ATOLL RESEARCH BULLETIN
No.
122MARINE TOXINS FROM THE PACIFIC 1 1 . THE CONTAMINATION OF WAKE ISLAND LAGOON
by A l b e r t H. Banner, J u d d C . Nevenzel and Webster R . Hudgins
I s s u e d by
THE SMITHSONIAN INSTITUTION Washington, D . C . , U . S . A.
March 30, 1969
MAHINE TOXINS FROM THE PACIFIC 11. T N E CONTAMINATION OF WAKE ISLAND LAGOON
'
by A l b e r t 1-1. Banner, Judd C . Nevenzel and Webster R . Hudgins- 2/
Wake I s l a n d l i e s a t 19" 18' N and 166' 38' E ; i t i s a small a t o l l about 6 . 5 ke long and 3 km wide. I t s s h a l l o w lagoon, w i t h a maximum depth of 25 m , h a s no connection with t h e s u r r o u n d i n g s e a a t low w a t e r , b u t a t high t i d e w a t e r may e n t e r and leave t h e lagoon e i t h e r a c r o s s t h e two-mile broad w e s t e r n r e e f o r through a p a s s a g e on t h e n o r t h e r n s i d e between Wake I s l a n d p r o p e r and Peale I s l a n d ( t h e o r i g i n a l passage between Wake and Wilkes I s l a n d s on t h e s o u t h w e s t e r n s i d e h a s been blocked by a causeway). The i s l a n d i s i n h a b i t e d by about 1500 people who a r e p r i m a r i l y employed i n t h e s e r v i c i n g of t r a n s - P a c i f i c a i r c r a f t . Observations
The evening o f Sunday, 20 June 1965 was u n u s u a l l y s t i l l with a complete c e s s a t i o n o f t h e normal t r a d e winds. P o l i c e O f f i c e r E a r l H a r r i s , on n i g h t d u t y , n o t i c e d an u n u s u a l , pungent and r e p u l s i v e odor about midnight, when h e was p a s s i n g o v e r t h e b r i d g e connecting Wake and Peale I s l a n d s . The odor was s o s t r o n g t h a t h e paused and f l a s h e d h i s s p o t l i g h t i n t o t h e w a t e r below. He was s u r p r i s e d t o f i n d t h a t w a t e r r u s h i n g i n t o t h e lagoon with a r i s i n g t i d e , i n s t e a d of b e i n g c l e a r a s normal, was milky i n appearance. A t 3 A. M . and a g a i n a t 6 A . M . he a g a i n checked t h e w a t e r , which remained pungent and milky.
L1
C o n t r i b u t i o n No. 305, Hawaii I n s t i t u t e o f Marine Biology, U n i v e r s i t y o f Hawaii, Honolulu, Hawaii 96822. The second i n a c o n t i n u i n g s e r i e s of p a p e r s on marine t o x i n s p u b l i s h e d by a group a t t h e HawaiiI n s t i t u t e of Marine Biology, U n i v e r s i t y o f Hawaii; t h e f i r s t 'Ad- vances i n t h e I n v e s t i g a t i o n of F i s h Toxins' by A l b e r t H . Banner i n Animal Toxins, 1967, e d s . R u s s e l l , F. E . , and P . R . Saunders, x i i i + 428, Pergamon P r e s s , Oxford.
2/
A. H . Banner, Department of Zoology, U n i v e r s i t y o f Hawaii;J . C . Nevenzel, Department of Biophysics and Nuclear Medicine,
U n i v e r s i t y o f C a l i f o r n i a , Los Angeles; W . R . Hudgins, Research Laboratory, A l l i e d Chemical C o r p o r a t i o n . A. 1-1. Banner r e s p o n s i b l e f o r g a t h e r i n g f i e l d d a t a and b i o l o g i c a l o b s e r v a t i o n s ; W. R . Hudgins f o r g e n e r a l c h e m i s t r y ; J . C . Nevenzel f o r d e t a i l e d chromatographic a n a l y s e s . Work a t Hawaii I n s t i t u t e of Marine Biology s u p p o r t e d i n p a r t by U . S . P u b l i c H e a l t h S e r v i c e Grant EF-00216 and O f f i c e of Naval Research C o n t r a c t Nonr 2289(00), NR 107-405; work a t
U n i v e r s i t y o f C a l i f o r n i a , Los Angeles, b y t h e Atomic Energy
Commission C o n t r a c t AT(04-1) GEN-12 between t h e Commission and t h e U n i v e r s i t y .
A t 8 o ' c l o c k t h a t morning he v i s i t e d t h e beach a t P e a l e I s l a n d w i t h t h e A s s i s t a n t I s l a n d Manager, Mr. George La C a i l l e , and t h e
r e s i d e n t p h y s i c i a n , D r . F r e d e r i c k Goff. They found a band of f l o a t - i n g m a t e r i a l , perhaps a meter wide and of t h e c o n s i s t e n c y o f a ' t h i n soup, r e a c h i n g along t h e lagoon beach o f Peale I s l a n d a s f a r a s t h e eye c o u l d s e e . Patches of t h e m a t e r i a l were f l o a t i n g f a r out i n t o t h e lagoon. The m a t e r i a l bias l i g h t i n c o l o r w i t h t i n g e s o f p i n k . Most c h a r a c t e r i s t i c of t h e m a t e r i a l was i t s sweet b u t f e t i d odor, v a r i o u s l y d e s c r i b e d a s " s i c k e n i n g l y s w e e t , " "a combination of a nol lasses r e f i n e r y and an exposed o y s t e r f l a t , " o r " l i k e sewage." During t h a t day and f o r s e v e r a l days t o come t h e odor permeated t h e whole i s l a n d .
By e a r l y a f t e r n o o n of t h a t day f i s h were observed t o e x h i b i t a b e r r a n t b e h a v i o r . A s t h e m a t e r i a l , now b e i n g moved from t h e lagoon towards Peale I s l a n d by a s l i g h t w e s t e r l y wind, d r i f t e d towards t h e s h o r e , t h e s m a l l lagoon f i s h behaved f r a n t i c a l l y - - moray e e l s l e f t t h e i r h o l e s and swam t o deeper w a t e r , w h i l e o t h e r s such a s w r a s s e s l e a p e d from t h e w a t e r t o t h e beach and l a y t h e r e f l o p p i n g i n t h e sand.
The s m e l l , t h e a c t i o n of t h e f i s h , and e s p e c i a l l y t h e unknown n a t u r e o f t h e f l o t s a m , caused t h e o f f i c i a l s t o ban a l l swimming, b o a t i n g , and f i s h i n g i n t h e a t o l l w a t e r s .
During t h e n e x t s e v e r a l days t h e contamination of t h e lagoon was watched c a r e f u l l y by D r . F r e d e r i c k Goff. The o r i g i n a l milky m a t e r i a l t u r n e d p i n k , and i n c o n c e n t r a t i o n s a l o n g t h e s h o r e l i n e , brownish r e d . So many f i s h were k i l l e d t h a t a s a n i t a r y p a t r o l was s e n t o u t ; i t
g a t h e r e d o v e r t h r e e b a r r e l s o f dead f i s h from t h e s h o r e o f P e a l e I s l a n d . The r e c e d i n g t i d e l e f t t h e exposed r o c k s and b a r s of t h e lagoon s t a i n e d r e d . Even t h e b i r d s were a f f l i c t e d and a number of s o o t y t e r n s i n t h e colony on P e a l e I s l a n d which were f e e d i n g i n t h e western s e c t i o n of t h e lagoon s i c k e n e d and d i e d . A f r i g a t e b i r d was s e e n t o d i v e i n t o a f l o a t i n g mass, presunlably a f t e r a s i c k f i s h ; b u t when i t s u r f a c e d it was u n a b l e t o f l y and f l o p p e d i t s way t o a small sand b a r . Within minutes it c o l l a p s e d and d i e d .
Over f o u r l i t e r s of t h e m a t e r i a l were d i s p a t c h e d f r o z e n t o t h e Hawaii I n s t i t u t e o f Marine Biology f o r s t u d y ; i t was r e c e i v e d on t h e a f t e r n o o n o f 24 June (Wake t i m e ) . Arrangements were completed f o r t h e s e n i o r a u t h o r t o v i s i t t h e i s l a n d a s a c o n s u l t a n t .
Late i n t h e a f t e r n o o n o f 26 June and t h e f o l l o w i n g day, t h e s e n i o r a u t h o r was a b l e t o i n s p e c t contaminated beaches and t h e s u r r o u n d i n g w a t e r s . No l o n g e r was t h e r e any f l o a t i n g m a t e r i a l i n t h e w a t e r
- -
i t was r e p o r t e d t o have d i s a p p e a r e d by 23 o r 24 J u n e . However, n e a r t h e h i g h t i d e zone on t h e beaches a t P e a l e I s l a n d were p a t c h e s o f r e d d i s h - brown creamy t o waxy m a t e r i a l . No p a t c h e s were e x c e s s i v e l y l a r g e , although some were up t o a meter wide and seven o r more meters long, and with a t h i c k n e s s of a m i l l i m e t e r o r s o . Other p a t c h e s were more c o n c e n t r a t e d , over a c e n t i m e t e r t h i c k f o r a diameter of more t h a n 30 c e n t i m e t e r s . On t h e lagoon s i d e of P e a l e I s l a n d t h e p a t c h e s were most common, b u t t h e y were a l s o found on t h e ocean s i d e n e a r t h e p a s s a g e a s w e l l and c o r r e s p o n d i n g l y n e a r both s i d e s o f t h e passage on Wake I s l a n d . On Wilkes I s l a n d , a c r o s s t h e lagoon, a few p a t c h e s were found. On sandy beaches t h e m a t e r i a l had p e n e t r a t e d i n t o t h e sand s o t h a t whent h e t o p l a y e r was d i s t u r b e d , t h e u n d e r l y i n g sand had a pink c a s t . The exposed sand i n t h e s e a r e a s was of l i g h t g r e y c o l o r r a t h e r than i t s c h a r a c t e r i s t i c w h i t e . S i m i l a r l y , t u f t s o f a t t a c h e d a l g a e on a s h a l l o w t i d e f l a t a t P e a l e I s l a n d were c o a t e d with t h e pink m a t e r i a l . The
waxy m a t e r i a l on t h e b e a c h , t h e s a n d , and t h e a l g a e a l l had t h e pungent, s i c k e n i n g b u t s w e e t , odor.
Along t h e s h o r e s of ll'ilkes I s l a n d were numerous dead f i s h t h a t obviously had been exposed f o r some time. One s i c k t e r n , unable t o f l y , was c o l l e c t e d on P e a l e I s l a n d on S a t u r d a y evening; during t h e n i g h t i t d i e d .
On t h e morning of 2 7 June t h e lagoon was i n s p e c t e d by b o a t and t h e lagoon and t h e s u r r o u n d i n g w a t e r s o f t h e i s l a n d by a i r from a U . S. Navy ampllibian p l a n e f l y i n g a t 150 m e t e r s . No f l o a t i n g m a t t e r was s e e n , and t h e c o r a l heads of t h e w e s t e r n s i d e o f t h e lagoon, exposed by t h e
extreme low w a t e r a t t h e time o f i n s p e c t i o n , seemed u n a f f e c t e d .
A t t h e recommendation of t h e s e n i o r a u t h o r t h e lagoon was opened t o swimming and b o a t i n g on Sunday. Bathers r e p o r t e d t h e w a t e r "had a bad t a s t e " b u t no i l l e f f e c t s were s e e n from swimming.
Laboratory Examination
The f r o z e n i n a t e r i a l s h i p p e d t o l-ionolulu was pink w i t h grey t r a c e s . I t was a c o n s i s t e n c y of t h i c k l a d l e d o r skimmed cream, w i t 1 1 some f r e e w a t e r on t h e bottom of t h e c o n t a i n e r . The waxy m a t e r i a l c o l l e c t e d from t h e beaches on t h e 26th and 27th had a c o n s i s t e n c y o f a t h i c k medicinal o i n t m e n t . Both would adhere t o t h e s k i n when touched.
M i c r o s c o p i c a l l y , t h e b u l k of b o t h m a t e r i a l s was w i t h o u t formed s t r u c t u r e s and was immiscible with w a t e r when squashed on a microscope s l i d e . I n a l l m a t e r i a l examined t h e r e were numerous o i l d r o p l e t s and an amorphous ground s u b s t a n c e . The pink masses were e q u a l l y f o r m l e s s and were s c a t t e r e d a s s m a l l u n i t s through t h e o t h e r m a t e r i a l . I n t h e w a t e r s u r r o u n d i n g t h e sample s h i p p e d t o t h e l a b o r a t o r y were numerous g r e e n , s p h e r i c a l c e l l s o f a u n i c e l l u l a r a l g a ; i n t h e t u f t s of contam- i n a t e d a l g a e c o l l e c t e d from P e a l e I s l a n d , t h e c e l l s of t h e f i l a m e n t s appeared a l i v e ; i n t h i s m a t e r i a l and i n t h e contaminated sand were numerous c i l i a t e p r o t o z o a n s . Ilowever, i n t h e c o n s o l i d a t e d masses o f t h e waxy cream from t h e beaches t h e r e was no l i f e .
The creamy m a t e r i a l d e l i v e r e d t o t h e I-lawaii I n s t i t u t e of Marine Biology was found, by d e h y d r a t i o n a t 45' C under reduced p r e s s u r e , t o c o n t a i n 6 8 . 8 p e r c e n t w a t e r and v o l a t i l e components, 31.2 p e r c e n t s o l i d s which o b v i o u s l y i n c l u d e d t h e s a l t s l e f t by t h e e v a p o r a t e d s e a w a t e r ( e s t i m a t e d a t perhaps 2 p e r c e n t of t h e weight o f t h e o r i g i n a l sample, a flame t e s t f o r sodiwn was p o s i t i v e ) .
Five hundred grams o f t h e same emulsion, undehydrated, was
e x t r a c t e d by o c c a s i o n a l s h a k i n g f o r one day i n e q u a l volume of 95 p e r c e n t e t h a n o l and f i l t e r e d ; t h e e v a p o r a t e d e t h a n o l i c e x t r a c t y i e l d e d 1 5 . 3 g of a b r i g h t yellow waxy s o l i d . A d r y i c e t r a p , followed an i c e t r a p , i n t h e d i s t i l l a t i o n l i n e y i e l d e d a small amount o f a c o l o r l e s s
v o l a t i l e l i q u i d w i t h an odor c h a r a c t e r i s t i c , i n p a r t , o f t h a t of t h e o r i g i n a l sample. The r e s i d u e from t h e e t h a n o l i c e x t r a c t i o n was i n t u r n e x t r a c t e d with 500 m l of d i e t h y l e t h e r , and y i e l d e d 35 g o f a b r i l l i a n t orange waxy s o l i d t h a t melted s l i g h t l y below 45" C and t h a t
c o n t a i n e d s c a t t e r e d m i c r o c r y s t a l s . I t was p o s s i b l e t o d i s s o l v e and r e c r y s t a l l i z e t h e c r y s t a l s i n e t h a n o l ; t h e y were white and p l a t e - l i k e , and had a s h a r p nrelting p o i n t a t 51" C . The r e s i d u e u n d i s s o l v e d by t h e d i e t h y l e t h e r e x t r a c t i o n was yellow and f i b r o u s ; i t gave a p o s i t i v e b i u r e t t e s t f o r p r o t e i n .
The t o x i c i t y of t h e mixture and sonie of i t s p r i n c i p a l conlpolients were checked t o s e e i f t h e observed d e a t h s of f i s h and b i r d s could be a t t r i b u t e d t o t h e m i x t u r e . The crude cream was e m u l s i f i e d and s t i r r e d i n t o an aquarium a t about 10 m l p e r l i t e r and t h r e e b u t t e r f l y f i s h
(Chaetodon s p . ) were added; t h e aquarium was c o n t i n u o u s l y a e r a t e d . I n 40 t o 60 minutes t h e f i s h , a f t e r showing marked r e s p i r a t o r y d i s t r e s s , d i e d . Five w h i t e mice, weighing from 21 t o 24 g , were f o r c e - f e d some 0 . 3 t o 0 . 6 m l of e m u l s i f i e d m i x t u r e ; a l l e x h i b i t e d heavy b r e a t h i n g , gasping and l i s t l e s s n e s s , and t h r e e d i e d o v e r n i g h t . S i m i l a r l y , when t h e s u p e r n a t a n t wax from t h e o r i g i n a l sample was f o r c e - f e d t o mice i n s i m i l a r amounts, t h r e e of t h e s i x mice d i e d o v e r n i g h t . However, when t h e e t h a n - o l i c o r e t h e r e a l e x t r a c t s were i n j e c t e d i n t r a p e r i t o n e a l l y i n t o mice a t t h e r a t h e r massive dosage o f 4 mg/g, no d e a t h s r e s u l t e d . The f i n a l f i b r o u s r e s i d u e caused no d e a t h s when f o r c e - f e d , b u t t h e same r e s i d u e when i n j e c t e d a t 4 mg/g caused one of t h e two mice t o d i e o v e r n i g h t .
A s t o p p e r e d v i a l of t h e s o f t beach m a t e r i a l w i t h a pink c a s t was k e p t a t room t e m p e r a t u r e ; a f t e r f o u r days it smelled s t r o n g l y of hydrogen s u l f i d e .
A n a l y s i s o f t h e
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The t o t a l l i p i d i n t h e o r i g i n a l creamy m a t e r i a l d e l i v e r e d t o t h e llawaii I n s t i t u t e of Marine Biology was o b t a i n e d by e x t r a c t i o n i n t o d i e t h y l e t h e r , u s i n g c e n t r i f u g a t i o n and methanol t o break e n ~ u l s i o n s . The crude l i p i d was s e p a r a t e d by a d s o r p t i o n colwnn chromatography on s i l i c i c a c i d ( F i l l e r u p and Mead, 1953) i n t o t h e f r a c t i o n s l i s t e d i n Table I . The i d e n t i f i c a t i o n o f t h e main c o n s t i t u e n t (60%) o f t h e Wake I s l a n d l i p i d a s wax e s t e r s ( t h a t i s , f a t t y a c i d s e s t e r i f i e d w i t h long-chain a l c o h o l s ) was confirmed by (a) d i r e c t comparison i n t h i n - l a y e r chromatography (TLC) w i t h s y n t h e t i c h e p t a d e c y l s t e a r a t e ; (b)
g a s - l i q u i d chromatography (GLC) o f tile unhydrolyzed e s t e r s ( c f . Nevenzel, e t a l . , 1966)
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t h e r e s u l t s a r e g i v e n i n Table 11; and ( c ) G d r o l y s i s- -
t o a c i d and a l c o h o l m o i e t i e s , which were analyzed by GLC
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t h e a c i d s a s t h e i r methyl and phenacyl e s t e r s and t h e a l c o h o l s a s t h e i r t r i f l u - o r o a c e t a t e d e r i v a t i v e s (Nevenzel, e t a l . , 1965)- -
- - s e e Table 111.R coinparison of t h e a n a l y s i s of t h e Wake I s l a n d l i p i d with t h e d a t a of Lederer,
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e t a l . , 1946, f o r ambergris ( a l s o given i n Table I ) makes i t c l e a r t h a t our m a t e r i a l was n o t ambergris. A d i r e c t comparison by TLC o f t h e crude Wake I s l a n d l i p i d w i t h a u t h e n t i c low-grade amber- g r i s from llawaii f u r t h e r emphasized t h i s p o i n t : on B i o - S i l A (Bio-Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a ) developed with 10 p e r c e n t d i i s o p - ropy1 e t h e r i n petroleum e t h e r ( b . p . 60-70' C) t h e 1Vake I s l a n d l i p i dshowed one main s p o t o f I< 0 . 6 8 ( i d e n t i c a l t o a u t h e n t i c wax e s t e r s ) and a f a i n t s p o t o f llf 0 . 6 7 wiiile t h e H a w a i i a n a m b e r g r i s h a d o n l y a f a i n t s p o t a t Rf 0 . 6 9 f o r wax e s t e r o r s t e r o l e s t e r , a n o t h e r ininor corn- p o n e n t o f Rf 0 . 3 1 , and t h e main s p o t o f Rf 0 . 2 5 w i t 1 1 e x t e n s i v e t a i l i n g ; a u t h e n t i c o c t a d e c a n o l h a d an Itf of 0 . 1 3 and c h o l e s t e r o l a b o u t 0 . 0 5 i n tilis s y s t e m .
The s t e r o l - c o n t a i n i n g f r a c t i o n o f T a b l e I was f u r t h e r c l i a r a c t e r i z e d by ?'LC, which c o n f i r m e d t h e p r e s e n c e o f f r e e l o n g - c h a i n a l c o h o l s o r c h o l e s t e r o l . By GLC a n a l y s i s o f t h e t r i f l u o r o a c e t a t e d c r i v a t i v e s o f t h e s e h y d r o x y l - c o n t a i n i n g compounds s t r a i g h t c h a i n a l c o l i o l s C16-CZ5 were i d e n t i f i e d t o g e t h e r w i t h a t r a c e o f a h i g h e r m o l e c u l a r w e i g h t con~pound, t h o u g h t t o be a t r i t e r p e n e a l c o i ~ o l o r a s t e r o l o t h e r t h a n c h o l e s t e r o l . TLC e x a m i n a t i o n o f t h e most p o l a r l i p i d f r a c t i o n e s t a b - l i s l l e d t h e p r e s e n c e o f t r a c e s o f f r e e f a t t y a c i d s and p h o s p l i o l i p i d
( p r o b a b l y l e c i t h i n ) , b u t t h e b u l k o f t h e i n a t e r i a l a p p e a r e d a s a s t r e a k w i t h no d e f i n e d s p o t s and was n o t c h a r a c t e r i z e d f u r t h e r . 'The y e l l o w c o l o r o f t h i s s a m p l e was due t o s t r o n g end a b s o r p t i o n from a peak below 230 mu; no i d e n t i f i a b l e pigment was p r e s e n t .
O i s c u s s i o n
To o u r knowledge, no blooni o f a n a l g a , e i t h e r f i x e d o r p l a n k t o n i c , n o r any spawning o r o t h e r a c t i v i t y o f a m a r i n e i n v e r t e b r a t e o r f i s h h a s b e e n r e p o r t e d t o form a n amorphous mass o f f l o a t i n g l i p i d s o f s u c h volume and n a t u r e . A s i d e from t h e r e s u l t s o f illan's a c t i v i t y , t h e o n l y
~ n a s s i v e d e p o s i t i o n s of waxy m a t e r i a l i n t h e s e a i s t h e voided s e c r e t i o n o f t h e g u t o f t h e sperm !.illale, known c o i n m e r c i a l l y a s a m b e r g r i s . A s Wake I s l a n d l i e s i n t i l e t r a d i t i o n a l w h a l i n g ground f o r t h e sperin w h a l e , i t was o r i g i n a l l y t h o u g h t t h a t t h e c o n t a m i n a n t o f t h e l a g o o n was a huge inass o f a i n b e r g r i s , e m u l s i f i e d and a l t e r e d i n c o n s i s t e n c y i n i t s p a s s a g e t h r o u g l i t h e s u r f . The h y p o t h e s i s was r e n d e r e d even more a t t r a c t i v e by t h e ~ i l u s t y s m e l l n o t e d on t h e c o n t a m i n a t e d b e a c h e s . ilowever, t i l e l a b o r a t o r y s t u d i e s o f one o f us ( N e v c n z e l ) h a v e d e s t r o y e d t h e h y p o t h e s i s o f t h e o t h e r two: t h e m a t e r i a l d e f i n i t e l y i s n o t a m b e r g r i s . On t h e o t h e r h a n d , o n l y l i p i d s o f t h e sperm whales
( f a m i l y P h y s e t e r i d a e , l i i l d i t c h arid I V i l l i a m s , 1 9 6 5 ) , t h e beaked w h a l e s ( f a m i l y i i p h i i d a e , Mori, - - e t a l . , 1 9 6 4 ) , and some p e l a g i c copepods I N e v e n z e l . u n ~ u b l i s h e d ) and f i s h e s ( f a m i l i e s k l v c t o o h i d a e . Nevenzel ,
.
,and Rodegker, 1965; G a d i d a e , Koii~ori and Agawa, 1955; Gempylidae, 1963;
and T r a c h i c h t h y i d a e , Kaufn~ann and G o t t s c h a l k , 1 9 5 6 ) , and t h e l i v i n g f o s s i l ( f a m i l y L a t i m e r i d a e , ~ J e v e n z e l , e t a l . , 1966) a r e l a r g e l y wax
- -
e s t e r s . Of s e v e n s o e c i e s whose wax e s t e r s h a v e b e e n examined i n d e t a i l , o n l y t h o s e o f t h e sperin whale h a v e as lo^^ an a v e r a g e c h a i n l e n g t h a s t l l o s e o b s e r v e d h e r e : IVake I s l a n d l i p i d h a s 85 p e r c e n t e s t e r s s h o r t e r t h a n C34, s p e r m a c e t i wax 90 p e r c e n t , b u t t h e wax e s t e r s from f i s h h a v e o n l y 4-20 p e r c e n t o f t h e t o t a l s h o r t e r t h a n C34. 'IXe a v a i l a b l e d a t a on t h e c o ~ n p o s i t i o n o f s e v e r a l sperin w h a l e l i p i d s a r e g i v e n i n t h e T a b l e s f o r c o m p a r i s o n w i t h t h e c o r r e s p o n d i n g v a l u e s f o r t h e Wake I s l a n d l i p i d . Note t h a t s p e r m a c e t i i s t h e
n l a t e r i a l whicii c r y s t a l l i z e s from t h e t o t a l s p e r m wliale o i l on c h i l l i n g and c o n s i s t s o f t h e inore s a t u r a t e d , l o n g e r c h a i n wax e s t e r s ; i t
c o n t a i n s o n l y t r a c e s o f t r i g l y c e r i d c s o r u n s a t u r a t e d coinponents.
The s i m p l e s t e x p l a n a t i o n f o r t h e o r i g i n of t h e Wake I s l a n d m a t e r i a l i s t h a t a mass of whale o i l w i t h some f i b r o u s c o n t e n t f l o a t e d i n t o t h e lagoon i n an e m u l s i f i e d c o n d i t i o n , where i t s e t t l e d out i n t o t h e f l o a t - i n g cream-like l a y e r . I n t h e p r o c e s s i t was a c t e d upon by micro-organisms and was s u b j e c t e d t o chemical changes ( h y d r o l y s i s , r e c o m b i n a t i o n , oxida- t i o n , e t c . ) . In g e n e r a l , t h e d i f f e r e n c e s i n composition between t h e Wake I s l a n d l i p i d and sperm whale o i l (Tables I and 111) a r e c o n s i s t e n t w i t h (a) p r o p o r t i o n a l l y g r e a t e r l o s s e s of t h e s h o r t e r c h a i n wax e s t e r s , due t o t h e i r g r e a t e r s o l u b i l i t y and g r e a t e r r a t e of h y d r o l y s i s ; (b) t h e e x t e n s i v e l o s s e s o f u n s a t u r a t e d components by a u t o x i d a t i o n ; and ( c ) t h e p r e f e r e n t i a l l o s s of t r i g l y c e r i d e s because of t h e i r h i g h e r c o n t e n t of u n s a t u r a t e d f a t t y a c i d s and g r e a t e r r a t e of h y d r o l y s i s . The n e t r e s u l t o f t h e s e p r o c e s s e s would be t h e p r o d u c t i o n of a l i p i d c o n s i s t i n g
l a r g e l y of s a t u r a t e d wax e s t e r s w i t h a reduced c o n t e n t of CZ6 and CZ8 components:
- -
i . e . , something approaching s p e r m a c e t i . Tables I 1 and I11 confirm t h e s i m i l a r i t i e s o f Wake I s l a n d l i p i d w i t h s p e r m a c e t i , although i n t h e former t h e c o n t e n t o f decanoic a c i d (10:O) i s t e nt i m e s a s much a s expected f o r whale o i l , and t h e p e r c e n t a g e o f p a l m i t i c a c i d (16:O) i s a l s o t o o h i g h . Nor does t h i s e x p l a n a t i o n account f o r t h e t o x i c i t y o f t h e samples.
The t o x i c i t y probably i s t h e r e s u l t o f b a c t e r i a l a c t i o n upon t h e m i x t u r e . When t h e m a t e r i a l was f i r s t s e e n , i t was e m u l s i f i e d i n t o a
t h i n milky s u s p e n s i o n . I n such a s t a t e t h e s u r f a c e a r e a f o r b a c t e r i a l a t t a c k would have been tremendous and a s t h e f a t t y m a t e r i a l r e c o n s o l - i d a t e d i n t h e "cream" found f l o a t i n g on t h e s u r f a c e , t h e b a c t e r i a were a l s o i n c o r p o r a t e d . Some probably worked on t h e l i p i d s , b u t o t h e r s could have worked upon t h e f i b r o u s p r o t e i n mixed i n t h e mass. By- p r o d u c t s from t h e b a c t e r i a l decomposition of t h e mixture c o u l d be t o x i c and account f o r t h e d e a t h o f f i s h and p o s s i b l y t h e b i r d s . An example of such a p r o d u c t i s t h e hydrogen s u l f i d e noted both i n t h e f i e l d and t h e l a b o r a t o r y . The e x h a u s t i o n o f oxygen i n t h e w a t e r by b a c t e r i a l a c t i o n under t h e f l o a t i n g "cream" c o u l d a l s o account f o r some d e a t h s o f f i s h , b u t i t cannot account f o r t h e d e a t h of t h e b u t t e r f l y f i s h i n t h e w e l l - a e r a t e d aquarium. The i n a b i l i t y of t h e b i r d s t o f l y prob- a b l y was n o t from t h e t o x i c i t y o f t h e mass but from t h e o i l s c o a t i n g t h e i r f e a t h e r s .
I t i s more d i f f i c u l t t o account f o r t h e observed l a r g e amount (17.5%) o f decanoic a c i d (ClO), s i n c e i n sperm whale o i l C I Q i s a minor c o n s t i t u e n t ; c f . Table 111. One p o s s i b l e s u g g e s t i o n i s t h a t t h e mass reforming f r o m y h e t h i n emulsion t r a p p e d and i n c o r p o r a t e d C10
a c i d s from a bloom o f b l u e - g r e e n a l g a , f o r O s c i l l a t o r i a (Trichodesmium) e r y t h r a e u n i s known t o c o n t a i n t h e s e ( P a r k e r , and Van Baalen, p e r s o n a l communication). I-lowever, from i n t e r v i e w s of t h e r e s i d e n t s , t h e r e was no i n d i c a t i o n of a p r e v i o u s bloom of a l g a i n t h e lagoon, and t h e c o i n c i d - ence o f a major bloom o f b l u e - g r e e n p h y t o p l a n k t o n , a r a t h e r r a r e o c c u r r e n c e , a t t h e moment of t h e contamination of t h e lagoon by waxes s t r a i n s o n e ' s c r e d u l i t y .
Conclusions
As sperm whales do n o t g i v e up t h e i r wax e s t e r s v o l
3Yntarily ( u n l i k e t h e i r ambrein and s t e r o l s ) we f i n d t h e b e s t way- t o account f o r t h e contamination of Wake I s l a n d lagoon i s t o p o s t u l a t e t h a t some unknown whale f a c t o r y s h i p , c r u i s i n g t h e whaling grounds d i s c h a r g e d i t s r e n d e r i n g r e t o r t s f o r unknown r e a s o n s . This crude oil-wax
c o n c e n t r a t e , s t i l l c o n t a i n i n g t h e f i b e r s o f t h e a d i p o s e t i s s u e , f l o a t e d i n a c o h e r e n t mass and probably f o r a r e l a t i v e l y s h o r t t i m e , u n t i l i t was c a r r i e d by c u r r e n t s t o t h e r e e f o f f t h e n o r t h e r n c o a s t o f Wake
I s l a n d . I t a r r i v e d i n time f o r t h e c u r r e n t s o f t h e incoming t i d e a t t h e s h a l l o w e n t r a n c e t o t h e lagoon. Here t h e c u r r e n t s c a r r i e d i t through t h e s u r f ( i n s p i t e of t h e calmness of t h e n i g h t , t h e r e would be c o n t i r ~ u i n g and p o s s i b l y heavy s u r f on t h e windward r e e f f a c e ) . Once t h e crude mixture was e m u l s i f i e d by t h e s u r f , t h e g r e a t s u r f a c e a r e a p e r m i t t e d r a p i d b a c t e r i a l a c t i o n on a l l components, s p e e d i n g a massive a l t e r a t i o n of t h e l i p i d s and decomposing t h e p r o t e i n s . A chemical by-product by t h i s a c t i o n could b e t h e l o s s of some l i p i d f r a c t i o n s , t h e r e b y c o n c e n t r a t i n g o t h e r f r a c t i o n s (such a s c e t y l a l c o h o l ) , and t h e s y n t h e s i s of C10 a c i d . The r e d c a s t t o t h e mass observed i n t h e w a t e r and on t h e beaches probably was t h e r e s u l t o f growth of pigmented s u l f u r b a c t e r i a , which were u t i l i z i n g t h e H2S given o f f i n o t h e r b a c t e r i a l decomposition.
\lie f u r t h e r p o s t u l a t e t h a t t o x i c by-products o f t h e b a c t e r i a l a c t i o n k i l l e d t h e f i s h and rendered t h e mixture t o x i c t o l a b o r a t o r y mice when f e d o r i n j e c t e d with i t . A t t h e same t i m e , t h e p r e s e n c e o f b a c t e r i a caused t h e c i l i a t e p r o t o z o a n s , which f e e d on b a c t e r i a , t o
i n c r e a s e and t h e b a c t e r i a l r e l e a s e of n u t r i e n t s a l t s encouraged t h e growth o f u n i c e l l u l a r a l g a e . P o s s i b l y some C10 a c i d s were i n c o r p o r a t e d i n t o t h e mass from t h e phytoplankton. Otherwise we cannot account f o r t h e i r p r e s e n c e .
F i n a l l y , t h e e m u l s i f i e d d r o p l e t s , f l o a t i n g t o t h e s u r f a c e , reformed i n t o t h e waxy mass and was c a r r i e d t o t h e beaches by t h e l i g h t a i r s .
3/
The s e n i o r a u t h o r a l o n e , having h i s ambergris h y p o t h e s i s r u t h l e s s l y d e s t r o y e d by f a c t , wishes t o emphasize t h a t t h e chromatography proved t h a t t h i s p a r t i c u l a r combination of wax e s t e r s could have come from no known animal o r p l a n t . Ile s u g g e s t s t h a t we must t h e r e f o r e look f o r some y e t unknown animal, and t h a t perhaps t h e Great S e a - S e r p e n t , s o convincingly d e s c r i b e d by Oudemans, might be a wax e s t e r producer. Presumably even t h e s e a - s e r p e n t does n o t shed i t s a d i p o s e t i s s u e s , b u t perhaps t h e m a g n i f i c e n t c r e a t u r e might d i s c h a r g e wax e s t e r a s a medium f o r i t saura
s e m i n a l i s . He begs t o r e c a l l t h e d e s c r i b e d a u r a of t h e Wake I s l a n d lagoon.-
iVe w i s h t o t h a n k t h e F e d e r a l A e r o n a u t i c s A d m i n i s t r a t i o n and i t s a c t i n g R e g i o n a l F l i g h t S u r g e o n , D r . P. M. Corboy, f o r t h e s a m p l e s and t h e o p p o r t u n i t y t o v i s i t Wake I s l a n d ; D r . F r e d e r i c k Goff and P o l i c e O f f i c e r E a r l i . l a r r i s f o r t h e i r a i d and i n f o r i n a t i o n on t h e e a r l y con- d i t i o n s t h e r e on \\lake I s l a n d ; and biiss B e t t y J a n e S t e p h e n s f o r r u n n i n g l a b o r a t o r y t e s t s a t t h e Hawaii I n s t i t u t e o f Marine B i o l o g y .
UIBLIOGRAPHY
F i l l e r u p , U. L . , and J . F . Mead. 1953. C h r o m a t o g r a p h i c S e p a r a t i o n o f t h e Plasma L i p i d s .
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P r o c S o c . Exptl.- - -
B i o l . Med., 8 3 , 574-77.H i l d i t c h , T. P . , and P. N . Williams. 1965. THE CHEMICAL CONSTITUTION OF i\IATURAL FATS. 4 t h e d . New York, i v i l e y , p p . 72-74.
Kaufmann, l i . P . , and E. G o t t s c h a l k . 1956. The Body O i l o f t h e F i s h I - l o p l o s t e t h u s i s l a n d i c u s , 1 1 . F e t t e , S e i f e n , A n s t r i c h m i t t e l ,
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5 8 , 411-16.Komori, S . , and T . Agawa. 1 9 5 5 . O i l o f Laemonenia morosuil. I . Re- s e a r c h on t h e Docosenol F r a c t i o n . J . Am. O i l C h e m i s t s ' S o c . , 3 2 ,
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525-28.
L e d e r e r , E . , R . Marx, D . M e r c i e r , and G . P e r o t . 1946. S u r l e s c o n s t i t u a n t s de l ' a m b r e g r i s 11. A n ~ b r e i n e e t C o p r o s t a n o n e t l e l v . Chim. A c t a , 2 9 , 1354-65.
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Mori, M . , Y . I w a k i r i , A . Ozawa, and S . S h i b a t a . 1964. A n a l y s i s o f F a t t y Acid and F a t t y A l c o h o l C o m p o s i t i o n s o f S p e r n Whale O i l by G a s - l i q u i d Cnromatography. Nippon S u i s a n G a k l t a i s h i ,
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3 0 , 161-69.N e v e n z e l , J. C . 1963. The Wax E s t e r s o f Lepidocybimn f l a v o - b r u n n e u m . USAEC r e p o r t UCLA-520, p . 22.
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and IV. Rodegker. 1965. The L i p i d s o f blidwater Marine F i s h e s . USAEC r e p o r t UCLA-12-576, p p . 23-25.,
W. Rodegker, and J. F. Mead. 1965. 'Ihe L i p i d s o f R u v e t t u s p r e t i o s u s Muscle and L i v e r . B i o c l ~ e m i s t r y ,-
4 , 1589-94.,
W. Rodegker, J . F. Mead, and M. S . Gordon. 1966. L i p i d s o f t h e L i v i n g C o e l a c a n t h , L a t i i n e r i a chalunmae. S c i e n c e ,-
152, 1753-55.Oudemans, A. C . 1892. TllE GREAT SEA-SERPENT. L e i d e n , E. J . B r i l l . xv+592.
P a r k e r , P . L . 1966. I n s t i t u t e o f Marine S c i e n c e , U n i v e r s i t y o f
Texas, P o r t Aransas, Texas. P e r s o n a l conununication, r e p o r t i n g on work done with Dr. C . Van Baalen.
T a t e i s h i , T . , M. Fujiwara, and H . S a k u r a i . 1958. S e p a r a t i o n o f Sperm CVhale Head O i l i n t o h'ax and G l y c e r i d e s by E l u t i o n Chromatography.
Kagaku Z a s s h i , 6 1 , 1580-82.
-
TABLE I
Composition of Wake I s l a n d L i p i d (Weight %)
Component Wake I s l a n d
l i p i d s l i p i d
Hydrocarbons
Ketones
-
Wax e s t e r s
T r i g l y c e r i d e s
I
9 . 5 Free s t e r o l sand a l c o h o l s Free f a t t y a c i d s
and p o l a r l i p i d s
(1) Normalized d a t a of L e d e r e r , e t a l . , 1946.
- -
(2) T a t e i s h i , e t a l . , 1958.
- -
TABLE I 1
Composition o f Wax E s t e r s of Wake I s l a n d L i p i d (Weight %)
Source
Homologue (2) 26:O 27:O 28:O 29:O 30:O 30 : 1 31 :O 32 :O 32 : 1 33:O 34:O 3 4 : l 36:O 36 : 1
Wake I s l a n d L i p i d Spermaceti (1)
(1) "Spermaceti, U.S.P.", F i s h e r S c i e n t i f i c Company, F a i r Lawn, N . J . ( 2 ) The number b e f o r e t h e colon i n d i c a t e s t h e t o t a l number of carbon
atoms, t h e number f o l l o w i n g t h e colon d e n o t e s t h e t o t a l num- b e r o f double bonds i n t h e molecules.
Component F a t t y Acids and Alcohols of Wake I s l a n d Wax E s t e r s (Weight %)
(1) "Spermaceti, U.S.P.", F i s h e r S c i e n t i f i c Company, F a i r Lawn, N . J .
( 2 ) Data of T a t e i s h i , e t a l . , 1958, r e c a l c u l a t e d t o e s t i m a t e v a l u e f o r
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1 8 : 2 . (3) Data of Mori, e t a l . , 1964, f o r A r c t i c Sperm Whale, head o i l .--
( 4 ) The number b e f o r e t h e colon i n d i c a t e s t h e number o f carbon atoms, t h a t f o l l o w i n g t h e number of double bonds i n t h e a c i d o r a l c o h o l .
F i g u r e 1. I n t e r t i d a l a r e a a t P e a l e I s l a n d , showing contamination of beach.
The normal beach c o l o r i s d a z z l i n g w h i t e , a s shown i n t h e f o r e - and back- ground; t h e c r e s c e n t i c darkened a r e a , reddish-brown i n hue, i s o f t h e waxy m a t e r i a l w e l l over a meter wide b u t
o n l y a few m i l l i m e t e r s t h i c k .
F i g u r e 2 . I n t e r t i d a l a r e a a t Peale I s l a n d , showing a "glob" o f contaminant.
The t h i c k m a t e r i a l , dark red-brown i n c o l o r , i s o f t h e c o n s i s t e n c y of a t h i c k ointment and covers t h e s h e l l s and c o r a l fragments on t h e beach v a r i o u s l y from 1 t o 4 cm t h i c k . The s c a l e may b e i n t e r - p r e t e d from t h e wrist watch on t h e upper
l e f t .
