HELGOL~NDER M:EERESUNTERSUCHUNGEN
Helgol~inder Meeresunters, 33,272-277 (1980)
Determination and localization of oil components in
living benthic organisms by fluorescence microscopy
E. Zeeck
Universitfit Oldenburg, Projekt Umweltentwicklung und -planung im kfistennahen
Gebiet; D-2900 Oldenburg, Federal Republic of Germany
ABSTRACT: Microspectrofluorometric measurements are made to determine uptake and distribution of oil in marine organisms after exposure to crude oil. Equipment combining fluorescence
microscopy with spectral analysis of the fluorescence emission is described. After contamination
with oil, the intestine content of Lumbricillus lineatus, Nereis diversicolor and Anaitides mucosa
shows a fluorescence emission at long wavelengths with a maximum at about 550 nm; this is in
contrast to the fluorescence emission of these organisms without oil contamination. There is
evidence that aromatic hydrocarbons are metabolized in the intestine of the worms-studied.
INTRODUCTION
F l u o r e s c e n c e spectroscopy is k n o w n as a very sensitive tool for the d e t e r m i n a t i o n of
m a n y substances. W h e n restricted to i n v e s t i g a t i o n s w i t h i n the visible or the n e a r UV
r e g i o n of the spectrum, most aromatic hydrocarbons show fluorescence e m i s s i o n of h i g h
intensity. These s u b s t a n c e s are c o m p o n e n t s of crude oil. Fluorescence spectroscopy
therefore has b e e n proposed for i d e n t i f y i n g traces of oil i n sea w a t e r (e.g. G o r d o n &
Keizer, 1974).
At least as i n t e r e s t i n g as the proof of oil i n sea water is the identification of oil i n the
tissue of organisms. Some a d v a n t a g e s a n d results as well as difficulties i n the use of
fluorescence spectroscopy for the d e t e r m i n a t i o n of oil i n the tissue of b e n t h i c organisms
will b e o u t l i n e d here.
METHOD
Since there are powerful a n a l y t i c a l methods (e.g. gas chromatography i n c o n j u n c tion with mass spectrometry} for the analysis of s a m p l e s c o n t a i n i n g traces of hydrocarb o n s a n d their metabolites, the q u e s t i o n arises w h e t h e r there is a n e e d for such a m e t h o d
as fluorescence spectroscopy w h i c h gives m u c h less d e t a i l e d information with respect to
the identification of single chemical c o m p o u n d s w i t h i n complex mixtures.
Optical spectroscopy c o m b i n e d with microscopy offers the following a d v a n t a g e s to the biologist (see also K o h e n et al., 1978): (a} It e n a b l e s the biologist to inspect
small organisms. It is impossible for gas c h r o m a t o g r a p h y to a n a l y z e single organisms
from m e i o f a u n a or microfauna. U s i n g fluorescence microscopy it is possible to d e t e r m i n e
the distribution of several toxic s u b s t a n c e s i n different b o d y parts of these organisms.
9 Biologische Anstalt Helgoland
0017-9957/80/0033/0272/$ 02.00
D e t e r m i n a t i o n a n d l o c a l i z a t i o n of oil c o m p o n e n t s
273
T h e s m a l l e s t a r e a for m e a s u r e m e n t w i t h the e q u i p m e n t u s e d in the i n v e s t i g a t i o n s
p r e s e n t e d h e r e has a d i a m e t e r of only 0 . 5 / a n . (b) It is p o s s i b l e to a n a l y z e the c o n t a m i n a tion of l i v i n g o r g a n i s m s w i t h this m e t h o d . After d e a t h the d i s t r i b u t i o n of toxic s u b s t a n c e s
w i t h i n the o r g a n i s m m a y c h a n g e .
T h e m a i n r e s t r i c t i o n s of t h e m e t h o d are: (a) T h e c o m b i n a t i o n of f l u o r e s c e n c e
s p e c t r o s c o p y a n d m i c r o s c o p y is r e s t r i c t e d to the d e t e r m i n a t i o n of s u b s t a n c e s w h i c h g i v e
f l u o r e s c e n c e e m i s s i o n in the v i s i b l e or n e a r UV r e g i o n of the spectrum. T h e r e f o r e only a
r a t h e r l i m i t e d - n e v e r t h e l e s s i m p o r t a n t - g r o u p of c o m p o n e n t s , e.g. of crude oil, c a n b e
d e t e c t e d . (b) Tissue itself shows fluorescence. The shorter the w a v e l e n g t h of the
e x c i t a t i o n light, t h e m o r e the tissue itself emits f l u o r e s c e n c e light. It is difficult to d e c i d e
b e t w e e n e m i s s i o n of tissue s u b s t a n c e s a n d oil c o m p o n e n t s at w a v e l e n g t h s shorter t h a n
450 n m (Hodgins et al., 1977).
E v e n at l o n g e r w a v e l e n g t h s it is in most cases n e c e s s a r y to p e r f o r m s p e c t r a l a n a l y s i s
of t h e e m i t t e d light to d i s t i n g u i s h b e t w e e n f l u o r e s c e n c e of oil a n d the tissue itself.
S o m e t i m e s direct i n s p e c t i o n of t h e f l u o r e s c e n c e p i c t u r e m a y b e u s e d to d e t e r m i n e w h i c h
p a r t of the tissue contains oil a n d w h i c h p a r t does not, b u t t h e r e a r e m a n y situations
w h e r e this is not p o s s i b l e w i t h o u t s p e c t r a l analysis. In the f o l l o w i n g s u i t a b l e e q u i p m e n t
is d e s c r i b e d .
EQUIPMENT
F i g u r e 1 g i v e s a s c h e m a t i c r e p r e s e n t a t i o n of 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 unit. A
Zeiss f l u o r e s c e n c e m i c r o s c o p e w i t h N e o f l u a r optics is fitted w i t h filter c o m b i n a t i o n s for
the e p i f l u o r e s c e n c e m e t h o d , e.g. t h e filter c o m b i n a t i o n H 365 w i t h f l u o r e s c e n c e excitation at 365 n m a n d o b s e r v a t i o n of f l u o r e s c e n c e e m i s s i o n at w a v e l e f i g t h s l o n g e r t h a n
397 nm. The l i g h t source is a s t a b i l i z e d h i g h p r e s s u r e m e r c u r y (or xenon) lamp. A
p h o t o m e t e r 03 serves for s e l e c t i n g the m e a s u r e m e n t a r e a b y a s u i t a b l e d i a p h r a g m . It is
f o l l o w e d b y a z o o m lens (Zeiss No. 474 340 a n d 477 156) for o p t i c a l fitting of the
m o n o c h r o m a t o r a n d t h e p h o t o m u l t i p l i e r , the m o n o c h r o m a t o r (Jobin Yvon g r a t i n g m o n o c h r o m a t o r H 10) a n d the p h o t o m u l t i p l i e r . T h e l a t t e r s h o u l d s h o w only little d e p e n d e n c e
of s e n s i t i v i t y on w a v e l e n g t h in t h e r e g i o n studied, as e.g. the t y p e RCA C 31 034; it is
f o l l o w e d b y a p r e a m p l i f i e r (Ortec 9301) a n d a n a m p l i f i e r (an Ortec B r o o k d e a l p h o t o n
c o u n t e r 501/5014 is u s e d h e r e b u t for most p u r p o s e s it is not n e c e s s a r y to u s e p h o t o n
c o u n t i n g techniques). A strip c h a r t r e c o r d e r m a y b e the e n d of t h e chain.
As t h e s p e c t r u m o b t a i n e d b y this c o m b i n a t i o n of i n s t r u m e n t s is a s u p e r p o s i t i o n of
the f l u o r e s c e n c e s p e c t r u m on t h e one h a n d a n d t h e s p e c t r u m of the optics of the
i n s t r u m e n t a n d of the p h o t o m u l t i p l i e r on the other, it is h e l p f u l to use a c o m p u t e r for
u n f o l d i n g t h e s e c o m p o n e n t s . To do this, the d a t a are t r a n s f e r r e d b y a m i c r o p r o c e s s o r a n d
a t a p e r e c o r d e r to a m a g n e t i c tape.
RESULTS A N D D I S C U S S I O N
Initial results w e r e o b t a i n e d b y s t u d y i n g s e l e c t e d o r g a n i s m s of the t i d a l flat of the
G e r m a n N o r t h S e a coast: A n a i t i d e s m u c o s a , N e r e i s d i v e r s i c o l o r a n d L u m b r i c i l l u s 11neatus. T h e s e w o r m s w e r e e x p o s e d for five d a y s to o i l - i n - w a t e r d i s p e r s i o n s (0.5 %0 A r a b i a n
l i g h t oil in s e a water). E x p e r i m e n t s w i t h e x p o s u r e to different c o n c e n t r a t i o n s of the
w a t e r - s o l u b l e fraction of crude oil are b e i n g c o n t i n u e d a n d w i l l b e r e p o r t e d e l s e w h e r e .
2?4
E. Z e e c k
computer
11
osci,,oscope
II
t
tope recorder
recorder
II
amplifier
photomu[tiplie r
monochromator
photo met or
fluorescence
microscope
Fig. 1. Schematic description of the fluorescence spectrometer
\
bm]
600
sb0
\
~60
Fig. 2. Fluorescence spectra of the intestine content of ~ a i t i d e s mucosa; solid line: contamination
with petrol~ b r o k e n line: u n c o n t a m i n a t e d
Determination and localization of oil components
275
Figure 2 shows spectra of the intestine content of A n a i t i d e s m u c o s a with and without
exposure to oil. Spectra of analogous regions of N e r e i s diversicolor and L u m b r i c i l l u s
l i n e a t u s are similar.
The spectra in Figure 2 are superpositions of the fluorescence spectra and of the
spectrum of the instrument, as m e n t i o n e d above. The maximum at 490 nm is given by a
high translucence of the microscope optics together with a maximum of sensitivity of the
photomultiplier.
The most interesting differences b e t w e e n the spectra are located in the region
b e t w e e n 500 and 600 nm. The maximum at about 550 nm only in the spectrum of the oilexposed organisms indicates accumulation or generation of substances with long wavelength fluorescence in the intestine. The fluorescence spectrum of crude oil itself shows
a maximum at a much shorter wavelength.
Differences in the spectra in the region above 650 nm are of no interest in this
context. They are caused by different contents of chlorophyll or chlorophyll metabolites
in the intestine from the food of the worms (diatoms) and show no reproducible
d e p e n d e n c e on oil contamination.
Using gas chromatographic analysis, van Beruem (personal communication} found
that after exposure to oil larger organisms (Arenicola marina] accumulated perylene,
benzo-b-fluoranthene, benzo-c-phenanthrene, benzo-b,c-fluorene, pyrene, fluoranthene
and p h e n a n t h r e n e s in addition to benzo-a-pyrene, benzo-e-pyrene, methylchrysene and
chrysene; the latter substances were also found (at much lower concentrations) in the
uncontaminated organisms. Many other authors, too, report organismic u p t a k e of polynuclear aromatic hydrocarbons from petrol (e.g. Anderson, 1977; Varanasi & Malins,
1977).
But the maximum of fluorescence emission at about 550 nm cannot be e x p l a i n e d by
accumulation of aromatic hydrocarbons. There must be polar substances, too, probably
by metabolizing the aromatic hydrocarbons. This metabolic process seems to occur
within the intestine of these organisms. The intestine content of Lumbricillus l i n e a t u s
showed drops of different colour under the fluorescence microscope (excitation at
365 nm), the colour ranging from blue over light and dark yellow to brown. Spectroscopically the emission of these drops differed by the relative height of the maximum at
550 nm, the brownish drops showing the highest relative intensity at this point, indicating the highest content of metabolized compounds.
The assumption that this maximum at 550 nm is caused by metabolized compounds
of crude oil is b a s e d on the following facts: Crude oil itself shows relatively low intensity
of fluorescence emission at wavelengths longer than 500 nm (fluorescence excitation at
365 nm). A fluorescence emission at long wavelengths is due to compounds with large
pi-electron systems as polycyclic aromatic hydrocarbons. Examples for fluorescence
emission of these hydrocarbons are: chrysene 387 nm, pyrene 397 nm, p h e n a n t h r e n e
367 nm, b e n z o - a - p y r e n e 405 nm, perylene 465 nm, fluoranthene 477 nm, tetracene
511 nm (average w a v e l e n g t h of the fluorescence spectrum, according to Berlman, 1971).
Obviously, it is not possible to construct a maximum of fluorescence emission at
550 nm with these substances. But the introduction o f the amino or hydroxyl group
causes bathochromic shifts in the spectra of the aromatic hydrocarbons which can be
e n l a r g e d by additional groups bound to the same ring system as for example the
carbonyl group.
276
E. Z e e c k
As e x a m p l e s for t h e s e effects d a t a m a y b e g i v e n for b e n z e n e a n d some b e n z e n e
d e r i v a t i v e s (Berlman, 1971): b e n z e n e 282 nm, p h e n o l (in m e t h a n o l ) 300 nm, 1,4-dimet h o x y - b e n z e n e 324 nm, 2 , 3 - d i h y d r o x y b e n z o i c a c i d 431 nm, a n i l i n e (in ethanol) 341 nm,
1 , 3 - d i a r n i n o b e n z e n e 439 nm.
For p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s , the situation is of course m o r e c o m p l i c a t e d
b u t the t e n d e n c y is the same. Thus the w a v e l e n g t h of the fluorescence e m i s s i o n of
f l u o r a n t h e n e (477 nm) is shifted b y the i n t r o d u c t i o n of a n a m i n o g r o u p to 552 nm (3a m i n o f l u o r a n t h e n e in ethanol). T h e r e will b e no difficulty constructing a m a x i m u m of
fluorescence e m i s s i o n at a b o u t 550 n m b y a c o m b i n a t i o n of h y d r o c a r b o n d e r i v a t i v e s
c o n t a i n i n g h y d r o x y or a m i n o or o t h e r p o l a r groups. At present, t h e r e are not e n o u g h
s p e c t r a l d a t a a v a i l a b l e to p r o d u c e a c o m p l e t e c a t a l o g u e of all c o m p o u n d s w h i c h c o u l d
contribute to such f l u o r e s c e n c e emission, a n d m o r e o v e r a d d i t i o n a l d a t a from other
a n a l y t i c a l m e t h o d s s h o u l d b e u s e d to select the r e a l l y i m p o r t a n t compounds.
H o w e v e r , it is p o s s i b l e to state that not the h y d r o c a r b o n s t h e m s e l v e s b u t t h e i r
d e r i v a t i v e s w i t h p o l a r substituents give rise to the f l u o r e s c e n c e e m i s s i o n at 550 n m
w h i c h w a s o b s e r v e d in t h e i n t e s t i n e of o i l - c o n t a m i n a t e d organisms.
In p r i n c i p l e it is p o s s i b l e that s u b s t a n c e s w i t h t h e s e spectral p r o p e r t i e s w h i c h are
p r e s e n t in crude oil in s m a l l concentrations a r e a c c u m u l a t e d b y the organisms, b u t it is
not clear b y w h a t m e c h a n i s m this a c c u m u l a t i o n occurs in the intestine. T h e fact that the
p o l a r d e r i v a t i v e s are m o r e s o l u b l e in w a t e r t h a n the p u r e h y d r o c a r b o n s can b e e x c l u d e d
for such a n a c c u m u l a t i o n process, as the f l u o r e s c e n c e w a s not e m i t t e d b y a h o m o g e n e o u s
content of the i n t e s t i n e of Lumbricillus lineatus b u t b y s i n g l e drops, different drops
s h o w i n g differr
r e l a t i v e i n t e n s i t i e s of the fluorescence m a x i m u m at 550 nm, as
m e n t i o n e d above.
On the other h a n d it is w e l l k n o w n that m e t a b o l i s m of p o l y c y c l i c a r o m a t i c h y d r o c a r bons in o r g a n i s m s l e a d s to d e r i v a t i v e s of t h e t y p e d i s c u s s e d here. H y d r o x y l g r o u p s are
i n t r o d u c e d into the h y d r o c a r b o n s v i a the formation of e p o x y d e r i v a t i v e s (Sims & Grover,
1974; Malins, 1977; J e r i n a et al., 1979; Sch~fer-Ridder, 1979). This w a s not only o b s e r v e d
in m a m m a l i a n tissue b u t also in i n v e r t e b r a t e s (Varanasi & Malins, 1977; A n d e r s o n , 1978;
Ernst, 1979). A n d e r s o n r e p o r t e d the m e t a b o l i s m of b e n z o - a - p y r e n e b y aryl h y d r o c a r b o n
h y d r o x y l a s e s in Spodoptera, in Mytilus edulis a n d in Geukensia demissa; Ernst s t u d i e d
the m e t a b o l i c transformation of 1-(1-14C)naphthol in Mytilus edulis a n d f o u n d m e t a b o l i tes of h i g h polarity. W h e r e a s m a m m a l s initiate the o x i d a t i o n of a r o m a t i c h y d r o c a r b o n s
b y the i n c o r p o r a t i o n of one a t o m of m o l e c u l a r o x y g e n to form a r e n e o x i d e s a n d further
m e t a b o l i z e t h e s e o x i d e s to t r a n s - d i h y d r o d i o l s , b a c t e r i a i n c o r p o r a t e b o t h atoms of m o l e c u l a r o x y g e n a n d c i s - d i h y d r o d i o l s are formed. F u r t h e r o x i d a t i o n l e a d s to the formation of
catechols (Gibson, 1976).
It therefore s e e m s l i k e l y that the fluorescence e m i s s i o n o b s e r v e d in the intestine of
the o i l - c o n t a m i n a t e d s p e c i e s m e n t i o n e d a b o v e is d u e to s u b s t a n c e s f o r m e d b y m e t a b o l i s m of oil components. Still o p e n is the q u e s t i o n w h e t h e r this m e t a b o l i s m is c a u s e d b y
e n z y m e s of the worms t h e m s e l v e s or b y o i l - d e g r a d i n g bacteria. T h e s e b a c t e r i a w i l l b e
p r e s e n t in the o i l - i n - w a t e r d i s p e r s i o n s a n d m a y h a v e e n t e r e d the i n t e s t i n e of the worms
t o g e t h e r w i t h oil drops. I n v e s t i g a t i o n s are c o n t i n u i n g in this field.
Acknowledgements. Some of the studies described in this manuscript were carried out in collaboration with C. Matuschek. I am indebted to M. Gewers and J. Krollpfeiffer for technical assistance, to
D e t e r m i n a t i o n a n d l o c a l i z a t i o n of oil c o m p o n e n t s
277
Dr. L. Trapp, Carl Zeiss Comp., for technical information a n d to Dr. O. Giere for m a k i n g available to
me the species Lumbricillus lineatus after exposure to crude oil. The studies were supported by the
U m w e l t b u n d e s a m t , Berlin.
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