:145

 Kawahara, F. K.,  Santner,  J.  F.,  and Julian, E. C.  (1974):  "Characterization of
      Heavy Residual  Fuel Oils and Asphalts  by  Infrared  Spectrophotometry Using
      Statistical  Discriminant Function Analysis",  Anal.  Chem..  46:266-273

 Koelle, W. (1974):   "Mineral  Oil  Loading of Lake  of Constance  Sediments",  Kern-
      forschungszentrum Karlsruhe  (Berlin) ,  KFK1969UF:8

 Lamontagne,  R. A., et al.  (1974):  "Cj-C^ Hydrocarbons  in the North  and South
      Pacific", Tellus, 26:71

 Ledet, E. J., and Laseter, J. L.  (1974):   "Alkanes  at the Air-Sea  Interface from
      Offshore Louisiana  and Florida", Science, 186:261

 Lee,  C. C.,  Craig, W.  K.,  and Smith,  P.  J.  (1974):   "Water-Soluble Hydrocarbons
      from Crude Oil",  Bulletin of Environmental Contamination and  Toxicology,
      J_2:212-217

 Lewis, B. W., et  al.  (1974):  "Hydrocarbons Identified in Extracts  from Estuarine
      Water Accomodated No. 2  Fuel  Oil by  Gas Chromatography-Mass Spectrometry",
      NASA, Hampton,  Virginia

 Lordi, R., Manci, C.,  and  Petronio,  B. M.  (1974):   "Studies on  Industrial Waters
      Containing Oil  Emulsions", Inquinamento, 16:31

 LysyJ,I- and Russel,  E. C. (1974):   "Dissolution of Petroleum-Derived Products
      in Water", Water  Resources (G.B.),  8^:863

 Majori, L.,  et al. (1974):  "Marine  Pollution by Hydrocarbons in the Northern
      Adriatic Sea",  Rev. Int. Oceanog. Med., 31:137

 Mallevialle, J.  (1974):  "Measurement of  Hydrocarbons in Water: Application to
      Cases of Surface Water Pollution", Water Research, j}: 1071-1075

 Masimi, M., et al. (1974):  "Hydrocarbon  Components of Floating Oil Pollutants of
      Sea Water",  Bull. Jap. Soc.  Sci. Fish, 40:111

Mayo, D.  W,,  et al.  (1974):   "Long Term Weathering  Characteristids of Iranian
     Crude Oil: The Wreck of  the "Northern  Gulf"", NBS Special Publication 409:201

McAuliffe, C. D.  (1974):  "Determination  of Cj^-C^Q  Hydrocarbons in Water", In the
     Proceedings  of  the Marine Pollution Monitoring Symposium, NBS  Special Publi-
      cation 409:121

-------
McGlynn, J. A.  (1974):   "A Review of Techniques for the Characterization and
     Identification of  Oil Spillages", In:   Examination of Waters:  Evaluation
     of Methods for Selected Characteristics;  Australian Water Resources
     Council Technical  Paper No.  8:85-89

Medeiros, G. C., and Farrington,  J.  W. (1974):  "IDOE-5 Intercalibration Sample:
     Results of Analysis After Sixteen Months  Storage", NBS Special Publication
     409:167

Mitra, G. D., et al.  (1974):  "Gas Chromatographic Analysis of Complex Hydro-
     carbon Mixtures",  Journal Chromatography, 91;633

Mommessin, P. R. ,  and Raia, J. C. (1974):   "Chemical and Physical Characterization
     of Tar Samples from the Marine Environment", U.S.  Coast Guard, Washington, D.C

Novotny, J. (1974):  "Applications of Gas  Chromatography for Analyses of Water
     Polluted by Petroleum Products", Vodni Hospodarstvi, 24:45

Osgood, J. 0. (1974):  "Hydrocarbon Dispersion in Ground Water: Significance and
     Characteristics",  Ground Water, 12:427

Polak, J., and Lu, B. C.-Y. (1974):   "Determination of  the Total Amount of Volatile
     but Slightly  Soluble, Organic Materials Dissolved  in Water from Oil and Oil
     Products", Anal. Chim. Acta., 68:231

Pozdnyshev, G.  N. , et al. (1974):  "Extraction Separation of Petroleums into Oils,
     Tars and Asphalts", Khimiya  Tekhnologiya  Topliv i  Masel,  10:54

Rashid, M. A. (1974):  "Degradation of Bunker  C Oil Under Different Coastal Envi-
     ronments of Chedabucto Bay,  Nova Scotia", Estuarine & Coastal  Mar. Sci., ^:137

Ray, S. M., Oja, R. K., Jeffrey,  L.  M., and Presley, B. J. (1974):   "A Quantitative
     and Qualitative Survey of Oils and Tars Stranded on Galveston  Island Beaches",
     Available from the National  Technical Information  Service, Springfield,
     Virginia:   Report  No. CG-D-10-75

Rijks, J. A., et al.  (1974):  "Characterization of Hydrocarbons in  Complex Mixtures
     by Two-Dimensional Precision Gas Chromatography",  Journal Chromatography,
     9^:603

Sackett, W. M., and Brooks, J. M. (1974):   "Use of Low Molecular-Weight-Hydrocarbon
     Concentrations as  Indicators of Marine Pollution", NBS Special Publication
     409:171

Sleeter, T. D., et al.  (1974):  "Quantitative  Sampling  of Pelagic Tar in the North
     Atlantic,  1973", Deep Sea Res., 21:773

Straughan, D. (1974):  "Field Sampling Methods and Techniques for Marine Organisms
     and Sediments",  NBS Special  Publication 409:183

Sutton, C., and Calder, J. A. (1974):  "Solubility of Higher-Molecular-Weight
     n-Paraffins in Distilled Water and Seawater", Environmental Science and Tech-
     nology, 8:654-657

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Suzuki, R., Yamaguchi, N., and Matsumoto, R. (1974):  "Determination of Trace
     Amounts of Dispersed Oil in Waste Water by Solvent Extraction - Infrared
     Analysis", Japan Analyst. 23; 1296

Swinnerton, J. W. , Lamontagne, R. A. (1974):  "Oceanic Distribution of Low-
     Molecular-Weight Hydrocarbons-Baseline Measurements",  Environmental
     Science and Technology, j^:657-663

Tu-Ching, T. (1974):  "The Infrared Studies of Santa Barbara Channel Oil Spill",
     Available from University Microfilms, Inc., Ann Arbor, Michigan, (Ph.D.
     Thesis)

U. S. Coast Guard (1974):  "Oil Spill Identification System-Interim Report",
     Available from the National Technical Information Service,  Springfield,
     Virginia:  Report No. CG-D-41-75

vonHellman, H., and Holeczek, M. (1974):   "Kohlenwasserstoffe in Quellwassern-
     Olverschmutzung oder Naturstoffe?",  Tenside Detergents, 11:197

Warner, J. S. (1974):  "Quantitative Determination of Hydrocarbons in Marine
     Organisms", NBS Special Publication  409:195

Wasik, S. P. (1974):  "Determination of Hydrocarbons in Sea Water Using an
     Electrolytic Stripping Cell", Jour^  Chromatog.  Sci. ,  12:845

Whitham, B. T. (1974):  "Marine Pollution by Oil, Characterization of Pollutants,
     Sampling, Analysis, and Interpretation", by Institute  of Petroleum, Oil
     Pollution Analysis Committee, Applied Science Publishers Ltd., Essex, England

Whittle, K., Mackie, P. R., and Hardy, R. (1974):  "Hydrocarbons in the Marine
     Ecosystem", South African Journal of Science, 70:141

Zeller, M. V. (1974):  "Infrared Analysis of Oil in Water Using  the Model 100",
     Perkin-Elmer Infrared Bulletin 22

Zeller, M. V. (1974):  "Oil in Water: Use of Non-Toxic Solvent and Importance of
     Acidification", Perkin-Elmer Infrared Bulletin 41
                   *
Zsolnay, A. (1974):  "Determination of Aromatic and Total Hydrocarbon Content in
     Submicrogram and Microgram Quantities in Aqueous Systems by Means of High
     Performance Liquid Chromatography",  In the Proceedings of the Marine Pollu-
     tion Monitoring Symposium, NBS Special Publication 409:119

Zsolnay,  A.  (1974):   "Determination  of Total Hydrocarbons in Sea Water at the
     Microgram Level with a Flow Calorimeter", Journal Chromatography, 90:79

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ASTM D3325-74T (1975):   "Tentative Method for Preservation of Waterborne Oil
     Samples", ASTM Standards, 31;565-567

ASTM D3325-74T (1975):   "Tentative Method of Teat for Preparation of Sample
     for Identification of Waterborne Oils", ASTM Standards, 31;561-564

ASTM D3327-74T (1975):   "Tentative Methods of Analysis for Selected Elements
     in Waterborne Oils", ASTM Standards. 31;568-576

ASTM D3328-74T (1975):   "Tentative Methods of Test for Comparison of Petroleum
     Oils by Gas Chromatography", ASTM Standards. 31:577-583

Clark, H. A., and Jurs, P. C. (1975):  "Qualitative Determination of Petroleum
     Sample Type from Gas Chromatograms Using Pattern Recognition Techniques",
     Anal. Chem., 47^374-378

Cramer, C. D. (1975):  "Detection and Characterization of Animal/Vegetable and
     Petroleum Oil in Municipal Wastewater by Thin Layer Chromatography", Dis-
     tributed during 1975 D-19.10 ASTM meeting; Available from Nalco Chemical
     Company, 2901 Butterfield Rd., Oak Brook, 111.  60521

Davis, C. E., Krc, A. E., Szakasits, J. J., and Hodgson, R.  L. (1975):  "Multi-
     element True Boiling Point Gas Chromatography for Monitoring Oil Pollution",
     Joint Conference on Prevention and Control of Oil Pollution, San Francisco*:
     93-97

Davis, S. J., and Gibbs, C. F. (1975):  "The Effect of Weathering on a Crude Oil
     Residue Exposed at Sea", Water Research, 9.:275-285

Dell'Acqua, R., Egan, J. A., and Bush, B. (1975):  "Identification of Petroleum
     Products in Natural Water by Gas Chromatography", Environmental Science and
     Technology, 2=38-41

Duewer, D. L., Kowalski, B. R., and Schatzki, T. F. (1975):   "Source Identifica-
     tion of Oil Spills by Pattern Recognition Analysis of Natural Elemental
     Composition", Anal. Chem., 47:1573-1583

Farrington, J. V. , and Medeiros, G. C. (1975):  "Evaluation of Some Methods of
     Analysis for Petroleum Hydrocarbons in Marine Organisms", Joint Conference
     on Prevention and Control of Oil Pollution, San Francisco*:   115-123

Frank, U. (1975):  "Identification of Petroleum Oils by Fluorescence Spectroscopy",
     Joint Conference on Prevention and Control of Oil Pollution, San Francisco*:
     87-93

Gruenfeld, M. (1975):  "Quantitative Analysis of Petroleum Oil Pollutants by
     Infrared Spectrophotometry", ASTM STP-573:290

Harrison, R. M., Perry, R., and Wellings, R. A. (1975):  "Polynuclear Aromatic
     Hydrocarbons in Raw, Potable, and Waste Waters", Water Research, 9^: 331-346

Harrison, W., Winnik, M. A., Kwong, P. T. Y., and Mackay, D. (1975):  "Disappear-
     ance of Aromatic and Aliphatic Components from Small Sea Surface Slicks",
     Environmental Science and Technology, 9^:231-234

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Regnier, Z. K., and Scott, B. F. (1975):   "Evaporation Rates of Oil Components",
     Environmental ScJence and Technology,  9_: 469-472

Smiley, C.  T., Montgomery, D. S.,  and Sawatzky,  H.  (1975):   "A Gas Liquid - Gas
     Solid  Chromatographic Method  for the Identification of Sources of Oil
     Pollution", ASTM STP-573:271

Smith,  W.  E.,  Napier,  B.,  and Home,  0. J.  (1975):   "Characterization of
     Petroleum Pitches Used for Coke  Production",  Symposium on Petroleum Derived
     Carbon Presented  Before the Division of  Petroleum Chemistry,  Inc. American
     Chemical  Society  Philadelphia Meeting, April  6rll,  1975:   369-375

Spiker, E.  C., and Rubin,  M.  (1975):   "Petroleum Pollutants in Surface and
     Graundwater as Indicated by the  Carbon-lA Activity  of  Dissolved Organic
     Carbon",  Science, 187:61-64

Walker, J.  0., Colwell, R. R., Hamming, M.  C., and  Ford, H.  T.  (1975):  "Extrac-
     tion  of Petroleum Hydrocarbons from  Oil-Contaminated Sediments", Bulletin
     of Environmental  Contamination and Toxicology,  13:245-248

Warner, J.  S.  (1975):   "Determination of  Sulfur-Containing  Petroleum Components
     in Marine Samples", Joint Conference on  Prevention  and Control of Oil
     Pollution, San Francisco*: 97-103

Weiss,  F.  T. (1975):   "Activities  of  Standardization in  the Identification of
     Waterborne Oils", Prepared for the National Bureau  of  Standards Workshop
     on Standard Reference Materials  for  Offshore  Drilling-Petroleum; Available
     from Shell Development Co., P- 0. Box  481,  Houston, Texas  77001

Yu, T.  S.,  and Coleman, W. H. (1975):  "A Quantitative Method  for  Determining
     Apparent Oil Concentration in Water  Containing  Detergents", Naval Ship
     Research and Development Center, Report  TM-28-75-10
**Bentz, A. P.  (1976):   "Oil Spill Identification",  Anal.  Chem.,  48:454A

**John, P., Soutar, I.  (1976):   "Identification of  Crude  Oils by  Synchronous
     Excitation Spectrofluorimetry",  Anal.  Chem., 48:520

**Mackay, D.,  Shiu, W.  Y.  (1976):   "Aqueous Solubilities  of Weathered  Northern
     Crude Oils",  Bulletin of Environmental Contamination and Toxicology,
     15:101
*Available from the American Petroleum Institute,  1801  K Street,  N. W. ,
     Washington,  D. C.   20006

**1976 search in progress

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Hargrave, B. T., and Phillips, G.  A.  (1975):  "Estimates of Oil in Aquatic
     Sediments by Fluorescence Spectroscopy",  Environmental Pollution, ji:
     193-215

Hunt, G., Horton, D., Levine,  J.,  Mayo, D.,  Donovan, D., Shelly, W. ,  Jiang, L. ,
     Grove, R. , and Johnson, R. (1975):  "The  Evaluation and Development of
     Passive Tagging Procedures for the Identification of Crude Oil Spilled
     on Water", Joint Conference on Prevention and Control of Oil Pollution,
     San Francisco*:  129-143

Hunter, L. (1975):  "Quantitation  of Environmental Hydrocarbons by Thin-Layer
     Chromatography, Gravimetry/Densitometry Comparison", Environmental Science
     and_ Technology, 9^:241-246

Jackson, B. W., Judges,  R.  W., and Powell,  J.  L.  (1975):  "Characterization
     of Australian Crudes and Condensates by Gas  Chromatographic Analysis",
     Environmental Science  and Technology,  9^:656-660

Ladner, L., and Hagstrom, A. (1975):   "Oil  Spill  Protection in the Baltic Sea",
     Journal Water Pollution Control Federation,  47:796-809

Lynch, P. F., Tang, S.,  and Brown, C. W. (1975):   "Application of Cryogenic
     Infrared Spectrometry  to the  Identification  of Petroleum", Anal.  Chem.,
     47:1696-1699

MacKay, D., Shiu, W. Y., and Wolkoff, A. W.  (1975):  "Gas Chromatographic
     Determination of Low Concentrations of Hydrocarbons in Water by  Vapor
     Phase Extraction",  ASTM STP-573:251

Miles, D. H., Coign, M.  J., and Brown, L. R. (1975):  "The Estimation of the
     Amount of Empire Mix Crude Oil in Mullet, Shrimp, and Oysters by Liquid
     Chromatography", Joint Conference on Prevention and Control of Oil
     Pollution, San Francisco*:  149-154

Mommessin, P. R., and Raia, J. C.  (1975):  "Chemical and Physical Character-
     ization of Tar Samples from the Marine Environment", Presented at the
     1975 Joint Conference  on Prevention and Control of Oil Pollution, San
     Francisco*:  155-167

Neff, J. M., and Anderson,  J.  W. (1975):  "An Ultraviolet Spectrophotometric
     Method for the Determination of Naphthalene  and Alkylnaphthalenes in
     the Tissues of Oil-Contaminated Marine Animals", Bulletin of Environmental
     Contamination and Toxicology, _L4:122-128

Pancirov, R. J., and Brown, R. A.  (1975):  "Analytical Methods for Polynuclear
     Aromatic Hydrocarbons  in Crude Oils, Heating Oils, and Marine Tissues",
     Joint Conference on Prevention and Control of Oil Pollution, San Francisco*;
     103-115

Pym, J. G., Ray, J. E.,  Smith, G.  W., and Whitehead, E. V. (1975):  "Petroleum
     Triterpane Fingerprinting of Crude Oils", Anal. Chem. , 4_7_: 1617-1622


                                                       *USGPO: 1977  757-056/6451 Region 5-11

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