HELGOL-K---~DER MEERESUNTERSUCHUNGEN
Helgolander Meeresunters. 33, 257-271 (1980)
A n a l y s i s for p e t r o l e u m p r o d u c t s in m a r i n e
environments
D. C. Malins, M. M. Krahn, D. W. Brown
W. D. MacLeod, Jr. & T. K. Collier
Environmental Conservation Divison, Northwest and Alaska Fisheries Center, National
Marine Fisheries Service, National Oceanic and Atmospheric Administration;
2725 Montlake Boulevard East, Seattle, Washington 98112, USA
ABSTRACT: Petroleum is composed of a complex mixture of hydrocarbons that readily undergo
chemical and biological conversions on entering aquatic environments. These conversions lead to
the formation of a host of oxygenated products, some of which are potentially toxic to marine life
and to the consumer of fishery products. State-of-the-art analytical methods, as employed in our
laboratories, utilize glass-capillary gas chromatography in conjunction with mass spectrometry to
analyze environmental samples containing trace amounts of aliphatic and aromatic petroleum
hydrocarbons. These procedures are applied on a routine basis to the analysis of seawater,
sediments and tissues of marine organisms. Despite this analytical proficiency, a need exists for
analyzing oxygenated and other polar petroleum products in environmental samples. For example,
techniques such as high-performance liquid chromatography (HPLC), in conjunction with on-line
fluorometric assay techniques and mass spectrometry, make possible the analysis of polar oxygenated compounds resulting from both chemical and biological conversions. These methodologies
are first steps toward the development of routine assay procedures for environmental samples.
Current techniques for hydrocarbon analyses and new methods for analyzing polar aromatic
compounds are discussed.
INTRODUCTION
Each year a n e s t i m a t e d four m i l l i o n metric tons of p e t r o l e u m e n t e r the m a r i n e
e n v i r o n m e n t t h r o u g h sea a n d l a n d - b a s e discharges, t a n k e r mishaps, atmospheric fallout, a n d various other h u m a n activities (Clark & MacLeod, 1977). The p e t r o l e u m is a
complex mixture of c o m p o u n d s such as aliphatic, alicyclic a n d aromatic hydrocarbons,
a n d a variety of polar aromatic c o m p o u n d s a n d h i g h m o l e c u l a r w e i g h t polymers (Clark &
Brown, 1977). Extracts of seawater, s e d i m e n t a n d tissues are often r o u t i n e l y a n a l y z e d b y
gas c h r o m a t o g r a p h y for aliphatic h y d r o c a r b o n s as well as for aromatic hydrocarbons
c o n t a i n i n g as m a n y as six b e n z e n e rings (Brown et al., 1980). Yet, certain components,
n o t a b l y polar aromatic c o m p o u n d s a n d h i g h m o l e c u l a r w e i g h t polymers, are not well
suited to gas chromatography. A n a l y s e s of these compounds, b e c a u s e of their low
volatility or i n s t a b i l i t y at e l e v a t e d temperatures, are a c o n t i n u i n g c h a l l e n g e to the
i n g e n u i t y of the a n a l y t i c a l chemist (Malins, 1980}.
Other factors add t r e m e n d o u s l y to the problem. Some of the p e t r o l e u m c o m p o n e n t s
such as aromatic hydrocarbons r e a d i l y u n d e r g o structural alterations i n a q u a t i c e n v i r o n ments. O x y g e n a t e d products are formed t h r o u g h p h o t o c h e m i c a l reactions, as well as b y
9 Biologische Anstalt Helgoland
0017-9957/80/0033/0257/$ 02.00
258
D . C . Malins et al.
enzymatic and other chemical conversions that occur in living systems as diverse as
microorganisms and fish (Karrick, 1977~ Varanasi & Malins, 1977). The transformations
of petroleum hydrocarbons generally yield pollutants of enhanced water solubility, and
their presence adds greatly to problems in studying the chemical composition of
petroleum-derived products in marine environments.
Because of these difficulties, it is obvious that conventional gas chromatographic
analyses for a narrow range of hydrocarbons in seawater, sediment and tissues may
provide only a limited, perhaps myopic, perspective of petroleum contamination.
Responding to the need for a detailed perspective of petroleum pollution, we have
developed sophisticated high-resolution gas chromatographic techniques for hydrocarbons (Ramos et al., 1979). We are also exploring ways to analyze the polar aromatic
compounds (e. g. oxygenated products) arising from chemical and biological processes.
I WET SEDIMENTI
Extract on tumbler with
2:1 CH2C12/CHgOH
Discard
CH2DI2/CH30H
EXTRACT
Combine
I TOTALEXTRACT 1
Wash with H20
Discard
WASHEDCH2C2
I
EXTRACT
Filter through silica gel
precol umn
PARTIALLYPURIFIED
CH2CI2 EXTRACT
Concentrate~and displace
CH2CI2 wiih n'CdHI4
CONCENTRATEDEXTRACT
IN n-CBHl4
1
Chromatographic separation with Silica Gel
I
.2__.
SATURATED
HYDROCARBONS
I
AROMATIC
HYDROCARBONS
b
Glass Capillary Gas Chromatography
:
i
GC/MSConfirmations~W
Fig. 1. Analytical scheme for hydrocarbons in sediment
Petroleum products in marine environments
259
In this paper, we describe our gas chromatographic procedures as applied to hydlocarbons in water, sediment, and tissues. In addition, we discuss our advances in developing
new methods (e. g., applications of high-performance liquid chromatography} for the
resolution and quantitation of complex mixtures of the polar aromatic compounds
present in environmental samples.
METHODOLOGY
Hydrocarbons and dibenzothiophenes
Sample collection
Special precautions were taken in collecting and storing marine environmental
samples for trace analyses of petroleum hydrocarbons (MacLeod et al., 1976). Sampling
devices (corers, bottles, etc.) were scrupulously cleaned to avoid extraneous organic
components that could invalidate the analyses. This was accomplished by usual laboratory washing procedures, followed by sequential rinsing with contaminant-free acetone
and dichloromethane.
Samples of sediments or mussels (Mytilus edulis) were stored in pre-cleaned glass
jars tightly sealed with Teflon-lined lids. These samples were refrigerated immediately
and frozen as soon as possible to inhibit evaporation and chemical and biological
oxidative changes.
Water samples were collected in pre-cleaned glass bottles containing 5 % v/v
contaminant-free dichloromethane, sealed with Teflon-lined lids, and refrigerated. The
dichloromethane served both as an extraction solvent and a biocide.
Sample preparation
Samples of mussels (at least 15 individuals) were thawed and homogenized. A 10-g
portion of the homogenate (wet wt) was digested with 4 N aqueous sodium hydroxide
BIOLOGICAL
SYSTEM
Fluids
J
(e.g., bile, urine)
Tissue
(e.g., liver, brain)
Homogenize with solvent
)
(
HPLC
Preparative HPLC
UV/UVF detection
(with UVF lex, Xem
selected for specific
aromatic hydrocarbon)
MS confirmation
of identities
Stop-flow
Emission or
excitation
spectra
l
Fig. 2. Analytical scheme for determination of polar aromatic compounds
260
D . C . M a l i n s et al.
o v e r n i g h t at 30 ~ (MacLeod et al., 1976). T h e d i g e s t e d s a m p l e w a s e x t r a c t e d w i t h two
portions of d i e t h y l e t h e r (ethanol- a n d peroxide-free). The e t h e r extract w a s concent r a t e d a n d f i l t e r e d t h r o u g h a silica g e l b e d p r e p a r e d in d i e t h y l ether. The e t h e r e l u a t e
w a s c o n c e n t r a t e d b y fractional d i s t i l l a t i o n a n d the e t h e r w a s d i s p l a c e d w i t h h e x a n e . The
r e s u l t i n g h e x a n e solution w a s t h e n c h r o m a t o g r a p h e d on silica g e l to isolate the satur a t e d a n d a r o m a t i c h y d r o c a r b o n s a n d other w e a k l y p o l a r c o m p o u n d s (e. g. d i b e n z o t h i o phenes). T h e s e fractions w e r e a n a l y z e d b y gas c h r o m a t o g r a p h y (GC).
S e d i m e n t s a m p l e s (100 g w e t wt) w e r e e x t r a c t e d first w i t h m e t h a n o l a n d t h e n w i t h
d i c h l o r o m e t h a n e / m e t h a n o l as s h o w n in F i g u r e 1 (Brown et al., 1979, 1980). O n e - l i t e r
w a t e r s a m p l e s w e r e a c i d i f i e d a n d e x t r a c t e d w i t h d i c h l o r o m e t h a n e . The extracts w e r e
concentrated, e x c h a n g e d into h e x a n e , a n d t h e n a n a l y z e d b y GC. If c o m p l e x p a t t e r n s of
p e t r o l e u m - r e l a t e d c o m p o u n d s w e r e found, the extract w a s c h r o m a t o g r a p h e d on silica
gel, as d e s c r i b e d above, a n d a n a l y z e d b y GC.
Sample analysis
G a s c h r o m a t o g r a p h y ( G C ). A n a l y s e s w e r e p e r f o r m e d u s i n g a m i c r o p r o c e s sor-controlled G C e q u i p p e d with (1) a n a u t o m a t i c s a m p l e injector, (2) a w a l l - c o a t e d ,
o p e n t u b u l a r (WCOT) g l a s s - c a p i l l a r y c o l u m n (20-30 m l o n g a n d 0.25 m m i. d.), a n d (3) a
h y d r o g e n f l a m e - i o n i z a t i o n d e t e c t o r (FID). The p a c k e d c o l u m n G C s a m p l e i n j e c t i o n port
w a s m o d i f i e d for splitless s a m p l e i n j e c t i o n as d e s c r i b e d b y Ramos et al. (1979). C o l u m n
conditions a n d o p e r a t i n g p a r a m e t e r s a r e s h o w n in the figure captions.
Gas chromatography/mass
spectrometry
(G C/MS).Theidentities
of c o m p o u n d s d e t e c t e d a n d m e a s u r e d b y G C w e r e c o n f i r m e d b y G C / M S analysis. A
m i c r o p r o c e s s o r - c o n t r o l l e d GC, s i m i l a r to that above, w a s i n t e r f a c e d d i r e c t l y to a q u a d m p o l e t y p e m a s s s p e c t r o m e t e r (Ramos et al., 1979).
Polar aromatic compounds
Sample collection and preparation
lq x p o s u r e s t u d i e s. R a i n b o w trout w e r e a c c l i m a t e d to e x p e r i m e n t a l conditions
for two w e e k s prior to exposure. T h e test a n i m a l s w e r e a n e s t h e t i z e d w i t h t r i c a i n e m e t h a n e s u l f o n a t e (50 gg/1) a n d force-fed a g e l a t i n c a p s u l e c o n t a i n i n g t h e test c o m p o u n d
d i s s o l v e d in ca. 50/~l of e t h a n o l (see figure captions for d e t a i l s of a m o u n t fed a n d time
e l a p s e d before sampling). Tissue a n d fluid s a m p l e s w e r e frozen at - 60 ~ prior to
analysis. Bile s a m p l e s w e r e i n j e c t e d directly into the HPLC columns.
Mussel
field studies
and "Amoco
Cadiz"
oil spill. Aspartofa
s t u d y b y Wolfe et al. (unpublished), m u s s e l s w e r e c o l l e c t e d from e s s e n t i a l l y p e t r o l e u m free w a t e r s a n d p l a c e d in c a g e s 1 m b e l o w the surface at two sites: (1) directly in the
p a t h w a y of t h e s p i l l e d p e t r o l e u m , a n d (2) r e m o v e d from the direct p a t h w a y of the s p i l l e d
p e t r o l e u m , b u t in a n a r e a that w a s s l i g h t l y polluted. S a m p l e s w e r e t a k e n for c h e m i c a l
a n a l y s e s a n d m i c r o s c o p y at the time t h e y w e r e p l a c e d in the c a g e s a n d after 5, 8, 12, a n d
25 days. M u s s e l tissues, w i t h 1.5 ml m e t h a n o l a d d e d p e r g r a m w e t tissue, w e r e
h o m o g e n i z e d , centrifuged, a n d the s u p e r n a t a n t was d e c a n t e d . T h e tissue p e l l e t was
r e s u s p e n d e d a n d r e e x t r a c t e d w i t h 1.0 ml m e t h a n o l p e r g r a m origina~ w e t tissue a n d
r e c e n t r i f u g e d . The s u p e r n a t a n t w a s d e c a n t e d , a n d the c o m b i n e d s u p e r n a t a n t s w e r e
P e t r o l e u m p r o d u c t s in m a r i n e e n v i r o n m e n t s
261
c o n c e n t r a t e d a n d u s e d for I-IPLC injections. The m u s s e l s a m p l e s w e r e s e p a r a t e l y
e x t r a c t e d for G C a n d G C / M S a n a l y s e s as d e s c r i b e d above. Tissues w e r e also e x a m i n e d
b y e l e c t r o n m i c r o s c o p y for e v i d e n c e of a b n o r m a l i t i e s (Wolfe et al., u n p u b l i s h e d ) .
Sample analysis
High-performance
liquid
chromatography
(HPLC).
W e use
HPLC to a n a l y z e for n o n v o l a t i l e or t h e r m a l l y u n s t a b l e o x y g e n a t e d c o m p o u n d s w h i c h
c a n n o t b e d e t e r m i n e d b y G C (Krahn et al., 1980). T h e a n a l y t i c a l p r o c e d u r e s u s e d to
s e p a r a t e a n d i d e n t i f y t h e s e p o l a r a r o m a t i c c o m p o u n d s a r e s h o w n in P i g u r e 2. S e p a r a tions w e r e c a r r i e d out w i t h a d u a l - p u m p l i q u i d c h r o m a t o g r a p h e q u i p p e d w i t h a r e v e r s e
p h a s e (10 ~m C- 18 h y d r o c a r b o n c o a t e d p a c k i n g ) a n a l y t i c a l c o l u m n a n d a g u a r d column.
The d e t e c t o r w a s a d u a l - m o n o c h r o m a t o r f l u o r e s c e n c e s p e c t r o m e t e r fitted w i t h a 20-~1
s q u a r e flow cell.
G r a d i e n t elutions w e r e p e r f o r m e d w i t h 0.5 % v / v acetic a c i d in w a t e r (Solvent A)
a n d m e t h a n o l (Solvent B) in a 15-min l i n e a r g r a d i e n t from 5 to 95 % Solvent B, f o l l o w e d
b y 5 m i n at final conditions.
F l u o r e s c e n c e e m i s s i o n or e x c i t a t i o n s p e c t r a w e r e r e c o r d e d on the f l u o r e s c e n c e
s p e c t r o m e t e r after t h e effluent c o m p o n e n t s w e r e t r a p p e d in t h e d e t e c t o r flow cell.
M a s s s p e c t r o m e t r y. The c h e m i c a l i o n i z a t i o n i n s t r u m e n t w a s u s e d for p l a s m a
d e s o r p t i o n c h e m i c a l i o n i z a t i o n m a s s s p e c t r o m e t r y (PD/CI MS) (Krahn et al., 1980).
S p e c t r a of c o m m e r c i a l 1 - n a p h t h y l - ~ - D - g l u c u r o n i c a c i d ( n a p h t h y l g l u c u r o n i d e ) a n d
n a p h t h y l g l u c u r o n i d e from fish b i l e w e r e o b t a i n e d b y PD/CI MS. A 2 - m m o. d. • 2 - c m
l o n g glass t u b e s e a l e d at one e n d a n d f i l l e d w i t h 0.25-mm o. d. silver w i r e w a s i n s e r t e d
into the s o l i d - s a m p l e probe. This insert s e r v e d as the s u p p o r t for the c o m p o u n d to b e
a n a l y z e d a n d w a s p l a c e d d i r e c t l y into the CI p l a s m a (ionized ammonia). H e a t w a s
c o n d u c t e d b y the silver w i r e to the s a m p l e as the p r o b e t e m p e r a t u r e w a s r a i s e d from 50 ~
to 400 ~ in a b o u t 90 s. A m u l t i p l e - o n d e t e c t i o n c o m p u t e r p r o g r a m w a s u s e d to o b t a i n a
s p e c t r u m of 100 n g of n a p h t h y l g l u c u r o n i d e s t a n d a r d .
RESULTS A N D D I S C U S S I O N
In the last few years, s i g n i f i c a n t a d v a n c e s h a v e b e e n m a d e in our a b i l i t y to a n a l y z e
c o m p l e x m i x t u r e s of h y d r o c a r b o n s in s e a w a t e r , s e d i m e n t , a n d b i o l o g i c a l s a m p l e s (MacL e o d et al., 1976~ Brown et al., 1979, 1980). M u c h of t h e p r o g r e s s was m a d e t h r o u g h the
use of g l a s s - c a p i l l a r y gas c h r o m a t o g r a p h y (Ramos et al., 1979). U s i n g this t e c h n i q u e one
Table 1. Phenols extracted from seawater-soluble fraction of Prudhoe Bay crude oil and identified
by GC/MS (adapted from Ramos et al., 1979)
No. of isomers present
ComPounds
No. of isomers present
1
2
5
7
4
Phenol
Cresols
C2-Phenols
C3-Phenols
C4-Phenols
2
1
2
1
1
Compounds
Cs-Phenols
Methylphenetole
C2-Bromophenols
C3-Bromophenol
Cyclohexylphenol
262
D . C . h 4 a l i n s e t al.
Table 2. Aromatic p e t r o l e u m hydrocarbon concentrations in sediments experimentally oiled with
Prudhoe Bay crude oil. Samples were t a k e n from the top 2 cm of a 5-cm s e d i m e n t layer (adapted
from M c C a i n et al., 1980)
%
composition
at day 0
Hydrocarbons
1, 2, 3, 4 - T e t r a m e t h y l b e n z e n e
Naphthalene
1-Methylnaphthalene
2-Methylnaphthalene
2,6-Dimethylnaphthalene
2,3,5-Trimethylnaphthalene
Fluorene
Phenanthrene
4
8
16
29
14
15
3
11
Days
0
2
7
16
30
concentration (ng/g dry wt.)
105
210
408
753
375
379
87
275
74
200
736
742
185
338
75
155
50
61
400
601
411
318
67
150
64
89
362
723
344
336
78
156
61
82
557
519
218
187
102
112
%
remaining
on day 30
58
39
137
69
58
49
117
41
is a b l e to s e p a r a t e h u n d r e d s of i n d i v i d u a l c o m p o u n d s i n l e s s t h a n o n e h o u r . U s i n g o u r
s a m p l e - p r e p a r a t i o n p r o c e d u r e s , t h e a p p r o x i m a t e l e v e l s of s e n s i t i v i t y a r e 0.1 n g / g for
s e a w a t e r a n d d r y s e d i m e n t , a n d 4.0 n g / g for d r y t i s s u e . G C / M S is e m p l o y e d i n i n s t a n c e s
w h e r e e l u c i d a t i o n of s t r u c t u r e i s r e q u i r e d . A l t h o u g h t h e G C a n d G C / M S p r o c e d u r e s a r e
u s u a l l y a p p l i e d to t h e a n a l y s i s of p e t r o l e u m h y d r o c a r b o n s , a n d w e a k l y p o l a r c o m p o u n d s , s u c h as b e n z o t h i o p h e n e s , it is a l s o p o s s i b l e to r e s o l v e a n u m b e r of t h e m o r e
volatile oxygenated organics by these techniques. For example, we have analysed a
w i d e r a n g e of p h e n o l s p r e s e n t i n t h e w a t e r - s o l u b l e f r a c t i o n (V~rSF) of P r u d h o e B a y c r u d e
oil ( T a b l e 1) ( R a m o s e t al., 1979).
T h e f o l l o w i n g e x a m p l e s e r v e s to i l l u s t r a t e t h e u s e of g a s c h r o m a t o g r a p h i c a n a l y s e s
of h y d r o c a r b o n s i n l a b o r a t o r y s t u d i e s r e l a t i n g to t h e f a t e a n d b i o l o g i c a l e f f e c t s of
Table 3. Petroleum hydrocarbons in the tissues of English sole exposed to oil-contaminated
s e d i m e n t {test, T) a n d to n o n o i l e d sediment (control, C) for two months continuously (adapted from
M c C a i n et al., 1978)
Polycyclic
aromatic
hydrocarbons*
1,2,3,4-Tetramethylbenzene
Biphenyl
Naphtalene
1-Methylnaphthalene
2-Methylnaphthalene
2,6-Dimethylnaphthalene
Skin Muscle Liver Skin
11 days
C T C T C T C T
concentration
33 - 44 - 922
- 156 - - - 307
82 - 20 - 100
- 1189 - 369 - 1940
- 888 - 279 - 3070
- 130 - - 69
Muscle Liver Skin Muscle Liver
27 days
51 days
C T C T C T C T C T
(ng/g dry weight) * *
863
278
1325
1500
124
60
* Arenes with aromatic rings r a n g i n g from one to six were determined; (-) refers to values below
limits of detection
* * Skin a n d liver samples were pooled from three fish at each analysis; muscle samples were
analyzed individually
P e t r o l e u m p r o d u c t s in m a r i n e e n v i r o n m e n t s
263
.I
f22
f:ZO.
I
--
--
>'>
i
'II
Fig. 3. Gas chromatogram of the seawater-soluble fraction of Prudhoe Bay crude oil. HewlettPackard 5840A GC with FID. J & W Scientific 30 m • 0.25 mm SE-54 WCOT glass column. Helium
carrier pressure 20 psi. Three ~1 splitless injection, with split valve opened after 18 s to a 20 : I split.
Isothermal for first 5 min at 50 ~ then programmed to 270 ~ at 4 ~
p e t r o l e u m . S t u d i e s w e r e c o n d u c t e d on the b i o a v a i l a b i l i t y to E n g l i s h sole (Parophrys
vetulus) of p e t r o l e u m from e x p e r i m e n t a l l y - o i l e d s e d i m e n t s ( M c C a i n et al., 1978). G a s
c h r o m a t o g r a p h i c a n a l y s e s of s e d i m e n t h y d r o c a r b o n s a n d h y d r o c a r b o n s a c c u m u l a t e d in
skin, muscle, a n d liver r e v e a l e d that t h e s e tissues c o n c e n t r a t e d c o m p o u n d s such as 1a n d 2 - m e t h y l n a p h t h a l e n e a n d 1 , 2 , 3 , 4 - t e t r a m e t h y l b e n z e n e from the s e d i m e n t , b u t d i d
not a c c u m u l a t e h i g h e r m o l e c u l a r w e i g h t c o m p o u n d s , such as fluorene a n d p h e n a n t h r e n e (Tables 2, 3) ( M c C a i n et al., 1978). As e x p o s u r e p e r i o d s i n c r e a s e d b e y o n d a b o u t
two w e e k s , h y d r o c a r b o n c o n c e n t r a t i o n s in tissues d e c r e a s e d . T h e s e c h a n g e s w e r e
a t t r i b u t e d to the i n d u c t i o n of m i x e d function o x y g e n a s e e n z y m e s that p r o m o t e the
c o n v e r s i o n of h y d r o c a r b o n s to m e t a b o l i t e s . The c h e m i c a l a n a l y s e s w e r e of critical
i m p o r t a n c e in r e l a t i n g the e x p o s u r e s of p e t r o l e u m to m o r p h o l o g i c a l c h a n g e s (e. g.,
h e p a t o c e l l u l a r l i p i d vacuolization) in the E n g l i s h sole a n d in d e t e r m i n i n g w h i c h h y d r o c a r b o n s h a d e n t e r e d the organisms. W h e n M c C a i n et al. (1978) c o n d u c t e d t h e i r study,
m e t h o d s h a d not y e t b e e n d e v e l o p e d for a n a l y z i n g n o n r a d i o a c t i v e m e t a b o l i t e s , a l t h o u g h
t h e r e w e r e i n d i c a t i o n s from our b i o c h e m i c a l studies that t h e s e c o n v e r s i o n p r o d u c t s w e r e
r e a d i l y f o r m e d a n d a c c u m u l a t e d in tissues a n d b o d y fluids of fish e x p o s e d to h y d r o c a r bons (Roubal et al., 1977; C o l l i e r et al., 1978; M a l i n s et al., 1979; V a r a n a s i et al., 1979).
W h e n w e a n a l y z e d the W S F of P r u d h o e Bay crude oil, a n u m b e r of a l k y l a t e d , lowm o l e c u l a r w e i g h t a r o m a t i c h y d r o c a r b o n s w e r e found to b e m a j o r c o m p o n e n t s (Fig. 3}.
Both t h e s e l e c t i v e d e p o s i t i o n of c e r t a i n h y d r o c a r b o n s a n d e x t e n s i v e b i o c o n v e r s i o n m a y
lead, in effect, to a " s c r a m b l i n g " of the r e l a t i v e p r o p o r t i o n s of p e t r o l e u m h y d r o c a r b o n s in
fish e x p o s e d to t h e W S F (Roubal et al., 1978}. Thus, s o m e t i m e s it m a y b e difficult to
r e l a t e the h y d r o c a r b o n a c c u m u l a t i o n s in o r g a n i s m s to a specific t y p e of p e t r o l e u m in the
environment.
264
D . C . M a l i n s et al.
~22324
[ ARGO MERCHANT CARGO
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5
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19
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29
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31
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Fig. 4. Gas chromatograms of saturated hydrocarbons from a sample of Argo Merchant cargo (upper)
and saturated hydrocarbons extracted from the stomach contents of cod collected in the region of the
spilled Argo Merchant oil (lower). Numbers denote n-alkane carbon chain lengths.
20 m x 0.25 mm SE-30 WCOT glass column. Other conditions same as Figure 3
N o t w i t h s t a n d i n g the effects of s c r a m b l i n g , it is s o m e t i m e s p o s s i b l e to e s t a b l i s h
r e l a t i o n s h i p s b e t w e e n p e t r o l e u m c o n t a m i n a t i o n a n d a c c u m u l a t i o n s of h y d r o c a r b o n s in
organisms. It w a s possible, for e x a m p l e , to show a p r o b a b l e c a u s e a n d effect r e l a t i o n s h i p
between" s p i l l e d A r g o M e r c h a n t oil a n d h y d r o c a r b o n s p r e s e n t in the stomachs of cod
c a u g h t n e a r the i m p a c t zone (MacLeod et al., 1978). U s i n g h i g h r e s o l u t i o n g a s
c h r o m a t o g r a p h y , m a t c h i n g compositions w e r e o b t a i n e d b e t w e e n both the a l i p h a t i c a n d
a r o m a t i c h y d r o c a r b o n s c o m p r i s i n g the cargo oil a n d the c o r r e s p o n d i n g h y d r o c a r b o n s in
the s t o m a c h (Fig. 4). F i n g e r p r i n t i n g of this t y p e is useful in i d e n t i f y i n g c o n t a m i n a t i o n of
m a r i n e o r g a n i s m s in p e t r o l e u m - i m p a c t e d areas.
W e h a v e a p p l i e d s i m i l a r a p p r o a c h e s in s t u d y i n g the i m p a c t on s e d i m e n t s a n d
m u s s e l s of a s m a l l oil s p i l l ' i n Port A n g e l e s harbor, W a s h i n g t o n . The r e l a t i v e proportions
of a r o m a t i c structures w e r e not e x a c t l y the s a m e in s e d i m e n t a n d o r g a n i s m s (Fig. 5);
however, the h i g h r e s o l u t i o n c h r o m a t o g r a m s of the m u s s e l a n d s e d i m e n t extracts left
little d o u b t that t h e a c c u m u l a t e d h y d r o c a r b o n s arose from p e t r o l e u m s i m i l a r to that
p r e s e n t in the s e d i m e n t .
H y d r o c a r b o n a n a l y s e s of water, s e d i m e n t , a n d tissues are useful w a y s of a s s e s s i n g
the p o s s i b l e i m p a c t of p e t r o l e u m p o l l u t i o n on m a r i n e life. W h e r e p e t r o l e u m h y d r o c a r bons are d e f i n i t e l y found in organisms, p e t r o l e u m from t h e e n v i r o n m e n t is i m p l i c a t e d ;
however, the a b s e n c e of p e t r o l e u m h y d r o c a r b o n s in tissues does not n e c e s s a r i l y prec l u d e t h e p o s s i b i l i t y that p o l a r c o m p o u n d s (e. g., o x y g e n a t e d products) from p e t r o l e u m
P e t r o l e u m p r o d u c t s in m a r i n e e n v i r o n m e n t s
c
1
!
265
Z~
.r
c~
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o_
~
o o ~
rn rv~ o.
Fig. 5. Gas chromatograms of the aromatic hydrocarbons from sediment (upper} and mussels (lower)
collected in the vicinity of a small oil spill in the harbor at Port Angeles, Washington, 1979.
Parameters and conditions as in Figure 3
m a y b e p r e s e n t in s i g n i f i c a n t concentrations. C e r t a i n of t h e o x y g e n a t e d c o m p o u n d s h a v e
b e e n i m p l i c a t e d as toxicants, some as m u t a g e n s a n d c a r c i n o g e n s (Sims & Grover, 1974).
E x a m p l e s are the p r o d u c t s of the b i o c o n v e r s j o n of b e n z o [ a ] p y r e n e a n d b e n z [ a ] a n t h r a c e n e . L a b o r a t o r y s t u d i e s h a v e s h o w n that a r o m a t i c h y d r o c a r b o n s r e a d i l y u n d e r g o
e x t e n s i v e c o n v e r s i o n in fish tissues (Varanasi & Malins, 1977), so it is r e a s o n a b l e to
c o n c l u d e that p o t e n t i a l l y d a m a g i n g m e t a b o l i t e s are f o r m e d in fish after e n v i r o n m e n t a l
e x p o s u r e to p e t r o l e u m products.
The t e n d e n c y for a r o m a t i c h y d r o c a r b o n s to oxidize a n d form p o l a r a r o m a t i c comp o u n d s in m a r i n e systems is of concern. H o w e v e r , b e c a u s e of t h e l i m i t a t i o n s of c o n v e n t i o n a l g a s c h r o m a t o g r a p h y in a n a l y z i n g most of t h e s e products, a l t e r n a t i v e s m u s t b e
e x p l o r e d . Efforts to w i d e n our p e r s p e c t i v e s of p e t r o l e u m p o l l u t i o n to e n c o m p a s s the
a n a l y s i s of the e s s e n t i a l l y n o n - v o l a t i l e p o l a r c o m p o u n d s h a v e r e s u l t e d in a t w o - p h a s e
a p p r o a c h to t h e p r o b l e m : (1} a s t u d y of w a y s to a n a l y z e m e t a b o l i c p r o d u c t s of s i n g l e
a r o m a t i c h y d r o c a r b o n s b y I-IPLC in c o n j u n c t i o n w i t h UVF, UV, or UV-UVF t e c h n i q u e s ,
a n d (2) a study of w a y s to a n a l y z e total a r o m a t i c p o l a r c o m p o u n d s in field s a m p l e s b y
HPLC in c o m b i n a t i o n w i t h UVF d e t e c t o r s o p e r a t i n g at w a v e l e n g t h s c h a r a c t e r i s t i c of
representative petroleum derived components.
In the first a p p r o a c h , K r a h n et al. (1980) r e p o r t a H P L C / U V F m e t h o d of a n a l y z i n g
fish a n d m o u s e b i l e s a m p l e s for n a n o g r a m a m o u n t s of l o w - f l u o r e s c e n t n a p h t h a l e n e a n d
its m e t a b o l i t e s . In t h e current study, t w o g r o u p s of r a i n b o w trout w e r e force-fed
266
D . C . M a l i n s et al.
NOH
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Ill
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N
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E
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hp t4
IV 9
,
".A._J ~
__.1
I
i
,
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I
0
I
5
10
li5
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20
Retention time (minutes)
Retention time (minutes)
Fig. 6. HPLC profiles of naphthalene and its
metabolites in the bile of rainbow trout. Upper:
Chromatogram of bile from fish which received
2.7 mg of naphthalene (11 mg/kg} in each of 3
force-feedings 12 h apart. Bile was collected
24 h after the last feeding. Lower: Chromatogram of the control bile. Compounds were detected at UVF wavelengths ~ex 305 nm and ~m
340 nm. Identification of the compounds: N,
naphthalene; NOH, 1-naphthol; NG, 1naphthyl-fl-D-ghicuronic
acid
(naphthyl
ghicuronide)~ NSO4, 1-naphthyl sulfate; and U,
unknown
Fig. 7. HPLC profiles of naphthalene and its
metabolifes in the bile of rainbow trout. Upper:
Chromatogram of bile from fish which received
2.7 mg (11 mg/kg) in one force-feeding. Bile
was collected 24 h later. Lower: Chromatogram
of the control bile. Compounds were detected at
UVF wavelengths Aex 305 nm and ~em 340 nm.
Identification of the compounds: N, naphthalene; NOH, 1-naphthol; NG, 1-naphthyl-fl-Dglucuronic acid (naphthyl glucuronide); NSO4,
1-nyphthyl sulfate; and U, unknown
n a p h t h a l e n e (11 mg/kg) i n our laboratories. O n e group received the dose i n each of
three feedings, a n d the other i n one feeding. The HPLC/UVH c h r o m a t o g r a m r e s u l t i n g
from the direct i n j e c t i o n of bile from fish fed n a p h t h a l e n e three times is s h o w n i n
Figure 6 a n d that from fish fed n a p h t h a l e n e once is s h o w n i n Figure 7. In each
c h r o m a t o g r a m the p a r e n t compound, n a p h t h a l e n e (N), a n d two metabolites, 1-napht h y l g l u c u r o n i d e (NG} a n d 1-naphthol (NOH), were detected. In addition, 1-naphthyl
sulfate (NSO4) was detected i n the c h r o m a t o g r a m from the m u l t i p l e - f e e d i n g study.
U n k n o w n polar aromatic c o m p o u n d s (U) were found i n both chromatograms. In the
m u l t i p l e - f e e d i n g study, the m a i n m e t a b o l i t e was 1-naphthyl g l u c u r o n i d e ; however, 1n a p h t h o l was the m a i n m e t a b o l i t e i n the s i n g l e - f e e d i n g experiment.
P e t r o l e u m p r o d u c t s in m a r i n e e n v i r o n m e n t s
267
c
o
u_
300
[
l
350
400
Wavelength (nm)
Fig. 8. Emission spectrum of naphthyl glucuronide (UVF Zex 294 nm). U p p e r spectrum: Naphthyl
glucuronide obtained from bile of rainbow trout force-fed n a p h t h a l e n e (see Fig. 6). Lower spectrum:
The commercially available standard of the sodium salt of naphthyl glucuronide
100
338
90
80
7O
8
~=
60
~
50
~
40
144
30
20
10
0
9 ' '
' ' = ' ' '
1O0
'
: ' ' ' ' ' ' 1
150
' ' ' ~
200
"-
;
I
250
"
" '
i
300
350
m/e
Fig. 9. Plasma desorption/chemical ionization mass spectrum of l-naphthyl-fl-D-glucuronic acid
using ammonia r e a g e n t gas at 0.9 tort. The probe was h e a t e d from 50 ~ to 400 ~ in ca. 90 s. A
multiple-ion-detection computer program was u s e d to emphasize the 144, 194, and 338 m / e ions
268
D . C . M a l i n s et al.
Structural d e t e r m i n a t i o n s of metabolites were reported b y K r a h n et al. (1980).
Structural information was o b t a i n e d by a stop-flow HPLC t e c h n i q u e l e a d i n g to the
fluorescence e m i s s i o n spectrum of 1-naphthyl g l u c u r o n i d e {Fig. 8). C o n f i r m a t i o n of the
n a p h t h y l g l u c u r o n i d e structure was o b t a i n e d b y PD/CI MS; a spectrum of the commercial s t a n d a r d is s h o w n i n Figure 9.
These t e c h n i q u e s for the analysis of o x y g e n a t e d c o m p o u n d s i n biological tissues
a n d fluids u s i n g HPLC/UVF a n d MS should have significant impact i n several areas of
research. For instance, the metaboli.sm a n d disposition of p e t r o l e u m hydrocarbons i n
biological systems can be s t u d i e d i n the laboratory without the use of isotopicallyl a b e l e d compounds. In addition, organisms s u b j e c t e d to xenobiotic c o n t a m i n a t i o n of
their n a t u r a l e n v i r o n m e n t can b e investigated. We are p r e s e n t l y c o n d u c t i n g studies of
\
>.
c
c
u_
\
j
L
I
I
I
~
0
10
20
30
0
Time (minutes)
I0
1
I
J
20
30
Time (minutes)
Fig. 10. HPLC profiles of methanol extracts of mussel samples~: Upper - directly in the path of the
"Amoco Cadiz" spilled oil; middle - out of the direct path of the "Amoco Cadiz" spilled oil; and
lower - mussels before exposure to "Amoco Cadiz" oil. The profiles on the left w e r e detected at "~ex
270 nm and ~em305 nm (characteristic of phenol); the profiles on the right side were detected at ,~ex
305 nm and ~ e m 340 nm (characteristic of naphthalene metabolites)
Petroleum products in marine environments
269
I
O
[h
O
Iii
I~r
d
I
o
i
i
25
50
Time (minutes)
Fig. i l . G C / M S total i o n c h r o m a t o g r a m of aromatic h y d r o c a r b o n fraction f r o m mussels: u p p e r -
directly in path of the "Amoco Cadiz" spilled oil; lower - out of direct path of the "Amoco Cadiz"
spilled oil. GC conditions same as Fig. 3, except that the capillary GC effluent was admitted to a
Finnigan 200 mass spectrometer. Intensities of both chromatograms adjusted to give same peak
height above base line for internal standard (I. STD.). Alkylated dibenzothiophenes (Cn-DBTs)
denoted according to carbon substitution, where n = number of carbons
this type. For e x a m p l e , a n a n a l y s i s for p o l a r a r o m a t i c c o m p o u n d s in field s a m p l e s is
p r e s e n t e d : W a v e l e n g t h s c h a r a c t e r i s t i c of n a p h t h a l e n e m e t a b o l i t e s a n d p h e n o l s (Schab r o n et al., 1979; K r a h n et al., 1980) w e r e u s e d to a n a l y z e s a m p l e s from a s t u d y of m u s s e l s
p l a c e d in c a g e s in t h e p a t h of t h e " A m o c o C a d i z " oil s p i l l off t h e Brittany coast.
Differences in H P L C / U V F profiles (Fig. 10) w e r e f o u n d in e x t r a c t s of m u s s e l s (1)
d e p l o y e d 1 m b e l o w the w a t e r surface d i r e c t l y in the p a t h of the spill, (2) from mussels.
s i m i l a r l y d e p l o y e d , b u t a w a y from the p a t h of the spill, a n d (3) from extracts of the
o r i g i n a l b a t c h of m u s s e l s before d e p l o y m e n t . Differences in H P L C / U V F r e s p o n s e s
correlate w e l l w i t h the p r o x i m i t y of the m u s s e l s to the p o l l u t i o n source. G a s chromatog r a p h i c a n a l y s e s of the w e a k l y p o l a r a r o m a t i c c o m p o u n d s also d i s t i n g u i s h e d b e t w e e n
m u s s e l s from i m p a c t sites a n d r e f e r e n c e sites, p a r t i c u l a r l y w i t h r e s p e c t to the d i b e n z o t h i o p h e n e s (Fig. 11). Thus, two t y p e s of a n a l y t i c a l p r o c e d u r e s , in a c o m p l e m e n t a r y
way, d e m o n s t r a t e d the a p p a r e n t a c c u m u l a t i o n of " A m o c o C a d i z " p e t r o l e u m p r o d u c t s in
m u s s e l s from the i m p a c t zones. Wolfe et al. ( u n p u b l i s h e d ) h a v e s h o w n that c e l l u l a r
c h a n g e s (e. g., i n c r e a s e s in l y s o s o m a l granules) o c c u r r e d in m u s s e l s from i m p a c t sites in
r e l a t i o n to m u s s e l s from r e f e r e n c e areas. A c c o r d i n g l y , t h e c h e m i c a l d a t a a n d t h e
270
D . C . M a l i n s et al.
o b s e r v e d h i s t o l o g i c a l a l t e r a t i o n s a p p e a r to b e a s s o c i a t e d w i t h e x p o s u r e to "Amoco
C a d i z " off.
To s u m m a r i z e , l a r g e q u a n t i t i e s of p e t r o l e u m - b a s e d p o l l u t a n t s a r e e n t e r i n g t h e
m a r i n e e n v i r o n m e n t e a c h year, c r e a t i n g a n e e d for a c c u r a t e a n a l y t i c a l d e t e r m i n a t i o n s of
t h e s e c o m p o u n d s in w a t e r , s e d i m e n t , tissues, a n d o t h e r s a m p l e s . P r e s e n t l y , m a n y of t h e
p e t r o l e u m h y d r o c a r b o n s a r e r o u t i n e l y d e t e r m i n e d b y G C or G C / M S . H o w e v e r , a v a s t
a r r a y of p o l a r p e t r o l e u m p r o d u c t s - m a n y of w h i c h a r e k n o w n to b e g e n e r a t e d b y
chemical and biological processes - have not previously been determined. Our methods
for a n a l y s i n g t h e s e p o l a r a r o m a t i c c o m p o u n d s s h o w p r o m i s e . M e t a b o l i t e s for a r o m a t i c
h y d r o c a r b o n s c a n n o w b e d e t e r m i n e d in c o n n e c t i o n w i t h l a b o r a t o r y e x p o s u r e studies. In
addition, research on the polar compounds can be conducted on field samples taken from
p o l l u t e d areas. W e h o p e to e x p a n d o u r a b i l i t y to d e t e c t a n d q u a n t i t a t e i n d i v i d u a l p o l a r
p e t r o l e u m d e r i v e d c o m p o u n d s i n c o m p l e x m i x t u r e s f r o m b o t h t h e l a b o r a t o r y a n d field.
Acknowledgment. This study was supported in part by the Bureau of Land Management through
interagency agreement with the National Oceanic and Atmospheric Administration, which is
managed by the Outer Continental Shelf Environmental Assessment Program (OCSEAP) Office.
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petroleum hydrocarbons in marine sediments using a solvent/sluny extraction procedure. In:
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Brown, D. W., Ramos, L. S., Uyeda, M. Y., Friedman, A. J. & MacLeod, W. D., Jr., 1980. Ambienttemperature extraction of hydrocarbons from marine sediments-comparison with boiling-solvent extractions. (Advances in Chemistry Series, "Petroleum in the Marine Environment".)
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Clark, R. C., Jr. & Brown, D. W., 1977. Petroleum properties and analyses in biotic and abiotic
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marine environments and organisms. Ed. by D. C. Malins. Acad. Press, New York I, 91-223.
Collier, T. K., Thomas, L. & Malins, D. C., 1978. Influence of environmental temperature on
disposition of dietary naphthalene in coho salmon (Oncorhy-nchus kisutch}: Isolation and
identification of individual metabolites. - Comp. Biochem. Physiol. 61C, 23-28.
Karrick, N. L., 1977. Alterations in petroleum resulting from physico-chemical and microbiological
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Krahn, M. M., Brown, D. W., Collier, T. K., Friedman, A. J., Jenkins, R. G. & Malins, D. C., 1980.
Rapid analysis of naphthalene and its metabolites in biological systems: Determination by highperformance liqnid chromatography/fluorescence detection and by plasma desorption/chemical
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