Determination and localization of oil components in living benthic organisms by fluorescence microscopy

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.

Z e e c k

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 distribu- tion 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.

I N T R O D U C T I O N

F l u o r e s c e n c e spectroscopy is k n o w n as a v e r y s e n s i t i v e tool for the d e t e r m i n a t i o n of m a n y s u b s t a n c e s . 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 v i s i b l e or the n e a r UV r e g i o n of the spectrum, most a r o m a t i c h y d r o c a r b o n s show f l u o r e s c e n c e e m i s s i o n of h i g h i n t e n s i t y . T h e s e s u b s t a n c e s are c o m p o n e n t s of c r u d e oil. F l u o r e s c e n c e spectroscopy therefore h a s b e e n p r o p o s e d 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 s e a w a t e r is the i d e n t i f i c a t i o n of oil i n the tissue of o r g a n i s m s . Some a d v a n t a g e s a n d results as w e l l as difficulties i n the u s e of f l u o r e s c e n c e spectroscopy for t h e d e t e r m i n a t i o n of oil i n t h e tissue of b e n t h i c o r g a n i s m s w i l l b e o u t l i n e d here.

M E T H O D

Since there are p o w e r f u l a n a l y t i c a l m e t h o d s (e.g. gas c h r o m a t o g r a p h y i n c o n j u n c - tion w i t h mass spectrometry} for the a n a l y s i s of s a m p l e s c o n t a i n i n g traces of hydrocar- b o n s a n d their m e t a b o l i t e s , the q u e s t i o n arises w h e t h e r t h e r e is a n e e d for s u c h a m e t h o d as f l u o r e s c e n c e spectroscopy w h i c h gives m u c h less d e t a i l e d i n f o r m a t i o n w i t h r e s p e c t to the i d e n t i f i c a t i o n of s i n g l e c h e m i c a l c o m p o u n d s w i t h i n c o m p l e x mixtures.

O p t i c a l spectroscopy c o m b i n e d w i t h microscopy offers the f o l l o w i n g a d v a n - t a g e s to the b i o l o g i s t (see also K o h e n et al., 1978): (a} It e n a b l e s the biologist to i n s p e c t s m a l l organisms. It is i m p o s s i b l e for gas c h r o m a t o g r a p h y to a n a l y z e s i n g l e o r g a n i s m s from m e i o f a u n a or m i c r o f a u n a . U s i n g f l u o r e s c e n c e microscopy it is p o s s i b l e to d e t e r m i n e t h e d i s t r i b u t i o n of s e v e r a l toxic s u b s t a n c e s i n different b o d y parts of these o r g a n i s m s .

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 t h e e q u i p m e n t u s e d i n t h e 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 h a s a d i a m e t e r of o n l y 0 . 5 / a n . (b) It is p o s s i b l e to a n a l y z e t h e c o n t a m i n a - t i o n 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 . A f t e r d e a t h t h e 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 t h e 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 t h e 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 i n t h e v i s i b l e or n e a r UV r e g i o n of t h e s p e c t r u m . T h e r e f o r e o n l y 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 c r u d e oil, c a n b e d e t e c t e d . (b) T i s s u e itself s h o w s f l u o r e s c e n c e . T h e s h o r t e r t h e w a v e l e n g t h of t h e e x c i t a t i o n light, t h e m o r e t h e t i s s u e itself e m i t s 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 t i s s u e 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 s h o r t e r t h a n 450 n m ( H o d g i n s 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 m o s t c a s e s 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 l i g h t 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 t h e t i s s u e itself. S o m e t i m e s d i r e c t 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 t h e t i s s u e c o n t a i n s oil a n d w h i c h p a r t d o e s not, b u t t h e r e a r e m a n y s i t u a t i o n s 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 a n a l y s i s . In t h e 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 .

E Q U I P M E N T

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 t h e 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 Z e i s s 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 f i t t e d w i t h f i l t e r c o m b i n a t i o n s for t h e 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 e x c i t a - t i o n 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. T h e l i g h t s o u r c e 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 x e n o n ) l a m p . A p h o t o m e t e r 03 s e r v e s for s e l e c t i n g t h e 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 l e n s (Zeiss No. 474 340 a n d 477 156) for o p t i c a l f i t t i n g of t h e 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 , t h e 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 t h e 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 o n l y l i t t l e 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 i n t h e r e g i o n s t u d i e d , as e.g. t h e 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 O r t e c 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 m o s t 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 t e c h n i q u e s ) . A strip c h a r t r e c o r d e r m a y b e t h e e n d of t h e chain.

A s 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 t h e 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 t h e optics of the i n s t r u m e n t a n d of t h e p h o t o m u l t i p l i e r on t h e other, it is h e l p f u l to u s e 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, t h e d a t a a r e 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 t a p e .

RESULTS A N D D I S C U S S I O N

2?4 E. Z e e c k

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photo met or

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Fig. 1. Schematic description of the fluorescence s p e c t r o m e t e r

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~60

D e t e r m i n a t i o n 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 a n d 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 m a x i m u m at 490 nm is g i v e n b y a h i g h translucence of the microscope optics t o g e t h e r with a m a x i m u m of sensitivity of the photomultiplier.

The most i n t e r e s t i n g differences b e t w e e n the spectra are l o c a t e d in the r e g i o n b e t w e e n 500 a n d 600 nm. The m a x i m u m at about 550 nm only in the spectrum of the oil- e x p o s e d organisms indicates a c c u m u l a t i o n or g e n e r a t i o n of substances with long w a v e - l e n g t h fluorescence in the intestine. The fluorescence spectrum of crude oil itself shows a m a x i m u m at a much shorter w a v e l e n g t h .

Differences in the spectra in the r e g i o n above 650 nm are of no interest in this context. They are c a u s e d b y different contents of chlorophyll or chlorophyll m e t a b o l i t e s in the intestine from the food of the worms (diatoms) and show no r e p r o d u c i b l e d e p e n d e n c e on oil contamination.

Using gas c h r o m a t o g r a p h i c analysis, van Beruem (personal communication} found that after exposure to oil l a r g e r organisms (Arenicola marina] a c c u m u l a t e d p e r y l e n e , b e n z o - b - f l u o r a n t h e n e , b e n z o - c - p h e n a n t h r e n e , benzo-b,c-fluorene, pyrene, fluoranthene a n d p h e n a n t h r e n e s in a d d i t i o n to b e n z o - a - p y r e n e , b e n z o - e - p y r e n e , m e t h y l c h r y s e n e a n d chrysene; the latter substances w e r e also found (at much lower concentrations) in the u n c o n t a m i n a t e d organisms. M a n y other authors, too, report organismic u p t a k e of poly- n u c l e a r aromatic hydrocarbons from petrol (e.g. Anderson, 1977; V a r a n a s i & Malins, 1977).

But the m a x i m u m of f l u o r e s c e n c e emission at about 550 nm cannot b e e x p l a i n e d b y accumulation of aromatic hydrocarbons. There must be p o l a r substances, too, p r o b a b l y b y m e t a b o l i z i n g the aromatic hydrocarbons. This m e t a b o l i c process seems to occur within the intestine of these organisms. The intestine content of L u m b r i c i l l u s l i n e a t u s

s h o w e d drops of different colour u n d e r the fluorescence microscope (excitation at 365 nm), the colour r a n g i n g from b l u e over light a n d d a r k y e l l o w to brown. Spectroscopi- cally the emission of these drops differed b y the relative h e i g h t of the m a x i m u m at 550 nm, the b r o w n i s h drops showing the h i g h e s t relative intensity at this point, indicat- ing the h i g h e s t content of m e t a b o l i z e d compounds.

The a s s u m p t i o n that this m a x i m u m at 550 n m is c a u s e d b y m e t a b o l i z e d compounds of crude oil is b a s e d on the following facts: Crude oil itself shows r e l a t i v e l y low intensity of fluorescence emission at w a v e l e n g t h s longer than 500 nm (fluorescence excitation at 365 nm). A fluorescence emission at long w a v e l e n g t h s is due to compounds with l a r g e p i - e l e c t r o n systems as polycyclic aromatic hydrocarbons. Examples for fluorescence emission of these hydrocarbons are: chrysene 387 nm, p y r e n e 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, p e r y l e n e 465 nm, fluoranthene 477 nm, tetracene 511 n m (average w a v e l e n g t h of the fluorescence spectrum, according to Berlman, 1971).

276 E. Z e e c k

A s 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 s o m e 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 - d i m e - t 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 , t h e s i t u a t i o n is of c o u r s e m o r e c o m p l i c a t e d b u t t h e t e n d e n c y is t h e s a m e . T h u s the w a v e l e n g t h of the f l u o r e s c e n c e e m i s s i o n of f l u o r a n t h e n e (477 nm) is s h i f t e d b y t h e 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 n m (3- a m i n o f l u o r a n t h e n e in ethanol). T h e r e w i l l b e no difficulty c o n s t r u c t i n g a m a x i m u m of f l u o r e s c e n c e 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 g r o u p s . A t p r e s e n t , t h e r e a r e n o t 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 c o n t r i b u t e to s u c h f l u o r e s c e n c e e m i s s i o n , 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 o t h e r 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 s e l e c t t h e r e a l l y i m p o r t a n t c o m p o u n d s .

H o w e v e r , it is p o s s i b l e to state t h a t not t h e 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 s u b s t i t u e n t s g i v e rise to t h e 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 o r g a n i s m s .

In p r i n c i p l e it is p o s s i b l e t h a t s u b s t a n c e s w i t h t h e s e s p e c t r a l p r o p e r t i e s w h i c h a r e p r e s e n t in c r u d e oil in s m a l l c o n c e n t r a t i o n s a r e a c c u m u l a t e d b y the o r g a n i s m s , b u t it is not c l e a r 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 t h e i n t e s t i n e . T h e fact t h a t t h e p o l a r d e r i v a t i v e s a r e m o r e s o l u b l e in w a t e r t h a n t h e p u r e h y d r o c a r b o n s c a n b e e x c l u d e d for s u c h a n a c c u m u l a t i o n process, as t h e 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 c o n t e n t of t h e i n t e s t i n e of Lumbricillus lineatus b u t b y s i n g l e drops, d i f f e r e n t d r o p s 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 t h e f l u o r e s c e n c e m a x i m u m at 550 nm, as m e n t i o n e d a b o v e .

O n t h e o t h e r h a n d it is w e l l k n o w n t h a t 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 - b o n s 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 h e r e . H y d r o x y l g r o u p s a r e i n t r o d u c e d into t h e h y d r o c a r b o n s v i a t h e f o r m a t i o n of e p o x y d e r i v a t i v e s (Sims & Grover, 1974; M a l i n s , 1977; J e r i n a et al., 1979; S c h ~ f e r - R i d d e r , 1979). This w a s not o n l y o b s e r v e d in m a m m a l i a n t i s s u e b u t also i n i n v e r t e b r a t e s ( V a r a n a s i & M a l i n s , 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 t h e 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 a r y l 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 i n Geukensia demissa; Ernst s t u d i e d t h e m e t a b o l i c t r a n s f o r m a t i o n of 1 - ( 1 - 1 4 C ) n a p h t h o l 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 p o l a r i t y . W h e r e a s m a m m a l s i n i t i a t e t h e 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 t h e i n c o r p o r a t i o n of o n e 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 f u r t h e r 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 a t o m s 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 a r e formed. F u r t h e r o x i d a t i o n l e a d s to t h e f o r m a t i o n of c a t e c h o l s (Gibson, 1976).

It t h e r e f o r e s e e m s l i k e l y t h a t t h e f l u o r e s c e n c e e m i s s i o n o b s e r v e d in t h e i n t e s t i n e of t h e 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 c o m p o n e n t s . Still o p e n is t h e 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 w o r m s 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 b a c t e r i a . 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 i n t h e 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 t h e i n t e s t i n e of t h e w o r m s 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 a r e c o n t i n u i n g in this field.

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 o i l c o m p o n e n t s 2 7 7 Dr. L. Trapp, Carl Zeiss Comp., for t e c h n i c a l information a n d to Dr. O. G i e r e for m a k i n g a v a i l a b l e to m e the species L u m b r i c i l l u s l i n e a t u s after exposure to c r u d e oil. The studies w e r e s u p p o r t e d b y t h e U m w e l t b u n d e s a m t , Berlin.

L I T E R A T U R E C I T E D

Anderson, J. W., 1977. Responses to s u b l e t h a l levels of p e t r o l e u m h y d r o c a r b o n s : Are t h e y s e n s i t i v e i n d i c a t o r s a n d do t h e y correlate w i t h tissue c o n t a m i n a t i o n ? In: Fate a n d effects of p e t r o l e u m h y d r o c a r b o n s i n m a r i n e o r g a n i s m s a n d ecosystems. Ed. b y D. A. Wolfe. P e r g a m o n Press, Oxford, 95-114.

A n d e r s o n , R. S., 1978. D e v e l o p i n g a n i n v e r t e b r a t e m o d e l for c h e m i c a l carcinogenesis: M e t a b o l i c a c t i v a t i o n of carcinogenesis. - Comp. Pathobiol. 4, 11-24.

B e d m a n , I. B., 1971. H a n d b o o k of fluorescence spectra of aromatic molecules. Acad. Press, N e w York, 379 pp.

Ernst, W., 1979. M e t a b o l i c transformation of 1-(1-14C)naphtho1 in b i o c o n c e n t r a t i o n studies w i t h the c o m m o n m u s s e l M y t i l u s edulis. - Ver6ff. Inst. Meeresforsch. B r e m e r h a v e n 17, 233-240. Gibson, D. T., 1976. Initial reactions i n the b a c t e r i a l d e g r a d a t i o n of aromatic hydrocarbons. - Zbl.

Bakt., ParasitKde. (Abt. 1., Reihe B) 162, 157-168,

Gordon, D. C. & Keizer, P. D., 1974. E s t i m a t i o n of p e t r o l e u m h y d r o c a r b o n s i n s e a w a t e r b y fluorescence spectroscopy: I m p r o v e d s a m p l i n g a n d a n a l y t i c a l methods. - Tech. Rep. Fish. Res. Bd Can. 481, 1-29.

Hodgins, H. O., G r o n l u n d , W. D., Mighe11, J. L , Hawkes, J. W. & Robish, P. A., 1977. Effect of c r u d e oil on trout reproduction. In: Fate a n d effects of p e t r o l e u m h y d r o c a r b o n s i n m a r i n e o r g a n i s m s a n d ecosystems. Ed. b y D. A. Wolfe. P e r g a m o n Press, Oxford, 143-150.

Jerina, D. M., Yagi, H., T h a k k e r , D. R., Karle, J. M., Mah, H. D., Boyd, D. R., G a d a g i n a m a t h , G., Wood, A. W., B u e n i n g , M. et al., 1979. Stereoselective m e t a b o l i c a c t i v a t i o n of polycyclic aromatic hydrocarbons. In: Pharmacology: i n t e r n a t i o n a l congress proceedings. Ed. b y Y. Cohen. P e r g a m o n Press, Oxford, 7 (11), 53-62.

Kohen, E., Kohen, C., Hirschberg, J. G., Wouters, A. & Thore11, B., 1978. Multisite t o p o g r a p h i c microspectrofluorometry of i n t e r c e l i n l a r a n d e x o g e n e o u s fluorochromes. - Photochem. Photo- biol. 27, 259.

Malins, D. C., 1977. M e t a b o l i s m of aromatic h y d r o c a r b o n s i n m a r i n e organisms. - Ann. N. Y. Acad. Sci. 298, 482-486.

Schfifer-Ridder, M., 1979. C a r c i n o g e n e s e d u r c h polycyclische a r o m a t i s c h e Kohlenwasserstoffe. - Nachr. C h e m . Tech. 27, 4-6.

Sims, P. & Grover, P. I., 1974. Epoxides i n polycyclic aromatic h y d r o c a r b o n m e t a b o l i s m a n d carcinogenesis. - Adv. C a n c e r Res. 20, 165-172.