Organic Marine Geochemistry - ACS Publications - American

18 Polybromomethanes A Year-Round Study of Their Release to Seawater from Ascophyllum nodosum and Fucus vesiculosis Philip M. Gschwend and John K. MacFarlane Ralph M . Parsons Laboratory for Water Resources and Hydrodynamics, Department of Civil Engineering, Massachusetts Institute of Technology, Cambridge, M A 02139

Polybromomethanes (CHBr, CHBrCl, and CHBr) were released to seawater in laboratory incubations by Ascophyllum nodosum and Fucus vesiculosis at nanograms to micrograms each compound per gram dry algae per day throughout the year. This biological source of halogenated compounds was detectable in nearshore seawater during high tides when these littoral algae were submerged. The weak seasonality of polybromomethane formation, together with various evidence from the literature, suggest that these brominated organic compounds may arise from fungal epiphytes closely associated with these algae.

Downloaded by TUFTS UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: April 21, 1986 | doi: 10.1021/bk-1986-0305.ch018

3

2

2

2

I t has long been recognized that marine macroalgae contain halogenated metabolites Ο - 3 ) . A few investigations have provided evidence that certain of the v o l a t i l e halogenated compounds are released from these algae into coastal seawater (4 - ])· In p a r t i c u l a r , of a l l the v o l a t i l e halogenated compounds observed, three polybromomethanes (CHBr , CHBr Cl, and CH Br ) were released In the greatest quantities i n a survey of several temperate macroalgae ( 7 ) · This natural source of halogenated organic compounds to the marine environment i s especially Important In l i g h t of our concern for emissions of halogenated solvents and haloforras i n coastal discharges (8 - 9) and to the atmosphere ( 1 0 ) . Such natural sources must be quantified to place man's Impact In perspective and to suggest natural sink mechanisms which may have evolved In response to these sources. In this report, we describe a seasonal study of polybromomethane releases from two common fucold algae, Ascophyllum nodosum and Fucus v e s l c u l o s l s . We performed this investigation to evaluate the yearround variations i n a l g a l Impact on coastal seawater chemistry. Further, we hoped to observe seasonal correlations between polybromomethane releases and factors i n the l i f e cycles of these algae to provide insights as to the genesis and function of these natural products. 3

2

2

2

0097-6156/86/0305-0314$06.00/0 © 1986 American Chemical Society

Sohn; Organic Marine Geochemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

18. G S C H W E N D A N D M A C F A R L A N E

Polybromomethane Release to Seawater


METHODS A l g a l Incubations, Our procedures f o r quantifying polybromomethane releases from macroalgae to seawater have been described previously (7). A b r i e f description follows. Ascophyllum nodosum and Fucus v e s i c u l o s l s specimens were collected approximately monthly from the rocky i n t e r t i d a l shoreline of the Cape Cod Canal, Sagamore MA. Algae were collected by breaking o f f a piece of the rock to which the plants were attached, thereby not causing any tissue lesions, and were held i n a bucket of seawater u n t i l delivery to the laboratory within 2 hours. Plants supporting v i s i b l e epiphytes were avoided, but i t was not possible to exclude closely associated microscopic organisms. Seawater temperature and t i d a l stage were recorded a t the time of sampling and water samples were taken f o r nutrients and stored frozen u n t i l analysis. Nltrate-N was determined by the cadmium reduction method and phosphate-P was assesed by the ascorbic acid or stannous chloride methods (11). At the laboratory, the algae were placed In a 3 4 - l i t e r aquarium, f i l l e d with seawater and maintained without headspace by a t i g h t l y f i t t i n g glass l i d . The aquarium was kept i n a temperature and l i g h t - c o n t r o l l e d Incubation r e f r i g e r a t o r set to mimic the current environmental conditions. Water motion was maintained using a two-inch magnetic s t i r bar. Algal incubations t y p i c a l l y lasted one day. After incubating, seawater was withdrawn from the aquarium, sρiked with a v o l a t i l e halogenated organic Internal standard, and analyzed f o r polybromomethane content. Analysis of Polybromomethanes In Seawater. Polybromomethanes In aquarium water and Cape Cod Canal seawater samples were determined by a hybrid procedure of the "purge and trap" method (12) and the Grob closed-loop-stripping analysis (13). B r i e f l y , headspace a i r was bubbled through a 1.8 l i t e r water sample a t a rate of 650 mL/mln f o r 10 minutes. The e f f l u e n t vapors pass through a condenser maintained at 10°C to reduce the water content of the gas stream, and then through a Tenax trap (65 mm long χ 3 mm i . d . , containing about 20 mg Tenax s o l i d adsorbent) where the nonpolar organic compounds are c o l l e c t e d . The "cleansed" a i r stream then recycles v i a a metal bellows pump to the water sample. After stripping the water, the Tenax trap was immediately transferred to the hot i n j e c t i o n port of a Hewlett Packard 5995B benchtop gas chromatograph-mass spectrometer (GC-MS). The v o l a t i l e s were then thermally desorbed from the Tenax and carried with the gas stream onto the front of a glass c a p i l l a r y column (coated with SE54). The f i r s t loop of the c a p i l l a r y column was dipped i n a Dewar containing l i q u i d nitrogen and served to cryogenlcally focus the v o l a t i l e concentrate a t the column front. After 5 minutes of thermal desorptlon and transfer, the l i q u i d nitrogen Dewar was removed and chromatography was begun. Data from the GC-MS was corrected f o r instrument response factors and stripping e f f i c i e n c i e s to ultimately derive the amounts of polybromomethanes present i n the sample. Seawater held i n the aquarium without macroalgae never showed any polybromomethane formation. 1-Chloropentane, added to the seawater as an Internal standard to monitor stripping e f f i c i e n c y , was recovered with a precision of 74 ± 18% (N-200). 1-Chlorobenzene added d i r e c t l y to the Tenax trap p r i o r to thermal desorptlon was


315

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O R G A N I C M A R I N E GEOCHEMISTRY

r e c o v e r e d w i t h a p r e c i s i o n o f 89 ± 12% (N=120). G i v e n the c o n d i t i o n s of our a l g a l i n c u b a t i o n s and a n a l y t i c a l c a p a b i l i t i e s , our lower l i m i t of d e t e c t i o n f o r a l g a l r e l e a s e s was about 50 ng polybromomethane/g dry algae»d. Polybromomethanes c o u l d be d e t e c t e d i n C a n a l seawater down t o a p p r o x i m a t e l y 10 ng/L.


RESULTS AND

DISCUSSION

The s e a s o n a l v a r i a t i o n s i n temperature and n u t r i e n t c o n c e n t r a t i o n s i n the C a n a l where the a l g a e were growing a r e shown i n T a b l e I . Water temperature v a r i e d by about 20°C b e i n g l o w e s t i n January and h i g h e s t i n August. N i t r a t e and phosphate were d e t e c t a b l e y e a r r o u n d , undoubtedly r e f l e c t i n g the i n f l u e n c e o f n u t r i e n t - r i c h Cape Cod Bay seawater. I n l i g h t o f the l i t e r a t u r e and g i v e n these e n v i r o n m e n t a l c o n d i t i o n s , these f u c o i d s grew o p t i m a l l y i n the s p r i n g , summer, and f a l l , and i n the w i n t e r grew s l o w e r due t o decreased l i g h t and c o o l e r temperatures (14 - 1 7 ) . Polybromomethanes were r e l e a s e d from b o t h A^ nodosum and JF. v e s i c u l o s l s t o l a b aquarium seawater throughout the y e a r ( F i g u r e 1 ) . Tribromomethane ( o r bromoform) was e m i t t e d by b o t h a l g a e a t r a t e s between 0.1 and 10 μg CHBr /g«d. R e l e a s e was h i g h i n f a l l o f 1983, b u t d i d n o t show any subsequent s e a s o n a l i t y . Dibromochloromethane was always observed from A^ nodosum, b u t was u n d e t e c t a b l e i n s e v e r a l F. v e s i c u l o s l s i n c u b a t i o n s . Dibromomethane was o n l y produced and r e l e a s e d by A^ nodosum, a l t h o u g h n o t d u r i n g the w i n t e r . T h i s dihalo-compound was r e l e a s e d a t g e n e r a l l y lower r a t e s ( n o t e s c a l e d i f f e r e n c e i n F i g u r e 1) than the two h a l o f o r m s . Dibromochloromethane and dibromomethane appear t o be r e l e a s e d somewhat more e x t e n s i v e l y d u r i n g the summer and f a l l . Cape Cod C a n a l seawater samples c o l l e c t e d a t h i g h t i d e ( i . e . , when b o t h f u c o i d p o p u l a t i o n s would be submerged) always c o n t a i n e d d e t e c t a b l e bromoform, a l t h o u g h no s e a s o n a l p a t t e r n c o u l d be seen ( F i g u r e 2 ) . Low t i d e water samples g e n e r a l l y showed u n d e t e c t a b l e bromoform, e x c e p t f o r a few s p r i n g and summer samples. C l e a r l y these f u c o i d p o p u l a t i o n s imparted s u b s t a n t i a l bromoform to the s u r r o u n d i n g seawater, and t h i s polybromomethane t y p i c a l l y had no o t h e r d e t e c t a b l e s o u r c e s as r e f l e c t e d by most low t i d e samples. Dibromochloromethane and dibromomethane were o n l y observed i n two C a n a l water samples each. S i n c e dibromochloromethane i s r e l e a s e d by these a l g a e a t comparable r a t e s t o bromoform, i t appears t h a t some s i n k i s r e l a t i v e l y more i m p o r t a n t f o r t h i s compound. Kaczmar e t a l . (18) r e p o r t t h a t C H B r C l v o l a t i l i z e s about 50% f a s t e r than C H B r , and p o s s i b l y i t I s t h i s mechanism which m a i n t a i n s C H B r C l c o n c e n t r a t i o n s below our d e t e c t i o n l i m i t s i n h i g h t i d e Cape Cod C a n a l seawater. Dibromomethane was o n l y r e l e a s e d from ^. nodosum and then a t s e v e r a l times lower r a t e s than bromoform, thus I t s n o n d e t e c t i o n i n the C a n a l water I s n o t s u r p r i s i n g . A s i m p l e c a l c u l a t i o n suggests t h a t the a l g a l i n c u b a t i o n r e l e a s e r a t e s we observed i n the l a b o r a t o r y c o u l d r e a d i l y a c c o u n t f o r the C a n a l water c o n c e n t r a t i o n s . I f A. nodosum and vesiculosls biomasses a r e about 1 0 g d r y a l g a e per m ( 1 7 ) , they w i l l r e l e a s e about 100pg o f bromoform/m «hr. Thus, a 20-50 cm deep l a y e r o f water c o v e r i n g the a l g a e a t h i g h t i d e w i l l take o n l y s e v e r a l minutes to b u i l d up the c o n c e n t r a t i o n s we o b s e r v e . Thus our f i e l d o b s e r v a t i o n s of 20-80 ng bromoform/L seem c o n s i s t e n t w i t h the l a b o r a t o r y r e s u l t s . 3

2

3

2

3

2

2


18.

GSCHWEND AND MACFARLANE

Polybromomethane Release to Seawater317

TABLE I . Temperature and n u t r i e n t d a t a f o r Cape Cod C a n a l seawater (September 1983-December 1984).

Cape Cod C a n a l Seawater


Date

Temp(°C)

9.10.83 9.20 9.25 9.29 10.05 10.18 10.26 11.01 11.08 11.15 11.22 12.01 12.06 12.13 12.22

17.5 15.0 15.8 15.0 14.5 14.5 11.0 10.0 9.7 9.2 9.0 8.5 6.5 7.0

1.04.84 3.01 3.07 3.15 3.21 4.03 5.03 5.10 5.15 5.22 6.04 6.18 7.10 7.24 8.07 8.22 9.12 10.04 10.24 11.15 12.12

1.5 3.5 3.7 1.5 3.8 5.0 8.0 10.0 9.2 10.3 12.0 13.5 16.5 14.5 21.0 21.5 14.3 14.4 14.1 9.5 7.2

-

N0

(μΜ)

3

Ρ0

(μΜ)

4

3.5

0.16*

1.1 11.2 3.4

0.99t l.OOt 0.93t

-

-

2.4 4.3 4.5 4.6/2.6

0.95t 0.93t 1.12t 0.79t

8.0

1.68t

-

2.4 4.0 3.7 5.2 3.2 0.7/1.4 3.5 5.5 4.9 7.8 2.2 3.3 1.4

-

-

0.25* 0.22*

-

0.12* 0.16*

0.44/0.13* 0.19/0.15* 0.15/0.19* 0.18±0.01* 0.19/0.34*

-

0.34/0.35* 0.34* 0.34/0.34*

- no d a t a a v a i l a b l e f o r t h i s sample d a t e . 3

* ΡΟΐψ " a n a l y z e d by the Stannous c h l o r i d e method ( S t a n d a r d Methods, APHA, 1980). 3

t ΡΟΐψ " a n a l y z e d by the A s c o r b i c a c i d method ( S t a n d a r d Methods, APHA, 1980).


318

O R G A N I C M A R I N E GEOCHEMISTRY

14

~l—ΓΊ

1 Algal

12

1

1

release

1

»

I

t o seawater

10 Ascophyllum

8

Fucus

3s

nodosum

vesiculosis

6


PC

4

0 12

Γ."

H

i ϊ ι — ι

κ

h-

ί

GO

PQ 33 Ο

&0 3.

F l g u r e 1. S e a s o n a l r e l e a s e r a t e s o f C H B r , C H B r C l , and C H B r t o seawater by the brown a l g a e , A s c o p h y l l u m nodosum and Fucus v e s i c u l o s l s . Open symbols I n d i c a t e no d e t e c t a b l e r e l e a s e . 3

2

2


2


GSCHWEND AND MACFARLANE


120 h

1983

1984

F i g u r e 2. S e a s o n a l C H B r c o n c e n t r a t i o n s (ng-ΙΓ ) i n n e a r s h o r e seawater o f t h e Cape Cod C a n a l , Sagamore, MA. 1

3



320

ORGANIC MARINE GEOCHEMISTRY

A l g a l v e r s u s E p i p h y t i c Source. Our o b s e r v a t i o n s may a l s o p r o v i d e some I n s i g h t to the g e n e s i s o f the polybromomethanes. Foremost i n t h i s r e g a r d i s whether the a l g a e o r some a t t a c h e d microorganisms s y n t h e s i z e these halogenated compounds. Gschwend e t a l . (7) p r e v i o u s l y reasoned t h a t a l g a e were most l i k e l y r e s p o n s i b l e ; however, the absence o f a s t r o n g s e a s o n a l i t y i n the polybromomethane r e l e a s e s from ^. nodosum and F\ v e s i c u l o s l s i n d i c a t e d t h a t t h i s c o n c l u s i o n may be wrong, s i n c e n e a r l y e v e r y o t h e r process a s s o c i a t e d w i t h these macrophytes does v a r y y e a r r o u n d . F i r s t , numerous s t u d i e s o f temperate f u c o i d a l g a e have demonstrated major changes i n t h e i r growth r a t e s and r e p r o d u c t i v e p h y s i o l o g y throughout the y e a r (15, 1 7 ) . I n v e s t i g a t i o n s o f a l g a l m e t a b o l i t e s , i n c l u d i n g the halogen I , a l s o show r e g u l a r s e a s o n a l i t y (_16, ^19» 20)· Further a l g a l peroxidase enzymes, which c o u l d r e a s o n a b l y be proposed to o x i d i z e h a l l d e s p r i o r t o t h e i r a d d i t i o n to o r g a n i c s u b s t r a t e s , have a l s o been found to v a r y g r e a t l y I n a c t i v i t y i n b o t h A. nodosum and F. v e s i c u l o s l s ( 2 1 ) . Both s p e c i e s e x h i b i t e d the g r e a t e s t a c t i v i t y i n the s p r i n g and summer; A. nodosum p e r o x i d a s e a c t i v i t y d i m i n i s h e d to one t e n t h maximal l e v e l s by September and P\ v e s i c u l o s l s showed more than f i f t y times decrease i n t h i s e n z y m a t i c a c t i v i t y by J u l y - S e p t e m b e r . C l e a r l y we d i d n o t see these magnitudes o f changes I n polybromomethane r e l e a s e on a s e a s o n a l b a s i s . F u r t h e r , Hewson and Hager (3) and V i l t e r (22) r e p o r t t h a t p e r o x i d a s e p r e p a r a t i o n s from ^. nodosum and v e s i c u l o s l s could not o x i d i z e bromide (a p r e r e q u i s i t e s t e p to polybromomethane f o r m a t i o n ) . Thus i t appears t h a t these a l g a e do n o t b i o s y n t h e s i z e and r e l e a s e the polybromomethanes we o b s e r v e . We have attempted to s u p p o r t t h i s c o n c l u s i o n by i n o c u l a t i n g seawater w i t h s u r f a c e m a t e r i a l g e n t l y s c r a p e d o f f the s u r f a c e s o f A. nodosum and F\ v e s i c u l o s l s p l a n t s and m o n i t o r i n g f o r polybromomethane formation. I n b o t h cases bromoform c o n c e n t r a t i o n s i n the water were observed to i n c r e a s e ( F i g u r e 3 ) . M e r c u r i c c h l o r i d e p o i s o n i n g , 0.2 pm f i l t e r i n g , and a u t o c l a v i n g the i n o c u l a t e d seawater prevented t h i s bromoform p r o d u c t i o n . Thus, some component o f the e p i p h y t i c community may be i n v o l v e d . The e p i p h y t i c commun!tes o f ^A. nodosum and F. v e s i c u l o s l s i n c l u d e b a c t e r i a , y e a s t s , o t h e r f u n g i , and p h y t o p l a n k t o n . Most o f these microorganisms appear to undergo y e a r r o u n d v a r i a t i o n s i n t h e i r abundance. U s i n g v i a b l e count t e c h n i q u e s , Chan and McManus (23) found the f e w e s t b a c t e r i a on A. nodosum d u r i n g the summer months o f J u l y and August. S i e b u r t h and T o o t l e (24) extended t h i s r e s u l t by e x a m i n i n g b o t h A. nodosum and J?. v e s i c u l o s l s w i t h s c a n n i n g e l e c t r o n microscopy ( S E M T . They found b a c t e r i a l c o l o n i z a t i o n o f these a l g a e was g r e a t e s t i n November and A p r i l and d i m i n i s h e d to v i r t u a l l y c l e a n I n May t o J u l y . S e s h a d r l and S i e b u r t h (25) a l s o enumerated the y e a s t s growing on these f u c o i d a l g a e and a g a i n d i s c o v e r e d a s t r o n g s e a s o n a l v a r i a t i o n . F u r t h e r , these same y e a s t s p e c i e s were most abundant on a r e d a l g a , Chondrus c r i s p u s , w h i c h e x h i b i t s l i t t l e o r uo polybromomethane p r o d u c t i o n ( 7 ) . F i n a l l y , some m i c r o a l g a e grow as e p i p h y t e s on f u c o i d s ( 2 4 ) ; b u t no b r o m i n a t i o n c a p a b i l i t i e s have e v e r been demonstrated i n a m i c r o a l g a e ( 7 ) . A l t h o u g h c u l t u r i n g and SEM approaches t o e v a l u a t i n g microorganisms on f u c o i d s may be somewhat crude f o r our purpose, these a s s a y s c o n s i s t e n t l y demonstrate s t r o n g s e a s o n a l t r e n d s i n the dominant m i c r o b i a l s u b p o p u l a t l o n s l i v i n g on A. nodosum and F. v e s i c u l o s l s . C o n s e q u e n t l y , we s u s p e c t these microorganisms do n o t make the polybromomethanes.



18. G S C H W E N D AND M A C F A R L A N E


321

F i n a l l y , V i l t e r e t a l . (21) have suggested t h a t an ascoraycete, M y c o s p h a e r e l l a a s c o p h y l l l , growing s y m b i o t i c a l l y on A. nodosum may be r e s p o n s i b l e f o r the bromoperoxldase a c t i v i t y a t t r i b u t e d t o t h a t f u c o i d . T h i s fungus i s a p p a r e n t l y always p r e s e n t on t h i s brown a l g a ( 2 6 ) , c o n s i s t e n t w i t h the c o n t i n u o u s p r o d u c t i o n o f polybromomethanes. M. a s c o p h y l l l has been I s o l a t e d and c u l t u r e d by Pederson and F r i e s (27) and p r o d u c t i o n o f brominated phenols by the c u l t u r e was o b s e r v e d . A l t h o u g h the ascomycete I s thought t o be r a r e on f \ v e s i c u l o s l s ( 2 6 ) , the same bromophenols a r e observed from t h a t algae (28), suggesting a s i m i l a r source. S i n c e bromoperoxidases c a p a b l e o f f o r m i n g bromophenols would a l s o be a b l e t o y i e l d bromoform from the a p p r o p r i a t e o r g a n i c p r e c u r s o r s , M. a s c o p h y l l l u b i q u i t o u s on k* nodosum, o r s i m i l a r f u n g i on o t h e r a l g a e , appears t o be the most l i k e l y source o f the polybromomethanes. As we have noted p r e v i o u s l y (])» these o r o t h e r e p i p h y t e s a r e i n v o l v e d , the microorganisms must be q u i t e h o s t - s p e c i f i c t o a c c o u n t f o r the d i f f e r e n t c o m b i n a t i o n s and r a t e s o f polybromomethane r e l e a s e s observed from between v a r i o u s a l g a l species. I f ascomycetes l i v i n g i n m y c o p h y c o b i o t i c r e l a t i o n s h i p s w i t h f u c o i d s (25) a r e indeed the answer, i t i s tempting t o s p e c u l a t e on the f u n c t i o n o f t h e i r bromoperoxldase a c t i v i t y . Such a s t r o n g o x i d a t i v e e n z y m a t i c c a p a b i l i t y i s common t o f u n g i ( 2 9 ) , and i t c o u l d be used by the ascomycete t o brominate the p l a n t s u r f a c e c h e m i c a l s ( e . g . , forming bromophenols such a s l a n o s o l ) . These m e t a b o l i t e s may then p r o t e c t the a l g a e from h e r b i v o r e s and m i c r o b i a l c o l o n i z a t i o n , i n r e t u r n f o r macrophyte s u p p l y o f n u t r i t i o n t o the fungus. 3

Γ

January Seawater amended with A. nodosum s c r a p e

X

ο Unamended

2

3

Seawater

r

July

Ο

S e a w a t e r amended with F. vesiculosis scrape

00

X

Unamended

0

100

Seawater

200

Time (hours)

F i g u r e 3. P r o d u c t i o n o f C H B r i n seawater w i t h and macroalgae s u r f a c e m a t e r i a l added. 3

without


322

ORGANIC MARINE GEOCHEMISTRY

SUMMARY Polybromomethanes ( C H B r , C H B r C l , C H B r ) were found t o be r e l e a s e d y e a r r o u n d from two common rockweeds, A. nodosum and vesiculosls, or t h e i r c l o s e l y a s s o c i a t e d m i c r o f l o r a . This b i o l o g i c a l source appears s u f f i c i e n t t o s u p p o r t p a r t s p e r t r i l l i o n l e v e l s o f these h a l o g e n a t e d o r g a n i c compounds i n nearby seawater. S e v e r a l l i n e s o f e v i d e n c e i n d i c a t e t h a t e p i p h y t i c f u n g i may a c t u a l l y a c c o m p l i s h t h e polybromomethane b i o s y n t h e s i s . F u t u r e r e s e a r c h c l a r i f y i n g the g e n e s i s and f u n c t i o n s o f these v o l a t i l e brominated compounds i s s u r e l y warranted. 3

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