Distillation Procedures. The distillation of micro amounts

of organic compounds in the 200° -300° C boiling point range can be suc-
cessfully accomplished at low temperatures and at atmospheric pressure
by using a purge gas such as nitrogen as a carrier. Consequently, ex-
tensive preliminary work was conducted in developing a micro distillation
apparatus with the associated recovery traps that would perform success-
fully, Various existing designs of apparatus including distillation flasks
and traps were investigated. A laboratory-prepared water standard
containing benzene, toluene, ethyl benzene, paraxylene, meta-xylene,
ortho-xylene, and cumene was used in the evaluation of the distillation
system. The objective was to recover the distilled aromatic compounds,
which were in the l-Ug range, by the best possible means, Various
designs of traps, including U tubes, straight tubes, micro impingers,
etc., were investigated. The cooling media included ice-salt and dry
ice-acetone baths. Numerous distillations were performed at 100°C

with no success in recovering the aromatic compounds.

Difficulty was encountered with the traps using dry ice-acetone
coolant due to freezing and plugging. To overcome this problem, it was
necessary to go to a large trap which of course is undesirable in micro
analytical work. Consideration was then given to employing an absorbing
solution to trap the aromatic distillate. Since it is not possible to absorb

these compounds chemically as is the case with organic acids, amines,

-15-


-96-

CLASSIFICATION OF SHELLFISH HARVESTING
AREAS OF GALVESTON BAY --- JULY 1, 1969

Texas State Department of Health
Division of Marine Resources,

Scale; None Approv:

LE] wesoven ana

CONDITIONALLY APPROVED
AREA

POLLUTED AREA

oyster Sample Locations

Shrimp Sample Locations

CHSiai 4 ny

ig BOLIVAR PENINSULA *

ROBINSON
BAYOU

Figure 1, Locations of Marine Organisms Obtained From Galveston Bay


The column used for detecting toluene in the samples was a
12 ft x 1/8 in. Apiezon L packed column at a temperature of 110° C and
a flow rate of about 70 cc/min. The solvent used for extraction was

carbon tetrachloride.

The fish sample which was spiked with a known amount of
toluene and the unspiked fish sample were run simultaneously for control
purposes. The other fish samples, which were exposed to the different
concentrations of the contaminant, were also run in order to determine
the amount of toluene in each,

It was shown that a concentration in the range of 0.5 ppm of
toluene could be measured in a 5-g prepared fish sample. At the end of
the 96-hour exposure tests, the following results were obtained with

various levels of exposure:

Exposure level, Measured level,
mg/l toluene in water ppm toluene in fish sample
20 6.7
40 11.6
80 17.0
160 20.5

In the analysis of diethylene glycol (DEG), a 5 ft x 1/4 in.,

five percent Reoplex column (on 40/60 mesh Chromosorb G) was used
at a temperature of 200°C and flow rate of about 350 cc/min. The

analytical procedure was similar to that used with toluene, except that

-32-


Chromatogram of Spiked Oyster Sample

Figure 6.

-41-


The first step in the development of gas chromatographic tech-
niques was to obtain an adequate reference library of chromatograms
of different organic compounds that are most likely to be present as
industrial contaminants in water. After some literature search, and
based on previous experience, it was decided to use a number of columns
which would perform the desired function. The columns selected were:
a 12 ft x 1/8 in. OD, 10% Apiezon L packed on
Chromosorb P, 60/80 mesh
a 50 ftx 0.02 in. ID, silicone DC 550 open tubular column
. 100 ft x 0.02 in. ID, silicone DC 550 open tubular column
. 5 ft x 1/8 in. OD, silicone DC 550 packed on
Chromosorb P, 60/80 mesh
s 6 ft x 1/8 in. OD, 20% SE 30 packed on Chromosorb P,
60/80 mesh
a 5 ftx 1/4 in. OD, 15% silicone DC 550 coated on 12.5%
potassium hydroxide-treated Chromosorb P, 60/80 mesh
2 5 ft x 1/4 in. OD, 5% Reoplex 400 coated on acid-washed
G-Chromosorb, 40/60 mesh.
The last two chromatographic columns were especially developed
and prepared during this study for the detection and analysis of glycols
and amines, respectively. These columns will be discussed fully under

"Preparation of New Chromatographic Columns".


to determine quantitatively the percent of recovery in the spiked samples.
Recovery of n-docosane from the spiked shrimp sample was 73 percent
whereas recovery from the spiked oyster sample was 88 percent.

14. Amide —N,N-Dimethyl Formamide HCON(CHp )2

The column used for detecting n, n-dimethyl formamide was a
1l-m x 2.3-mm Porapak Q, 80/100 mesh packed column at a temperature
of 180° C and flow rate of 60 cc/min. The solvent used for extraction
was ethyl alcohol.

It was shown that a concentration of 1.0 ppm of n, n-dimethyl
formamide could be measured in 5-g prepared shrimp and oyster
samples. The percent recovery of the compound in the oyster sample
was 90 percent and in the shrimp sample was 100 percent,

15. Chlorinated Hydrocarbon — Trichloroethylene Cz2HCl,

In the analysis of trichloroethylene, a 6-ft x 1/8-in. SE30 on
60/80 mesh Chromosorb P column was used at a temperature of 215°C
and flow rate of 60 cc/min. It was found that good recovery was obtained
when the shrimp and oyster samples were extracted with carbon tetra-
chloride as was done in many cases previously. Spiked samples were
used which contained 2 ppm of trichloroethylene by weight based on a
5-g sample of the organism,

Recovery of trichloroethylene from the spiked shrimp sample
was 90 percent, and recovery from the spiked oyster sample was also

90 percent.

-51-


10. Heterocyclic Compound — Pyridine C,H; N

The column selected to detect pyridine in the samples was also
a l-m x 2,3-mm Porapak Q, 80/100 mesh packed column at a tempera-
ture of 175°C and flow rate of 175 cc/min. The solvent used for extrac-
tion was ethanol. Concentrations as low as 2.0 ppm in the samples
could be detected. The percent of recovery in the shrimp sample was
50 percent whereas in the oyster sample recovery was 89 percent,

ll. Butyric Acid — C3H,COOH

The column used for detecting butyric acid in the samples was
a 5-ft x 1/4-in. acid-washed, 40/60 mesh G-Chromosorb solid support
coated with 5 percent of Reoplex 400 at a temperature of 150°C and
flow rate of approximately 200 cc/min.

Ethanol proved to be a satisfactory solvent for the extraction of
butyric acid from the samples. Concentrations as low as 2.0 ppm in
the shrimp and oyster samples could be detected.

The percent of recovery in the shrimp sample was approximately
67 percent whereas in the oyster sample recovery was 83 percent.

12. Diethyl Sulfide (Cz Hs 2 S

In the analysis of ethyl sulfide, a 6-ft x 1/8-in. SE 30, on 60/80
mesh Chromosorb P, column was used at a temperature of 70°C and
flow rate of about 50 cc/min, It was found that better recovery was

obtained if the shrimp and oyster samples were extracted with ethanol

-49-


BIBLIOGRAPHY

General

Bobbit, J. M., Schwarting, A. E., and Gritter, R. J., Introduction
to Chromatography, Reinhold, New York, 1968.

Mills, Paul A., "Detection and Semiquantitative Estimation of Chlorinates
Organic Pesticide Residues in Foods by Paper Chromatography," J. of
A.O.A.C. 42, November 15, 1959.

Rhoads, J. W., and Millar, J. D., ''Gas Chromatographic Method
for Comparative Analysis of Fruit Flavors," J, Agr. Food Chem.
Vol. 13, p.5, January/February, 1965.

Burchfield, H. P., et al, ''Guide to the Analysis of Pesticide Residues,"
Department of H.E.W., Bureau of State Services, Washington, D. C.,
1965, Vol. I, II. A.5. (1).

American Society for Testing and Materials, Manual on Hydrocarbon
Analysis, 2nd ed., Philadelphia, 1968.

Perkin-Elmer Corporation, ''Gas Chromatography Application,"
Application No. GC-AP-008, August, 1966.

Perkin-Elmer Corporation, ''Analysis of Aromatic Hydrocarbons With
Open Tubular Columns,'! Gas Chromatography Application No. GC-
DS-005, 1964.

Andreatch, A, J., and Feinland, R., ''Continuous Trace Hydrocarbon
Analysis by Flame Ionization," Anal, Chem. 32, No. 8:1021-4, July,
1960.

Innes, W. B., et al, 'Hydrocarbon Gas Analysis Using Differential
Chemical Absorption and Flame Ionization Detectors,'' Anal, Chem.
34, No. 9:1198-1203, August, 1963.

Perkin-Elmer Corporation, ''Analysis of Straight Chain (Cg - Cig)
Alkylbenzenes With Open Tubular Columns," Gas Chromatography
Application No. GC-DS-026, September, 1965.

Jacobs, Emmet S., 'Rapid Gas Chromatographic Determination of C,

to Cio Hydrocarbons in Automotive Exhaust Gas,'' Anal. Chem, 38,
No. 1:43-38, January, 1966.

55


Dove, Ray A., "Separation and Determination of Aniline and the Tolui-
dine, Xylidine, Ethylaniline, and N-Methyltoluidine Isomers by Gas
Chromatography of Their N-Trifluoroacetyl Derivatives,'' Anal, Chem.
39, No. 10:1188-92, August, 1967.

Grossi, G., and Vece, R., ''The Gas Chromatographic Analysis of
Primary, Secondary, and Tertiary Fatty Amines, and of Corresponding
Quaternary Ammonium Compounds," J. Gas Chromatogr. 3, No. 5:
170-3, May, 1965,

Metcalfe, L. D., and Schmitz, A. A., ''The Gas Chromatography of
Long Chain Diamines and Triamines,'' J. Gas Chromatogr. 2, No. 1:
15-17, January, 1964.

O'Donnell, Jerry F., and Mann, Charles K., 'Gas Chromatographic
Separation of Amines and Amides,'! Anal, Chem. 36, No. 11:2097-9,
October, 1964,

Smith, Lindsay J. R., and Waddington, ''Gas Chromatographic Analysis
of Aliphatic Amines Using Aromatic Polymers," Anal. Chem. 40, No. 3:
522-27, March, 1968.

Glycols

Davis, Abram, Roaldi, Arthur, and Tufts, Lewis E., ''Determination of
Glycols by Gas Chromatography," J. Gas Chromatogr. 2, No. 9:306-8,
September, 1964.

Nadean, Herbert G., and Oaks, Dudley M., ''Determination of Ethylene
and Propylene Glycols in Mixtures by Gas Chromatography," Anal. Chem,
32, No. 13:1760-62, December, 1960.

Spencer, Samuel F., and Mikkelsen, Louis, ''The Analysis of Glycols,
Alcohols, and Related Compounds by Gas Chromatography,'' presented
at 10th Detroit Anachem Conf., Wayne State University, Detroit, Mich.,
October 22-24, 1962.

Urone, Paul, ''Gas Chromatographic, Behavior of C; - Cz, Saturated

Alcohols and Water on Polyethylene Glycol Substrate. Effect of Solid
Support Treatment," Anal. Chem. 35, No. 7:837-41, June, 1963.

2US, GOVERNMENT PRINTING OFFICE:1974 546-318/360 1-5 59


2,4-dimethyl-3-pentanone could be detected in the 0.5-ppm range in
spiked samples. The chromatographic column used in the analysis was
a 50 ft x 0.02 in. ID, silicone DC 550 column at a temperature of 180°C
or 100°C. Chromatograms of some sample extracts showed some
unknown peaks, and no attempt was made to identify them.
7. Amines

It was mentioned previously that amines could not be measured
with many known chromatographic columns because of absorption or
reaction on the column material. This difficulty was overcome by using
a column that was 5 ft x 1/4 in. OD, 15 percent silicone DC 550.coated
on a 12,5 percent KOH-treated 60/80 mesh Chromosorb P at a tempera-
ture of 120°C. Results using this column indicated that micro quantities
of diethylamine, di-n-propylamine, di-n-butylamine, and cyclohexylamine
could be measured in a manner similar to that used for other types of
organic compounds, and accuracy and sensitivity were similar. There~-
fore, measurement of these amines to determine pollution by this class
of organic compounds seemed to be assured, Spiked oyster and shrimp
samples were used in testing for these amines, which constitute an
important class of organic pollutants.
8. Glycols

Glycols had also been a very troublesome class of organic com-
pounds investigated, from the standpoint of analysis by gas chromato-

graphy. Since they constitute an important class of organic pollutants,

-47-


The solvent extract obtained from oyster, shrimp, or fish
tissues contained not only the added contaminants but also numerous
naturally occurring oils and different dyes of unknown composition. The
presence of these high molecular weight materials made the sample
extracts after concentration undesirable for gas chromatographic
analysis. Therefore, a cleanup procedure which would remove the
foreign materials without removing the organic contaminants was
essential at this point.

Description of ''Cleanup'' Column. Cleanup of the extracts

by adsorption of the heavy oils and dyes on a solid adsorbent was
carried out efficiently by liquid column chromatography. The column
used was a 0.5-cm ID glass tube, 25-30 cm in length and tapered at
the outlet to provide an orifice of 3mm ID. This column was packed
with Florosil (60/100 mesh) adsorbent and anhydrous sodium sulfate
as illustrated in Figure 3, Such a column was good for cleaning up
carbon tetrachloride tissue extracts. However, for best use with other
solvents such as ethyl alcohol and dioxane, the length, diameter, and
amount of Florosil would need to be changed for best results, When
water was used as a solvent, there was no need to add anhydrous sodium
sulfate to the column,

Other adsorbents such as magnesium carbonate plus silica
gel 1:1, silica gel alone, alumina, silica gel plus alumina 1:1, and

magnesium oxide plus celite 1:1 were tested for the removal of dyes

-19-


wr MEH

Nitrogen Flow 10 cc/min
125 ml Round Bottom Flask
Sample Slurry

Ice-Salt Bath

Rotameter

SoOonn

Teflon Tubing (1 mm ID)
Water Bath

Solvent Reservoir
Microtrap

Stand

Figure 2, Micro Distillation Apparatus

-37-


Figure

Table I

ILLUSTRATIONS

Locations of Marine Organisms Obtained
from Galveston Bay

Micro Distillation Apparatus

The "Cleanup" Column

Chromatogram of Carbon Tetrachloride Solvent
Chromatogram of Calibration Sample
Chromatogram of Spiked Oyster Sample

Sample Preparation Procedures

Summary Data Sheet of Chromatographic
Analysis of Samples

iii

Page
36

37
38
39
40
41

42


a check on the validity of the experiments conducted with spiked samples.

Small fish were exposed in the laboratory in water containing
known concentrations of contaminants for various periods of time.
Samples were then analyzed and the results were compared with tests
using spiked samples and analytical controls.

In the case of the long-term effects of the contaminants and their
detection in fish, toluene and diethylene glycol of different concentrations
were used. O-cresol was used as a contaminant, in one concentration
only, for a short period of time.

Exposure Conditions. The type of fish used for the study
was sheepshead minnow, and the average weight of each fish was two
grams, The source of the fish and the dilution water was Galveston Bay
near Seabrook, Texas. Salinity of the water used was 16 ppt.

The exposure of the fish to the contaminants was conducted
in rectangular glass jars 24 cm in width, 28 cm in length, and 38 cm
in height with a volume of 20 liters of bay water. A control jar anda
minimum of four different concentrations were used for toluene and
diethylene glycol,

Separate tests were made for each concentration of the con-
taminants by placing ten fish in each test jar containing 20 liters of bay
water and observing the fish over a 96-hour period for toluene and di-
ethylene glycol. This was done to maintain the recommended 2-g fish

weight per liter of water. The toluene dilution series contained 20, 40,

-30-


and other reactive compounds, a solvent absorption step was considered.
The solvent selected was carbon tetrachloride (CCL), which offered
numerous advantages over other solvents, particularly in that it is
heavier than water and thus will remain in the lower layer of the trap

as the water distillate condenses.

Eventually, a micro distillation apparatus was designed as
shown in Figure 2, This device consists of a 125-ml round bottom
distillation flask and a series of micro traps connected together with a
1-mm ID Teflon tube. The traps were made from 6-mm ID Y-glass
connectors which were sealed at the bottom to provide a small reser-
voir in which to place the solvent. To prevent thermal cracking of
tissue samples, the distillation flask was totally immersed in a liquid
bath rather than using a heating mantle, which usually produces hot
spots around the flask, A hot liquid bath also provides better tem-
perature control than other means. The nitrogen purge gas line was
connected to the distillation flask, and a flow meter was attached to the
outlet of the trap. This flow device is necessary to control the carrier
gas flow through the system.

Distillations were performed on the aromatic standards using
spiked oyster samples. It was established that at certain operating
conditions, such as proper temperatures and nitrogen flow, aromatic

compounds in the 1-ug range could be successfully recovered in a trap

-16-


columns, both classes could not be measured because of adsorption or
reaction on the column material,

To overcome this difficulty, various techniques were evaluated
to prepare special columns that would perform satisfactorily in the
separation and detection of micro amounts of amines and glycols.

Of the columns tested, the one that showed the most promise
for measuring amines was 5 ftx 1/4 in. OD, 15 percent silicone DC 550
coated on a 12.5 percent KOH-treated 60/80 mesh Chromosorb P, This
column was prepared by adding 12.5 percent of KOH, by weight, dissolved
in ethanol to a known weight of Chromosorb P. The mixture was evapo-
rated on a rotary evaporator to dryness. To this mixture, 15 percent
(w/w) of DC 550 dissolved in acetone was then added, mixed well, and
evaporated also to dryness. Further drying was accomplished by heating
the mixture in an oven at 110°C for one hour. After the chromatographic
column had been properly packed and installed in the instrument, it was
necessary to "condition" it prior to use by passing nitrogen gas through
it at 125 cc/min for a period of at least four hours. Proper packing of
the coated solid support in the column is very important. Particles, for
example, must be evenly distributed in the column so that there are no
voids. This could be achieved by vibrating the chromatographic tube in
order to distribute the coated support evenly.

A similar procedure was adopted for the preparation of a

-22-


were added to the mixture during the extraction. The tube was then
stoppered with a saran-wrapped rubber stopper and the sample shaken
for one minute, then centrifuged at about 1500 rpm for three minutes.
The solvent extract was then poured into a 100-ml test tube. The tissue
mixture was further extracted with an additional 10 ml of carbon tetra-
chloride and the extract combined with the previous one. The test tube
was then stoppered and refrigerated for further use.

Sometimes unspiked samples were also extracted simulta-
neously with the spiked samples for control purposes. The extraction
experiments employing these procedures have shown that organic pollu-
tants absorbed and retained by marine organisms can be extracted and
analyzed in micro quantities.

Water Solvent Procedures. The extraction procedure using

water as a solvent was identical to the organic solvent extraction. As

an extraction solvent, water offered some advantages over carbon tetra-
chloride since it is a better solvent for certain organic contaminants such
as glycols. However, the possibility of forming emulsions with water
and naturally occurring organic constituents, and the difficulty in clean-
ing up the extracts, made carbon tetrachloride more advantageous to use.
After a few experiments, the use of water as an extraction solvent was
discontinued.

Cleanup and Concentration of Tissue Solvent Extract

-18-


80, and 160 mg/l, respectively. The diethylene glycol dilution series
contained 8, 16, 32, and 64 g/l, respectively. in the case of the o-cresol
test, the water used in the test contained only one concentration, 16 mg/l.
Relatively high concentrations were chosen deliberately, to provide fish
samples for laboratory use containing substantial amounts of contami-
nant. This would simulate a condition in a bay or estuary involving a
substantial waste discharge that might endanger marine life.

Dissolved oxygen was maintained at or near the saturation
level in each test jar by aeration. A diaphragm-type air pump and car-
borundum diffusers were used in the aeration system.

Dead fish were removed from the various test jars as soon
as death occurred to prevent further contamination of the test environ-
ment by decaying fish. In the case of the toluene and diethylene glycol
tests, the fish survivors were taken, cleaned up externally by rinsing
them thoroughly with tap water, and then processed for the analytical
procedures, However, in the o-cresol test, only dead fish were cleaned
up and processed for analytical purposes because all the fish which were
exposed to a concentration of 16 mg/l of this contaminant died within
two hours.

Analytical Procedures, After exposure, fish samples were
processed using the procedures described previously and illustrated in
Figure 7. The resulting extracts were then analyzed by gas chromato-

graphy. The analytical procedures employed were as follows:

-31-


sample, this also represented 0.5 ppm by weight for each component.

On certain occasions, a 5-g sample was spiked with 1.2 Ug of components,
as was the case with o-ethyl phenol, to obtain a concentration of approxi-
mately 0.25 ppm by weight.

Fish Sample Preparation. After developing satisfactory

methods for the preparation of shrimp and oyster samples, further
laboratory experience indicated that the same procedure was suitable
for fish specimens. Most of the laboratory work was performed with
sheepshead minnows averaging two grams in weight. Specimens were
obtained from Galveston Bay near Seabrook, Texas, together with bay
water used in laboratory experiments and in maintaining live specimens
for later use. Salinity of the water used was about 16 ppt.

To prepare samples for analysis, tails and fins were removed
and discarded after which the remaining material was drained of excess
water, chilled, ground, and homogenized in the same manner as in the
treatment of shrimp and oyster samples. Ground samples were then
spiked with a known weight of the organic contaminants to be investigated,
and anhydrous sodium sulfate was used to avoid the effects of excess water
in the same manner as in the previous laboratory experiments, Through
spiking of samples and comparing the analytical results with unspiked
samples and control samples, it was found that the sensitivity and percent

recovery with most organic contaminants was similar to that obtained

-13-


Averell, W., etal, ''Gas Chromatographic Analysis of C, - C, Hydro-
carbons With Open Tubular Columns," Nature 196, No. 4860:1198-99,
December 22, 1962.

Bruderreck, Hartmut, Schneider, Werner, and Halasz, Istvan, 'Quanti-
tative Gas Chromatographic Analysis of Hydrocarbons With Capillary
Columns and Flame Ionization Detector. IV. Principles of a New
Splitting System," J. Gas Chromatogr. 5, No. 5:217-25, May, 1967.

Eggertsen, F. T., et al, ''Determination of Five to Seven Carbon
Saturates by Gas Chromatography,'' Anal. Chem. 30, No. 1:20-25,
January, 1958,

Ford, Donald C,, "Analysis of Light Hydrocarbons, Cz to Cs," J. Gas
Chromatogr. 1, No. 8:36, August, 1963.

Keulemans, A.I.M., Gas Chromatography, 2nd ed., Reinhold, New
York, 1959.

Merchant, Philip, Jr., ''Resolution of Cz, to Cyg Petroleum Mixtures
by Capillary Gas Chromatography,'' Anal. Chem. 40, No. 14:2153-8,
December, 1968. .

Perkin-Elmer Corporation, ''Analysis of Cy Olefins With Open Tubular
Column,'' Gas Chromatography Application No. GC-DS-023.

Sanders, W. N., and Maynard, J. B., "Capillary Gas Chromatographic
Method for Determining the Cg - Cig Hydrocarbons in Full-Range Motor
Gasolines,'' Anal, Chem. 40, No. 3:527-35, March, 1968.

Bombaugh, Karl J., et al, ''Gas Chromatographic of Traces of Ethanol
in Methanol,'' Anal, Chem. 35, No. 10:1452-4, September, 1963,

Martin, Glenn E,, et al, ''Determination of Methanol by Gas Liquid
Chromatography," J. Assoc. Offic. Agr. Chem. 46, No. 2:297-8,
April, 1963.

Porcaro, Peter J,, and Johnston, V. D., ''Primary Amyl Alcohols
Determined by Gas Chromatography," Anal. Chem. 33, No. 3:361-2,
March, 1961.

57


SECTION IIL
INTRODUCTION

Objective

The overall objective of this program was to develop methods
of sample preparation suitable for processing, extracting, purifying,
and concentrating specific organic pollutants found in marine organisms,
so that chemical analysis to measure these contaminants can be carried
out. These methods in turn can be useful in the development and appli-
cation of new techniques for water quality monitoring based on the
analysis of marine organisms,

This investigation was directed primarily at the measurement
of contaminants of industrial origin. Serious pollution problems exist
from this type of contamination, and previous work has not been adequate
to provide suitable methods of evaluating the effects of pollution on
marine resources or on human health.

Plan of Operation

Shrimp, oysters, and fish were used to obtain representative
samples of different types of organisms. These varieties occur commonly
in Galveston Bay and other estuaries along the Texas coast and represent
different degrees of mobility in that fish move readily, shrimp move
more slowly, while oysters are immobile throughout most of their life
cycle. This difference in mobility is important in view of the effects of

tidal action on pollution in bays and estuaries.


"tailing" effect, however, identifiable peaks can be observed for

each of the nine trace hydrocarbons in the sample.

Figure 6 shows a similar chart record obtained in
analyzing an oyster sample which had been spiked with the same
hydrocarbons, The Cg and Cig hydrocarbons were used at a level
of one part per million each, while the remaining hydrocarbons
shown were used at a level of 0.5 ppm. The baseline is less stable
than in Figure 5, due to minor interference from various constitu-
ents not completely removed in the sample preparation, However,
separate peaks can be identified for each hydrocarbon and a quan-
titative or semi-quantitative estimate can be made of the amount

present,

Calibration With Various Organic Compounds

With the availability of analytical techniques and satisfactory
chromatographic methods, tests were conducted with a large number
of spiked samples to obtain calibration data and confirm the validity
of the entire procedure with different classes of compounds. A
table listing pertinent data is included in the Appendix, and brief
comments concerning the columns used and other experimental

details are also included.

-28-


(c) The standard solution was then prepared from the stock
solution. This was done by mixing 0.75 ml of the stock
solution with 25 ml of carbon tetrachloride. The standard
solution was prepared to give the same concentration as
that of the extract; i.e., 1uUl-= 10 nanograms of each
compound investigated.

The peak area method was used as the standard method for
quantitative analysis in the chromatographic study. It has been found
that the weight concentration of a sample component is directly pro-
portional to its peak area, compared to the peak area of the compound
in a standard mixture of known composition tested under the same
operating conditions.

To use this method, an aliquot of the concentrated extract
was injected into the gas chromatograph and the chromatogram obtained
The relative recovery of each added compound in the extract was calcu~
lated from its peak area relative to that of the standard obtained by
injecting the same volume as that of the extract into the gas chromato-
graph. Under the same operating conditions, the relative recovery of
the extracted compound was computed from the peak areas produced by
the standard and the final extract solution as follows:

peak area of compound x 100
peak area of standard

percent of recovery =

-25-


ketones, phenols, esters, heterocyclic compounds, acids, sulfides,
amides, and chlorinated hydrocarbons. With most of these, recovery
of 70 to 90 percent of the amount present was obtained, indicating that
quantitative measurements are possible within the ranges stated above,

« Several methods of sample preparation can be used prior to
analysis by gas chromatography. The combination of methods which
provides the greatest flexibility appears to be an extraction procedure
with carbon tetrachloride, followed by a cleanup procedure using column
chromatography to separate the organic contaminants being measured
from naturally occurring oils and other interfering substances. Most
of the classes of compounds investigated can be measured with a single
sample using this combination of techniques. The greatest difficulties
were encountered in measuring amines and glycols, and variations in
extraction and cleanup procedure are necessary if best results are to
be obtained with these two classes of compounds.

= Similar methods of sample preparation could be used with
specimens of shrimp, oysters, or fish. Based on the results obtained,
the analysis of almost any variety of marine specimens should be possible

to measure trace organic constituents,


SECTION I
SUMMARY AND CONCLUSIONS

A two-year laboratory investigation has been completed to develop
laboratory methods for processing, extracting, purifying, concentrating,
and measuring specific organic pollutants found in marine organisms.
These methods provide new techniques for measuring organic contami-
nants in water to establish monitoring procedures, identify sources of
contamination, evaluate methods of treatment, or for other uses in water
quality management. Since individual chemical compounds can be mea-
sured, these methods are more specific than the conventional water
quality parameters such as BOD, COD, etc.

Major conclusions are as follows:

. Quantitative measurement of many organic contaminants is
possible in the range of 0.2 to 0.5 part per million in a 5-g sample.

This limit of detection could be extended by increasing the sample size.

. Qualitative detection is possible at concentrations below the
limit of quantitative measurement, thus providing a means of identifying
the presence of organic contaminants at levels far below any known thresh.
old of toxicity or other adverse effects for most organic compounds,

. Compounds tested in laboratory studies included saturated
hydrocarbons to Cgg, aromatics to Cg, alcohols to C,, amines to Ce,

glycols to Cg, unsaturated hydrocarbons to Cio, as well as various


with shrimp and oyster samples. Ona 5-g sample, measurement of
various organic compounds in the fractional part per million concentra-
tion range was ccomplished routinely.

Grinding With Dry Ice. As a possible alternate procedure,
laboratory experiments were conducted to evaluate the possibility of
grinding specimens with dry ice while frozen. Dry ice has the advantage
of subliming directly to gaseous carbon dioxide, thereby avoiding the
problem of draining or otherwise disposing of water with the risk of
losing a portion of the organic contaminants being measured. In all
experiments, however, the water present in the original specimen proved
troublesome, and it was not possible to keep the sample sufficiently
frozen while grinding to avoid some drainage of liquid water. If a large
amount of dry ice was used, this caused the water to freeze but did not
avoid the subsequent drainage which occurred when the sample was thawed.
Therefore, it appeared that the alternate procedure of tying up water
with sodium sulfate followed by extraction with an organic solvent was a
better method of avoiding emulsification and other problems inherent in
the disposition of water, and the dry ice grinding procedure was abandoned.

Separation Procedures

Techniques used for the removal of organic contaminants from
the prepared samples included distillation, organic solvent extraction,
and water extraction, Each of these methods proved useful under certain

circumstances, as discussed below.

14-


than that obtained by using the conventional water quality parameters
such as BOD, COD, DO, etc., to characterize waste streams or bodies
of water. Possible applications include the following:

1, Water quality monitoring networks in bays and estuaries
could be based at least in part on the analysis of marine organisms.
Methods based on analysis of water samples are difficult to use to
establish long-term patterns because of the combined effects of tide
and wind action on mixing, and the resulting effects on the distribution
of pollutants throughout the estuary.

2. Measurement of the direct effect of pollution on marine
life should make it possible to decrease the number of sampling stations
or the frequency of sampling. In addition, individual components res-
ponsible for taste and odor problems in fish and other marine organisms
could be identified and measured.

3. Individual pollutants could be traced back to their source,
in order to identify various sources that cause unusual adverse effects
on the receiving body of water. This is especially important if a body
of water receives waste effluents from a large industrial complex which
includes many different types of operations.

4, Different methods of waste treatment could be evaluated to
determine what treatment techniques can satisfactorily eliminate indi-

vidual compounds found to be toxic, odorous, or otherwise objectionable,


and oils in the tissue extracts. However, none of these adsorbents was
found to be as good as Florosil in providing the degree of separation
required.

The column was prewetted with the solvent, such as carbon
tetrachloride, and the tissue extract was eluted through the column into
a 50-ml beaker at a rate of 1/2 ml per minute. The elution was allowed
to proceed without applying any pressure to the top of the column. An
additional 5 ml of CCl, was added to the column after the tissue extract
had passed through the column. It was found that this volume of CC
was sufficient to elute all the components under study.

Solvent Evaporation Technique, The beaker containing the

eluate was then placed in a water bath and kept at atmospheric pressure
and a temperature approximately equal to the boiling point of the solvent
(77°C for CCl). The sample was evaporated nearly to 0.25-ml volume,
and then removed and cooled. Additional CCl, was added to the beaker,
which was then shaken and the contents transferred to a graduated micro
test tube. The contents of the tube were further subjected to evaporation
in a water bath until the final volume was 0,25 ml, stoppered with a
rubber septum, and set aside for investigation. This final sample,
containing a known volume of solvent and the residue, was used in the
chromatographic analysis

In the evaporation, it is usually necessary to adjust the sol-

vent to an accurately known volume so that an aliquot can be taken for

-20-


Ecological Research Series

Development of Sample Preparation
Methods For Analysis of

Marine Organisms

TED ST4>
Ss &

A
"Al prove”

nodlans

~)

&
0,
N agenct

i

Office of Research and Development

U.S. Environmental Protection Agency
Washington, D.C. 20460


APPENDIX

This Appendix presents a discussion of the calibration tests
that were performed with spiked samples, as described in the text
of the report. Where pertinent, information is given on procedures,
chromatographic columns used, results obtained, and other experi-
mental details. A table is included, presenting detailed data on
these calibration tests. Also, a Bibliography is included, listing

literature references pertinent to the work described.

~43-


. Extraction with organic solvents to concentrate the
contaminants in a smaller volume for subsequent
analysis,

a Stripping with air or nitrogen to remove volatile
materials from water or other substrate and thus
provide a preliminary separation.

. Column chromatography, frequently useful to separate
a mixture of trace organic materials from water and
other constituents so that the organic fraction can be
further analyzed.

. Gas chromatography, which provides a sensitive method

for the analysis of the organic fraction.

Various combinations of these methods were used; in particular,
extraction, column chromatography, and gas chromatography was the
most useful combination. Various portions of the investigation devoted
to the development of these techniques will be discussed separately later

in this report.


These three species of marine organisms were obtained from
various parts of Galveston Bay, particularly where high levels of con-
tamination are most likely to be present, as shown in Figure 1.* Per-
sonnel of the Texas Parks and Wildlife Laboratory in Seabrook, Texas,
have graciously given their assistance in this phase. Other species
could also have been used, but it was felt that these varieties repre-
sented a logical choice for this study.

The general plan for the study involved obtaining specimens
from a relatively polluted portion of Galveston Bay, for laboratory study
to evaluate sample preparation methods. Spiked samples were used for
much of the work in order to obtain quantitative results which would
indicate the degree of recovery of the various contaminants. The
specimens were treated to separate the organic contaminants prior to
actual analysis. The selection of specific techniques depended on the
nature of the contaminants which were measured and on their vapor
pressure, chemical polarity, and other characteristics. In general,
the various techniques which were used in the study fell into the following
major categories:

. Sample preparation and cleanup methods.

. Solution in water or other suitable solvents to remove

materials to be measured.

*Tables and Figures are placed at the end of this report,


Aromatics

Bauman, F., et al, Capillary Column Gas Chromatography of Cg - Ci
Aromatic Compounds. Synthesis of Selected Isomers for Identification
Purposes," J, Chromatogr. 26, No. 1:262-67, January, 1967.

Karr, Clarence, Jr., et al, "Analysis of Aromatic Hydrocarbons From
Pitch Oils by Liquid Chromatography in Gas Chromatography Analog,"
Anal. Chem. 36, No. 11:2105-8, October, 1964.

Klayder, T. J., 'Low Boiling Aromatics in Petroleum Fractions by
Gas Chromatography," J. of A.O. A.C. 47, No. 6:1146-53, December,
1964.

Mukai, Mitsugi, et al, "Aromatic Hydrocarbons Produced During Com-
bustion of Simple Aliphatic Fuels," Anal, Chem. 37, No. 3:398-403,
March, 1965,

Walker, J. O., and Ahlberg, D. L., "Quantitative Analysis of Aromatic

Hydrocarbons by Capillary Gas Chromatography,'' Anal. Chem. 35,
No. 13:2022-7, December, 1963.

Unsaturated Hydrocarbons

Anon,, ' The Standardization of Gas Liquid Chromatography for the

Analysis of Simple Hydrocarbon Mixtures,'' J. Chromatogr. 12, No. 3:
293-304, November, 1963.

Ford, Donald C., "Analysis of Light Hydrocarbons, Cz to Cs,"' J. Gas
Chromatogr. 1, No. 8:36, August, 1963.

Schneider, Werner, Bruderreck, Hartmut, and Halasze, Istvan, 'Gas
Chromatographic Separation of Hydrocarbons (C, to Cg) by Carbon
Number Using Packed Capillary Columns," Anal. Chem, 36, No. 8:
1533-1540, July, 1964.

Saturated Hydrocarbons

Abert, D. K., 'Determination of Cs to Cj, N-Paraffins and Hydrocarhkon
Types in Gasoline by Gas Chromatography," Anal. Chem. 35, No. 12:
1918-21, November, 1963.

56


PHILA, ‘ NO. 492000 LCEDS & NORTHRUP CO, PHILA,

Figure 4, Chromatogram of Carbon Tetrachloride Solvent

=39-


solvent remaining in the final sample. To illustrate this problem
and show typical chromatograms, a series of chart records is
presented from laboratory experiments using oyster samples spiked
with a number of aliphatic hydrocarbons. Pure compounds ranging
from Cg (n-hexane) to Cig (n-hexadecane) were used, and are indi-
cated on the charts by the number of carbon atoms. Figure 4 shows
a chromatogram obtained with the carbon tetrachloride solvent; the
abrupt changes in the recording are due to changes in attenuation
scale as noted on the chart (4X, 8X, etc.). The "tailing" effect
shown is typical of many columns when relatively large amounts of
carbon tetrachloride are injected into the instrument. In sucha
situation, trace components that are recorded before the beginning
of the CCl, peak are not affected, those recorded during the "tailing"
must be measured by measuring the peak area above the solvent
background, and any that appear at or near the maximum point of

the solvent peak may not be capable of measurement,

To illustrate this, Figure 5 shows the chart record
obtained in a calibration run with carbon tetrachloride containing
small amounts of nine hydrocarbons. Again, abrupt changes in the

chart record are due to changes in attenuation scale. Despite the

~27-


i. Aromatic Compounds

Extraction of the known aromatic compounds from spiked oyster
samples was carried out by two methods, microdistillation and solvent
extraction. The analysis of the concentrated extracts of each method
differed and thus will be discussed separately,

Micro Distillation Extract. The concentrate which contained

0.25 ml of CCL, and the aromatic distillate was analyzed by gas chromato-
graphy using a column 12 ft x 1/8 in. OD, 10 percent Apiezon on
Chromosorb P, 60/80 mesh, The separation was carried out at 120°C.
The sample chromatogram, when compared to a calibration blend
chromatogram, showed a high percentage of recovery,

Organic Solvent Extraction. The solvents considered for

the extraction of aromatic compounds through Cs from oyster samples
were carbon tetrachloride, n-hexane, and n-dodecane. Though all of
these solvents proved to be excellent solvents by preliminary extrac-
tion experiments of prepared water-aromatic standards, it was decided
to investigate only n-hexane and n-dodecane, Samples spiked with

known amounts of aromatic compounds were run simultaneously with

the unspiked samples for control purposes, It was shown by analyzing
the spiked samples that concentrations in the range of 0.1 ppm aromatics
through Cg could be detected in a 5-g prepared sample.

The extraction procedure using n-dodecane as a solvent was

-44_


The peak area of a chromatogram was obtained by multi-
plying the peak height by the width of the peak taken at half height.
This was done for sharp, symmetrical, and completely resolved

peaks, such as for members of a homologous series.

Theoretical recovery was not obtained on any of the com-
pounds investigated. In most cases, some losses were incurred
during purification of sample extracts or during chromatography.
Recoveries were relatively constant with all types of compounds
tested, and the losses were thought to be due primarily to mechanical

handling losses in sample processing.

Sensitivity is better with a flame ionization detector than
with many other detectors that can be used. The methods developed
in this study make it possible to detect as little as 2-4 nanograms
of a compound as it emerged from the column. Thus, the ultimate
limit of detection for most types of organic compounds tested was
in the range of 0:1 ppm based on a 5-g sample, and quantitative

results could be obtained on samples containing 0.3-0.5 ppm or more

One problem that may be encountered in trace analysis

using extraction with an organic solvent is that of interference from

-26-


SECTION IL
RECOMMENDATIONS

Since this report outlines the development of new methods of mea-
surement, final recommendations for further action are not presented,
However, the results obtained to date indicate profitable avenues for further
investigation which are now being pursued. In addition, several practical
applications of these methods of measurement are possible, and these
are outlined briefly to indicate valuable future uses for this work.

A major advantage of this method of measurement is that it is
based on the analysis of fish, shrimp, or other marine organisms col-
lected in a bay or estuary, rather than on the direct analysis of water
samples. This avoids many of the errors inherent in the sampling pro-
cedure since it is always difficult to be sure that a water sample is
representative of the body of water being studied over some known period
of time. However, marine organisms act as cumulative sampling devices,
storing certain organic contaminants so that they can be measured by
subsequent laboratory analysis. While some compounds may be decom-
posed or otherwise eliminated, many are accumulated and stored over an
extended period of time and thus can be measured by chemical analysis.

In addition to the advantages inherent in this method of sampling,
results are also obtained on individual organic compounds which are
present in the sample. Therefore, the information obtained should be

useful in many ways since this type of information is much more specific


containing 0.25 ml of carbon tetrachloride which was immersed in an
ice-salt bath. Under the proper distillation conditions, which were
established at a nitrogen flow of 10 cc per minute and distillation pro-
gramming of from 28° to 100°C in about 15 minutes, very little loss of
solvent from the trap occurred. The technique proved highly successful,
yielding excellent recovery of the aromatic components as determined
by gas chromatography. This technique will be discussed further in

the discussion concerning aromatic compounds in the Appendix,

Organic Solvent Procedures. Another method for extracting

and collecting organic contaminants from marine organisms prior to
analysis was extraction with organic solvents,

Some of the organic solvents used for extracting contami-
nants from oyster and shrimp samples were n-hexane, n-dodecane,
acetone, isopropyl ether, carbon tetrachloride, dioxane, chloroform,
benzene, and ethyl alcohol. It was found that carbon tetrachloride
offered more advantages than the other solvents for the compounds
investigated in the study, because it elutes rapidly from the columns
and gives sharp peaks with little tailing.

The extraction experiments involved the use of 100-ml
centrifuge tubes, Five grams of the prepared ground tissue were placed
in the centrifuge tube and extracted with a volume of 25 ml of the organic

solvent. To provide better phase separation, sodium sulfate crystals

-17-


TABLE I. SUMMARY DATA SHEET OF CHROMATOGRAPHIC ANALYSIS OF SAMPLES

(Continued)
Gas Chromatograph ‘Tiseue Extract
Conditions Theoret, Concentration ‘Sample
Tj. Man. Ne Air ppm of Comp. Size ul Percent
Column Flow Nz Temp. Temp. Pres. Pres. Pres. __ per 5-g sample Injected Recovery
Organic Compounds Formula Column Temp.°C mifmin °C 2c tb wb _lb__ Oyster Shrimp Fish Oyster Shrimp Fish Oyster Shrimp Fish
9. Ester
methyl hexanoate Clg (CFs CHHCH, =m x 2.3 mm, 220 125, 210 2403030 a re 88 se
_ Porapak ©, 80/100
mesh
10, Heterocyclic Com-
pound
pyridine CoN mx 2.3 mm, 175 125 210 © 200-3030 so 2 a 89 50
Porapak ©, 80/100
mesh
lL, Acid
butyric acid aH, COOH Sitx 1/4 in. , 5% 150 200 210 2003030 502 2 es 83 on oe
Reoplex on G
Chromosorb, 40/60
mesh
12, Sulfide
diethyl sulfide (Co¥e eS 6 ft x 1/8 in, OD 70 50 210 200 3030 50 8 8 <= 030 03 = 8 eo

20% SE 30 packed
on Chromosorb P,
60/80 mesh

13, High Mol. H's
n-docosane ©-Cra Pas 50 ft x 0.02 in, 175 50 210 200 58 30 50 2 2 = 03 03 88 Be
open tubular columa
packed with Carbo-

wax 20 M
14. Amide
1, n-dimethyl HCON(Ci& Je 1m x 2.3 mm, 180 60 240-210-6030 501 1 = 0.3 03 = gn 09
formamide Porapak @, 80/100
mesh
15. Chlorinated BC
trichloroethylene Ce Cle 6 fx 1/8 in.OD 20% 60 60 2152103030 502 2 -- 0.30 0.3 =e 90 900
(Fisher) SE 30, packed ow
Chromosorb P, 60/80
mesh
16, Organophosphorus
Compound (}) iw
tri-iso-propyl [(CFb ja CHO]s P 6 ft x 1/8 in.OD, 15% 150 60 250235 9034 5030) - rr oo.
phosphate PEG 20 M on Ghromo-
pak

(1) Two standard solutions were used. The lower standard was
10 ppm. The higher concentration standard, 30 ppm, was
used to determine quantitatively the percent of recovery in
the spiked sample.

54


The column used for detecting o-cresol in the samples was
a6 ftx 1/8 in., SE 30 column (on 60/80 mesh Chromosorb P) at a
temperature of 180° C and a flow rate of approximately 70 cc/min.
Carbon tetrachloride proved to be a satisfactory solvent for the extrac-
tion of o-cresol from fish samples. Concentrations as low as 0.5 ppm
in fish could be detected.

Fish samples were exposed to only one concentration of
o-cresol in bay water, 16 mg/l. At the end of two hours, all of the fish
died due to exposure at this level. Analysis of fish samples showed a
concentration of 0.62 ppm of o-cresol after this exposure, although this
was near enough to the limit of detection that numerical accuracy may
be only approximate.

Discussion of Results, Based on the results of these tests
and other tests using spiked samples, some general statements can be
made concerning the degree of recovery and the sensitivity of the methods
of sample preparation and analysis that have been used.

The general extraction and cleanup procedure shown in
Figure 7 was shown to be satisfactory for processing samples. Thus,
detailed laboratory investigations utilizing a variety of marine samples
could be conducted without using unduly complicated methods for pro-
cessing a variety of samples. Different columns were used for the final
analytical step in order to determine sensitivity and percent recovery

under ideal conditions, but screening tests could be made to identify a

34.


variety of contaminants (if present) in a single run,

Sensitivity and precision were similar with fish, shrimp,
or oyster samples, and there is no reason to believe that other species
would be substantially different in this respect. Based on comparisons
of spiked and unspiked samples, together with solvent control tests and
calibration chromatographs, the quantitative recovery of most constitu-
ents was in the range of 70 to 90 percent, with only few exceptions (see
detailed data in Appendix). The lower limit of detection for most con-
stituents is in the range of 0.2 to 1.0 ppm in marine specimens, again
with only a few exceptions. Laboratory exposure tests with fish con-
firmed the accumulation of organic contaminants in the fish as a function
of concentration in the water during exposure, and demonstrated the use

of the methods developed in this study to make valid measurements.

_35-


rather than with carbon tetrachloride as was done in many cases pre-
viously, The reason for this is not known, but may be related to chemi-
cal and physical characteristics of the solvent,

A standard solution was used which contained 6 ppm of ethyl
sulfide by weight, based on a 5-g sample of the organism. This proved
to be near the lower limit of detection, and thus ethyl sulfide is again
more difficult to detect at lower concentrations than most other com-
pounds tested previously.

Recovery of ethyl sulfide from the spiked shrimp sample was
approximately 78 percent, and recovery from the spiked oyster sample
was 83 percent,

13, High Molecular Weight Hydrocarbon — N-Docosane
_N-Cae2 Hig

The column used for detecting n-docosane in the samples was a
50-ft x 0.02-in. support-coated open tubular column packed with Carbowax
20M at a temperature of 175°C and flow rate of about 50 cc/min, The
analytical procedure was similar to that used with other compounds,
except that it was found that better recovery was obtained if the samples
were extracted with ethyl benzene rather than with carbon tetrachloride
or with ethanol as was done previously.

Two standard solutions were used which contained 0.5 ppm and
1.5 ppm of n-docosane by weight, respectively, based on a 5-g sam-

ple of shrimp or oyster. The lower concentration standard was used

-50-


new chromatographic column for separating and detecting micro amounts
of glycols. The column that gave the best results was 5 ft x 1/4 in. OD,
acid-washed 40/60 mesh G-Chromosorb solid support coated with 5 per-
cent Reoplex 400 (polypropylene-glycol adipate). The processes of mixing
and coating, evaporating and drying, packing and conditioning of the column
were the same processes used in the preparation of the column used in

the analysis of amines except that chloroform was used as a solvent.

Analysis of Sample Extracts. The concentrated extracts

obtained from the spiked samples either by solvent extraction or micro
distillation were analyzed by gas chromatography. The chromatograms
obtained were compared with known calibration chromatograms of the
same components that were originally added to the sample, The extract
chromatograms from the spiked tissue sample and the chromatogram

of the solvent, used in the extraction process, were also compared with
the calibration chromatograms to determine if any of the components were
present originally, in the sample or in the solvent.

In order to make a standard solution of an organic mixture,
the components of the mixture and their respective densities should be
known, Because the procedures for preparing such standards were
almost the same for each homologous series investigated, only a des-
cription of preparing a standard solution of a blend of ketones is mentioned

as an example. To make a 25-ml standard solution of this blend (con-

-23-


€s

washed G-Chromo-
sorb, 40/60 mesh

TABLE SUMMARY DATA SHEET OF CHROMATOGRAPHIC ANALYSIS OF SAMPLES
Gas Chromatography ‘Tissue Extract
Conditions ‘Theoret. Concentration Sample
Ty. Man. Ne Air ppm of Comp. Size wl Percent
Column FlowNg Temp. Temp. Pres. Pres, Pres. per 5-g sample injected Recovery
Organic Compounds Formula Column Temp°C mi/min °C °C tb 1b _tb_ “Oyster Shrimp Fish Oyster Shrimp Fish Oyster Shrimp Fish
Aromatics
benzene Coty 12 ft x 1/8 in. OD 1 - —- woe
toluene CoHe CH 100% Apiezon L 1 -- 89 --
ethyl benzene Co He Co Bs packed on 410 70 200 20030 30st coe -- 68 --
methyl xylene GH (CH be Chromosorb P 1 - - 69 _
para xylene Co He (CHs be 60/80 mesh 1 - -
ortho xylene Ce Hi (CF bo 1 - 66
cumene CoH CH(CHs Jo 1 - 40
Unsaturated
Hydrocarbons
2-pentene CHyCH=CHCHE CH 6 ft x 1/8 in.OD 0.5 0.5 oe er 47 we
2ohexene CHy CH= CH(CHe lo CHs 20% SE 30 packed 100 50 210 200280 3S O20 = 6 wa
2-oetene CH CH= CH(CRe }y CH on Chromosorb P 05 0.5 soe es
60/80 mesh
Saturated
Hydrocarbons
a-hexane Cathe 0.5 Os 7 93 --
n-heptane Gite 05 0.5 - - =
n-octane Core 10 10 -- a
n-nonane Geo 100 ft x 0.02 in.ID 180 60 250 230 2830 40S OS - a
n-decane Gots silicone DC 550 0.5 0.5 - oo
n-undecane Ci Hee 05 0.5 -- 200.
a-dodecane Caos 10 10 - oon
notridecane Gis kee 0.5 0.5 - 500
n-tetradecane Crate 0.5 0.5 - 8900
n-hexadecane Getes 0.5 0.5 - 8900
Alcohols
L-butanol Cty (CHeeCHeOH 6 ft x 1/8 in. OD 0.5
i-pentano} CH (CH )sCHeOH 20% SE 30 packed 125, 50 2000-21025 3 DS
I-hexanol Clb (Cie CHOH on Chromosorb P 0.5
1-heptanol CH (CHe CHeOH 60/80 mesh 0.5 = ~
Phenols
phenol Got OH 0.9 67 5
mecresol CH Ge Hy OW same as above 180 50 210-210 2530 8686
o-ethyl phenol Co He Ce Fa OH 0.2 78 62
prethyl phenol GG HOH La 83044
Ketones
2-butanone CH COCa He 0.8 7 85
2-pentanone Gly CO(CHe Ja CH 100 ft x 0.02 in. 1D 100 50 20020020 30S 86 60
2,4-dimethyl- (Cll 2 CHCOCH(CHs fz silicone DC 550 0.5 8040
3-pentanone
Amines 5 ft x 1/4 in, OD
diethylamine (CoH bNH 15% silicone DC. os 8060
@i-a-propylamine — (CHs CHe CHe Je NH 550 coated on 120 10 20021032 BS --
disn-butylamine (Cx Hp Jo NH 12. 5% Chromosorb 05 a7 --
cyclohexylamine Ga Fi NHe P, 60/80 mesh 0s 83 3a
Glycols 5 ftx 1/4 in, OD
ethylene glycol Cl OBC OH 5% Reoplex 400 200 70 250 235268 Lo 75 _
diethylene glycol HO(CHe Js O(CHe 2 OH coated on acid~ 10 800


Section

I

I

IV

TABLE OF CONTENTS

SUMMARY AND CONCLUSIONS
RECOMMENDATIONS
INTRODUCTION

STUDY PROCEDURE
APPENDIX

BIBLIOGRAPHY

ii


1 Sample
preparation

ty

[oyste: sample |

compositing :

| Filtration
cheesecloth
chilling

Grinding
| Waring blender

s

Master sample
refrigerated

~~ 2 Extraction

[

Transferring sample to
centrifuge tube

3 - Separation
and cleanup

Adding
20-25 ml CCL

ot,

Shaking for |
1 minute

Centrifuging
1500 rpm 2 minutes
Aliquot sample
5 grams
| Transfer Hssue extract]
to a container
Spiking known
amount, mixing |
; Re-extract with
10 ml CC
Na, SO, addition
1:2 ratio

Refrigeration |.)
2-3 hours

preparation

Wetting the
column CCl,

Separating
oils and color]

Washing the

column 5 ml CCl,

Clear
tissue extract

of

Concentrating the
tissue extract 1/4 ml
by evaporation*

‘Analysis |
gas chromatography

Identification by
comparing with standards

*Evaporation temperature depends on boiling points of contaminants investigated.

Figure 7

~42-

Sample Preparation Procedures


AEDS A NOETURUE CO, PHIEA,

2C00

Chromatogram of Calibration Sample

Figure 5.

-40-


These columns were installed in a Perkin-Elmer 900 dual flame
chromatograph instrument which was used for analysis throughout the
investigation,

In addition to obtaining or developing these columns, some work
was done to install a backflush system in the instrument, Such a sys-
tem would be useful when injecting solvent-extracted samples into the
chromatograph. It could provide a tool for backflushing heavy sample
oils that might be extracted with the solvent and are not pertinent to the
analysis and, also, might damage the columns. However, difficulties
developed and the system was discontinued. It became unnecessary
when a preliminary cleaning step was utilized prior to analysis.

Sample Preparation

At the beginning of the investigation, work on sample preparation
was limited to oyster samples. Subsequently, shrimp and fish samples
were used in modifying the methods so that any of the three could be used.

Oyster Sample Preparation, Several series of tests were

conducted to evaluate different methods and procedures for grinding,

extracting, and otherwise processing samples. From this experience,

the following procedure was developed and used for subsequent experiments
Fresh oyster samples collected at the same source were

removed from their shells and composited in a glass container. Usually,

these composite samples from each source were frozen unless they were

10-


16. Organophosphorus Compound — Tri-Iso-Propyl Phosphate
[(CHe je CHO]s P

Only oyster sample was used in detecting tri-iso-propyl phosphate.
The column used for detecting this compound in the sample was a 6-ft x
1/8-in. OD 15 percent PEG 20 M on Chromopak at a temperature of 150°C
and flow rate of approximately 60 cc/min. The selection of the column
came after trying different columns which were not satisfactory.

Different organophosphorus compounds were also used in other
oyster samples, but their detection was not satisfactory. This class
of organic compounds was extremely difficult to detect by means of gas
chromatography under the previously described conditions,

Ethanol proved to be a satisfactory solvent for the extraction of
tri-iso-propyl phosphate from the sample. Concentration as low as
30 ppm in the oyster sample was detected.

The percent of recovery was approximately 86 percent.

-52-


Suffis, Robert, and Dean, Donald E., "Identification of Alcohol Peaks
in Gas Chromatography by a Nonaqueous Extraction Technique," Anal.
Chem. 36, No. 4:480-3, April, 1962.

Phenols

Barry, J. A,, Vasishth, R. C., and Shelton, F. J., "Analysis of
Chlorophenols by Gas-Liquid Chromatography,'' Anal. Chem. 34,
No. 1:67-9, January, 1962.

Karr, C., Jr., Brown, P. M., and Estep, P. A., "Identification and
Determination of Low-Boiling Phenols in Low Temperature Coal Tar,"
Anal. Chem. 30, No. 8:1413-6, August, 1958.

Smith, J.R.L., Norman, R.O.C., and Radda, G.K.J., 'Quantitative
Determination of Isomeric Phenols,'! J. Gas Chromatogr. 2, No. 5:
146-9, May, 1964.

Stevens, M. P., and Percival, D. F., ''Gas Chromatographic Deter-
mination of Free Phenol and Free Formaldehyde in Phenolic Resins,"
Anal, Chem, 36, No. 6:1023-4, May, 1964.

Ketones

Gudzinowicz, B. J., et al, '2,3-Diketones,'' Anal. Chem. 37, No. 13:
1745, December, 1965.

Kawada, T., et al, ''Aldehydes, Ketones, Alcohols, and Lactones,"
J. of A.O.C.S. 43, No. 4:237, April, 1966.
Amines

Amell, Alexander R., etal, "Gas Chromatographic Separation of Simple
Aliphatic Amines," Anal. Chem. 33, No. 12:1805-6, November, 1961.

Arad, Yael, Levy, Moshe, and Vofsi, David, 'Gas Chromatographic

Determination of Amines in Aqueous Solution," J, Chromatogr. 13,
No. 2:565-7, February, 1964.

58


The final material was then refrigerated for two to three
hours before extraction, Sometimes an unspiked sample was prepared
simultaneously in the same manner to serve not only as a control but
also to determine if any of the contaminants which were added to the
spiked sample were present initially.

Shrimp Sample Preparation. The methods which were used

in the preparation of shrimp samples were generally similar to those
developed during the oyster study with some slight modifications.

On receipt of the shrimp samples, tissues were removed
from the shells and composited in glass jars. The heads and tails were
discarded. The procedures of draining the excess water, chilling,
grinding, and homogenizing the shrimp tissues were the same procedures
used in oyster sample preparation. However, the time required to grind
the shrimp sample was about twice that needed for homogenizing oyster
samples. This appeared to be the main difference between oyster and
shrimp sample preparation,

Spiking an aliquot shrimp sample slurry with a known weight
of the organic contaminants to be investigated and adding the 10 g of
anhydrous sodium sulfate were carried out by the same procedures used
in the oyster sample preparation,

In the series of experiments performed, the shrimp slurry

was spiked with 2.5 Ug of each of the components studied. Ona 5-g

-12-


EPA-660/3-74-026
January 1974

DEVELOPMENT OF SAMPLE PREPARATION

METHODS FOR ANALYSIS OF MARINE ORGANISMS

By

Herbert C. McKee
and
David S, Tarazi

Grant No. 16020 EGG
SwRI Project No. 01-2693
Program Element 1BA022

Project Officer

Dr. William S. Hodgkiss
National Marine Water Quality Laboratory
South Ferry Road
Narragansett, Rhode Island 02882

Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D. C. 20460

For sale by the Superintendent of Documents, U.8. Government Printing Office, Washington, D.C. 20402 - Price $1.05


it was found that better recovery was obtained if fish samples were
extracted with ethanol rather than with carbon tetrachloride as was done
previously. The reason for this is not known, but may be related to
chemical characteristics, Extraction of a polar compound such as
DEG appears to be more successful with a polar solvent such as ethanol,
whereas the relatively non-polar carbon tetrachloride had given best
results in the past with non-polar constituents such as hydrocarbons.

A standard solution was used which contained 2 ppm DEG
by weight, based ona 5-g sample of fish. This proved to be near the
lower lirait of detect.on, aad thus DEG is more difficult to detect at
lower concentrations than most other compounds tested previously. At
higher concentrations, however, recovery of DEG from spiked samples
was approximately 90 percent, in the same range with the better results
obtained previously with other constituents.

At the end of 96 hours, analysis of the fish samples showed

the following results:

Exposure level, Measured level,
mg/l] DEG ia water ppm DEG in fish sample
3 174
Jo 360
32 494
64 1630


the analytical step. Care must be taken that the solvent is not lost by
evaporation when the residue is made up to a standard volume and an
aliquot is withdrawn for analysis.

Analysis by Gas- Liquid Chromatography

Specifications for the Gas Chromatographic System Used.

Chromatography is one of the most useful techniques for organic com-
pounds available to the analytical chemist. By this method, complex
organic mixtures of compounds can be separated and the individual com-
ponents detected and sometimes identified.

The instrument employed in this study was a Perkin-Elmer
900 gas chromatograph with a dual ionization detector, This instrument
is extremely sensitive to micro amounts of organic compounds. It is
also fast, and gives good resolution of complex mixtures,

Almost all the organic compounds used in the experiments
were analytical standards obtained from Poly Science Corporation
Chemical Division, Evanston, [llinois.

Preparation of New Chromatographic Columns. Amines and

glycols were the most troublesome classes of organic compounds investi-
gated, from the standpoint of analysis by gas chromatography. This was
not surprising, since previous studies at Southwest Research Institute
and elsewhere have encountered difficulties in the measurement of trace

quantities of these compounds. With many available chromatographic


RESEARCH REPORTING SERIES

Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4, Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formaticn,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.

EPA REVIEW NOTICE

This report has been reviewed by the Office of
Research and Development, EPA, and approved for
publication. Approval does not signify that the
contents necessarily reflect the views and policies
of the Environmental Protection Agency, nor does
mention of trade names or commercial products consti-
tute endorsement or recommendation for use.


identical to n-hexane extraction. As an extraction solvent, n-dodecane
offered definite analytical advantages over n-hexane since it elutes after
the aromatic compounds to Cp using a silicone DC 550 open tubular
chromatographic column, The lower vapor pressure also made n-dodecane
an excellent solvent since there is less danger that it will evaporate

during the extraction procedures, The limitation in using n-dodecane

as a solvent was its purity. It would be necessary to obtain a solvent

of high purity in order to apply it successfully.

2. Unsaturated Hydrocarbons

Chromatograms were also obtained for unsaturated hydrocarbons
including 2-pentene, 2-hexene, and 2-octene. The column selected to
perform the analysis was also 6 ft x 1/8 in. OD, 20 percent SE 30 packed
on Chromosorb P, 60/80 mesh, at a temperature of 100°C. In the
series of experiments performed, the tissue sample was spiked with
2.5 ug of each of the components studied. This concentration was
measured under the conditions mentioned above, for each of the unsatu-
rated hydrocarbons tested.

3. Saturated Hydrocarbons

The chromatograms of this class of compounds were obtained by
analyzing a mixture of Cg-Cig saturated hydrocarbons. In the analysis,
a 100 ft x 0.02 in. OD, silicone DC 550 open tubular gas chromatographic
column was used at a temperature of 180°C. Basedona comparison

of the spiked oyster extract chromatograms and calibration chromato-

-45-


These results show that all of the various processing steps
described previously can be used for sample processing and analysis,
and that quantitative results can be obtained with many different types
of organic compounds. The combination of processing steps that
proved to be most useful included the following operations:

1. Grinding and treatment with sodium sulfate to avoid

problems in subsequent extraction due to the presence
of water.

2, Extraction with carbon tetrachloride.

3, Treatment by column chromatography to separate

naturally occurring interfering substances.

4, Analysis by gas chromatography.

Figure 7 shows a flow diagram illustrating the various steps
in processing samples by the preferred method, which was used in
subsequent experiments.

Fish Exposure Studies

To confirm the utility of the methods developed, a series of
laboratory tests was conducted in which fish were exposed to various
organic compounds and the accumulated contaminants were measured,
This made it possible to measure the absorption of contaminants during
various periods of exposure, and to determine the recovery of contami-

nants and the sensitivity of the method with actual exposed samples as

-29-


15.0 cm Florosil, 60-100 mesh

cotton
} steel wool

i2- 5 cm Nag SQ,

yd
qoy
7
y

Tye Se 858 o 8,5 goto OU a a LUT oa © STS IS NT Se
a

BS Pee ne mad” to op G by Steg Se pe Be Be

The "Cleanup" Column
-38-

Figure 3,


centration 1 U1 = 10 nanograms per component), the following procedures

were followed:

(a)

(b)

Only pure chromatographic compounds and pure chromato-
graphic solvents were used. The ketone blend used in this
study was obtained from Poly Science Corporation, Evanston,
Illinois. The solvent used, carbon tetrachloride, was ob-
tained from Burdick and Jackson Laboratories, Inc.,
Muskegon, Michigan.

A stock solution of the blend (concentration 1 ul = 1 ug for
each component) was then prepared, calculated from the
average density of the components of the mixture. In this
case, the solution was prepared by dissolving 31.3 ul of
the blend in 25 ml of carbon tetrachloride, based on an
average density of 0.80 g/ml.

In using this solution, the tissue sample was spiked with
2.5 ug of each of the components of the blend. Ona 5-g
sample, this represented 0.5 ppm by weight for each
component. This known amount of compound was dissolved
in an organic solvent and was then carried through the
extraction, cleanup, and analytical procedures, The

final volume of the solution was reduced to 0.25 ml, so
that 1 ul of this solution contained 10 nanograms of each

compound investigated,

-24-


SECTION IV
STUDY PROCEDURE

Literature Survey

A literature survey was conducted by reviewing available litera-
ture, especially for the last five years. Some selectivity was exercised
to include only those analytical procedures which appeared to be most
useful in the project. Because of the large volume of literature pub-
lished on organic contaminants in water and marine organisms, only
articles which covered the preparation, separation, and identification
of these contaminants were investigated. The Bibliography is included
in this report, to indicate available sources of information pertinent to
the work performed. (See Appendix)

Development of Analytical Procedures

It was decided to concentrate primarily on gas chromatographic
techniques for the analysis of organic contaminants. With the present
technology on gas chromatography, numerous classes of organic com-
pounds can now be analyzed, thus providing a broad capability with only
limited use of other analytical techniques.

A gas chromatograph, especially one equipped with a flame
ionization detector, is sensitive to extremely small amounts of organic
compounds, Measurement of nanogram quantities of many organic con-

stituents is routine for trace analysis applications.


SELECTED WATER 4. Report No.] 2.
RESOURCES ABSTRACTS

INPUT TRANSACTION FORM WwW

‘ie DEVELOPMENT OF SAMPLE PREPARATION METHODS FOR 5. Kepore Dare
ANALYSIS OF MARINE ORGANISMS &

8. Performing Orga. .zation
Report No.

Aurherfs)
McKee, Herbert C. and Tarazi, David S. fo Beee e
” Organication -
Southwest Research Institute Fh Gautrac/ Geant Ne
3600 Yoakum Boulevard 16020 EGG
Houston, Texas 77006 Li Type. Repo.: and

Period Covered

ceca OCR CAAT

1% Sponseriar Orgeziretion

18 Supplompatary Mores

Environmental Protection Agency report
EPA~660/3-74-026, January 1974

Io Abstract

A two-year laboratory investigation has been completed to develop laboratory methods
for processing, extracting, purifying, concentrating, and measuring specific organic
pollutants found in marine organisms. Major conclusions are as follows: a. Quantita-
tive measurement of many organic contaminants is possible in the range of 0.2 to 0.5
parts per million in a 5-g sample. b. Qualitative detection is possible at concen-
trations below the limit of quantitative measurement, thus providing a means of
identifying the presence of organic contaminants at levels far below any known thresh-
old of toxicity or other adverse effects for most organic compounds. c. Compounds
tested in laboratory studies included saturated hydrocarbons to C22, aromatics to Cg,
alcohols to C7, amines to Cg, glycole to Cg, unsaturated hydrocarbons to Cy, as well
as various ketones, phenols, esters, heterocyclic compounds, acids, sulfides, amides,
and chlorinated hydrocarbons. With most of these, recovery of 70 to 90 percent of the
amound present was obtained, indicating that quantitative measurements are possible
within the ranges stated above. d. Several methods of sample preparation can be used
peior to analysis by gas chromatography. e. Based on the results obtained, the
analysis of almost any variety of marine specimens should be possible to measure trace
organic constituents.

17a. Descriptors analytical teéhniques* Organic compounds
Gas chromatography* solvent extractions
Aromatic compounds Chlorinated hydrocarbon pesticides
Nitrogen compounds Marine animals*
Pesticides
17b, Identifiers
Sample preparation* Phenols
Hydrocarbons Esters
Ketones
My COWRE Pets & Grae O5C
19. Security Class: | 21, No.of | send To:
"Repo: } Pages .
0. Se ritgci ss [22 Prod | USTEREESOURGES selENTUTIG fagonMaTiON cenrEn
(Page) WASHINGTON. D.C. 20240

C. S. Hegre I National Marine Water Quality Laboratory



grams, the quantitative recoveries were between 75 and 80 percent.
This indicated that the sample preparation, extraction, cleanup, and
concentration methods developed were adequate to recover the com-
pounds investigated and provided semiquantitative results.

4. Alcohols

In the analysis of alcohols, which included 1-butanol, 1-pentanol,
l-hexanol, and 1-heptanol, a 6 ft x 1/8 in. OD, 20 percent SE 30 packed
on Chromosorb P, 60/80 mesh gas chromatographic column was used
at a temperature of 125°C. The concentration of each compound was
0.5 ppm by weight.

5. Phenols

The column used for detecting phenols in the samples was 6 ft x
1/8 in. OD, 20 percent SE 30 packed on Chromosorb P, 60/80 mesh,
at a temperature of 180°C.

Oyster and shrimp samples were spiked with known amounts of
phenols, including phenol, m-cresol, o-ethyl phenol, and p-ethyl phenol.
These samples were also run simultaneously with unspiked samples for
control purposes. It was shown by analyzing the spiked samples that
concentrations in the range of 0.2 ppm of o-ethyl phenol could be mea-
sured in a 5-g prepared sample,

6. Ketones
The investigation of the oyster and shrimp samples studied had

shown that some ketones, including 2-butanone, 2-pentanone, and

~46-


it was felt that some method of measuring them would be desirable.
Various available columns were evaluated to select one that would perform
satisfactorily in detecting them, but none was good enough. The one
column that showed the most promise was developed at Southwest
Research Institute, and it is described fully under Section IV, ''Prepara-
tion of New Chromatographic Columns". The column was 5 ft x 1/4 in.
OD, 5 percent Reoplex 400 coated on acid-washed G-Chromosorb, 40/60
mesh, Results using it at a temperature of 180° -200° C indicated that
micro quantities of glycols in the range of 1.0 ppm could be measured
in both oyster and shrimp samples, in about the same range of sensitivity
obtained in measuring other organic compounds.

Many solvents were used for extracting glycols from the spiked
samples. The two that showed the best results were ethyl alcohol and
carbon tetrachloride.

9. Ester — Methyl Hexanoate Cs Hi: COOCHs

The column used for detecting methyl hexanoate in the samples
was a l-m x 2,3-mm Porapak Q, 80/100 mesh packed column at a
temperature of 220°C and flow rate of 125 cc/min. The solvent used
for extraction was carbon tetrachloride.

It was shown that a concentration in the range of 0.5 ppm of
methyl hexanoate could be measured in 5-g prepared shrimp and oyster
samples. The percent recovery of the compound in both samples was

approximately 88 percent.

~4g-


used immediately in laboratory experiments. The composite sample,
which contained excess water, was transferred to a cheesecloth filter
which was supported on a 500-ml beaker. The excess water was allowed
to drain without exerting any pressure on the oyster specimen. The
draining procedure was continued until no more liquid drained. The
semi-dry oysters were chilled for one hour and then placed in a Waring
blender and homogenized for about 30 seconds. The tissue slurry was
poured into a glass container, covered, and refrigerated until further use.
The sample was kept cold at all times to avoid losing low boiling organic
constituents,

Prior to solvent extraction, a 5-g aliquot of the cold sample
slurry was "spiked" by thoroughly mixing with a known weight of the
organic contaminants to be investigated. In most of the experiments
performed, the oyster sample slurry was spiked with 2.5 Ug of each of
the components studied. Ona 5-g sample, this represented 0.5 ppm by
weight for each component, The noise level of the chromatographic
instrument is such that the limit of detection for many organic compounds
is around 0.1 ppm for a 5-g sample. This limit could be extended through
the use of larger samples.

Following the spiking, 10 g of anhydrous sodium sulfate (Nag SQ, )
was added to the mixture and thoroughly mixed. The purpose of the
sodium sulfate was to absorb any water present which might lead to

emulsion formation during the extraction procedure,

-ll-