Marine Chemistry in the Coastal Environment

36 Toxins and Bioactive Compounds in the Marine Environment GEORGE M. PADILLA

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Department of Physiology and Pharmacology, Duke University Medical Center, Durham, N . C . 27710 D E A N F. MARTIN Department of Chemistry, University of South Florida, Tampa, Fla. 33620

Biodynamic compounds are o r g a n i c products which exert a s p e c i f i c e f f e c t on o t h e r organisms i n t h e marine environment ( 1 ) . They may be o f n a t u r a l o r a r t i f i c i a l o r i g i n and may be m o d i f i e d by p h y s i c a l and b i o l o g i c a l processes. I t i s f r u i t f u l t o consider the i m p l i c a t i o n s o f t h i s concept, p a r t i c u l a r l y as i t a p p l i e s t o the c o a s t a l areas o f t h e ocean where communities o f organisms are h i g h l y interdependent. One could say they communicate w i t h one another through the e l a b o r a t i o n and r e l e a s e o f "active" compounds t h a t form a complex dynamic network. In s p i t e o f the f a c t t h a t these " a c t i v e " compounds occur a t v e r y d i l u t e c o n c e n t r a t i o n s , they n e v e r t h e l e s s may a t times a f f e c t t h e p h y s i o l o g i c a l w e l l being o f s p e c i f i c organisms, as i s the case w i t h sex a t t r a c t a n t s (2) and i c h t h y o t o x i n s (3). To f u l l y understand t h e r o l e o f b i o a c t i v e compounds i n t h e sea, we must c o n s i d e r how they are d i s t r i b u t e d throughout the marine environment, the types o f organisms which produce them, and f i n a l l y , the s p e c i f i c modes o f a c t i o n , i f any, t h a t these compounds have on t a r g e t organisms. To achieve a l l t h i s , the study o f biodynamic compounds must be approached from a v a r i e t y o f s c i e n t i f i c d i s c i p l i n e s . We are d e a l i n g not merely w i t h a problem o f chemical i s o l a t i o n and i d e n t i f i c a t i o n , but w i t h an a n a l y s i s o f t h e r e l a t i o n s h i p s between organisms, t h e i r e x t e r n a l environment, and one another. P h y s i o l o g i c a l , e c o l o g i c a l , and chemical a t t r i b u t e s which d e f i n e these r e l a t i o n s h i p s must be considered and e x p e r i m e n t a l l y evaluated. O r i g i n and Sources o f Biodynamic Compounds I t i s c l e a r t h a t t h e r e i s no s i n g l e o r i g i n o f b i o a c t i v e compounds. Numerous organisms from w i d e l y d i f f e r e n t c l a s s e s are known t o c o n t a i n o r r e l e a s e b i o l o g i c a l l y a c t i v e chemicals. Table I shows a s e l e c t i o n o f such compounds. Even i f such compounds were produced by a s i n g l e c l a s s o f animals (e.g. p h y t o p l a n k t o n ) , i t i s not c l e a r t o what extent these n a t u r a l products are m o d i f i e d 596

In Marine Chemistry in the Coastal Environment; Church, T.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

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Table I Some Unique Organic Compounds Derived from Marine Organisms Compound


Halogenated Sesquiterpeneid Betaine Aromatic terpenoid Indoleninones Brominated phenols Prostaglandins (15-epiPGA ) Carageenan (polysaccharide) Saponin Diethylamine-dithiolanes

Organism

Ref.

Red Alga (Laurecia elata)

(10)

Soft Coral (Pseudoterogorgia americana Alga (Taonia atomaria) Mollusc (Dicathrais orbita) Red Alga (Corallina o f f i c a l i s ) Coral (Plexaura homonalla)

(16) (11) (14) (4) (9)

Red Seaweed (Chondrus crispus)

(15)

Starfish (Asterias sp) Annelid (Lambriconereis heteropoda) Nemertine Worms (Paranemertes)

(13) (12)

2

Anabaseine

(17)

as they pass through the various trophic levels of the whole biotic community. In addition they are mixed with other organic compounds resulting from the continual enrichment and modification of the environment that occurs through the operation of a variety of cycles (e.g. carbon, nitrogen, etc.). These cycles also interact directly with the food chain forming a complex network of energy exchange. In i t s simplest form, the food chain denotes the passage of nutrients from the "producers" (e.g. phytoplankton) to the "consumers" which include the zooplankton and progressively larger organisms at various trophic levels. The carnivores of the sea, among which we may include ourselves, constitute the last link of the chain. In a recent a r t i c l e , Benson and Lee (5) stated that the food chain i s not only a mechanism for the distribution of organic matter but serves as a dynamic energy depot. They estimated that at least one-half of the organic material produced in the ocean by the primary producers i s converted and temporarily stored into waxes by copepods, a type of small, abundant crustaceans. The waxes are not only used as energy sources, they serve as well to regulate these organisms' activity and behavior. For example, a high degree of unsaturation in the wax allows copepods to inhabit polar regions by acting as a sort of biological "anti-freeze". Similarly, changes in an organism's specific gravity, reflecting an altered wax content, permit copepods to migrate v e r t i c a l l y for considerable distances. Such vertical migration patterns follow the complex developmental sequences from egg to adult (5).



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One usually does not consider food as a specific biodynamic product, but in this instance waxes serve more than a dietary role. Their distribution and modification as they pass through the food chain i s an example of the dynamic nature of the roles they play as bioactive compounds. Recently Banner (6) discussed at some length the food chain theory of Randall as i t applies to the origin of ciguatoxin. This toxin w i l l appear sporadically and as yet unpredictably as a contaminant of reef f i s h in the Pacific. The basic premise of Randall's theory i s that f i s h owe their intoxication to their mode of l i f e . That i s to say, the herbivorous f i s h feed upon certain toxigenic algae on coral reefs. It i s thought these algae are the primary producers of the toxin. Even though the toxin does not seem to affect these small herbivorous f i s h , i t is toxic to the larger carnivores which feed upon them. Two p o s s i b i l i t i e s are implicit in this transfer : (1) the principle may become toxin as i t i s metabolized by the herbivorous f i s h at the lower trophic levels or (2) the toxic principle i s not changed but i s merely concentrated in the flesh of the f i n a l host f i s h . It i s in this concentrated form lethal to man. The concentration and storage of ultimately toxic organic compounds by organisms in seemingly "inert" forms i s one of the more important aspects of the transfer of bioactive compounds through the food chain. This phenomenon i s not limited to biotoxins produced by members of the food chain, i t i s also applicable to compounds which have been added to the environment by man (e.g. pesticides, organo-metallic compounds, plasticizers, petroleum by-products, etc. [7]). Halstead (8) was one of the f i r s t to note the importance of this factor. He was particularly concerned with the impact this would have on the plans to derive a high protein " f i s h flour" from otherwide inedible "trash" f i s h . A high level of contamination in the ocean would of course destroy the usefulness of this natural resource. Marine organisms do not store only harmful products. They have in fact been used as source material for a wide variety of useful industrial and medicinal compounds ( 1). For example, the prostaglandins are found in soft corals in quantities up to 1.3% of the dry weight (1,9). Included i n this class are very potent pharmacological agents with a wide spectrum of physiological activity (1). Figure 1 shows the physiologically active ones. We have also listed i n Table I a selection of some unique compounds obtained from marine organisms. The l i s t i n g i s not comprehensive but i s indicative of the diversity of these natural products. Included are halogenated phenols (4), aromatic terpenes (10,11), t h i o l amines (12), saponins (13), indoleninones (14), polysaccharides (15), betaine (16), and the toxin anabaseine (17). As you can see from Table I, these compounds are derived from diverse organisms. A more extensive l i s t would no doubt increase the diversity of the plants and animals that yield such compounds.


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As discussed in this symposium, biodynamic compounds also originate from our highly sophisticated technological a c t i v i t i e s . Although pollution may not be entirely unavoidable, the impact which our industrial culture has on the environment i s now well recognized. The deposition of chemical by-products of technology into the coastal environment i s significant not only because of their long term harmful effects on biological communities, as was the case with certain pesticides (7\ 18), but because of their existence as chemical contaminants. This i s of particular con cern to those studying natural products from marine sources. There i s no guarantee that complex organic molecules from natural sources can be isolated in the absence of contaminating exogenous materials. This i s particularly true for widely used industrial compounds such as phthalates which are now reported to be widely distributed in the terrestial and aquatic environment (19,20). We became aware of this problem during a recent analysis of a red-tide sample o f f the coast of Florida (21). We were trying to isolate and identify the toxins produced by the red-tide dinoflagellate Gymnodinium breve. The isolation procedure involved the extraction of a large volume of sea water with organic solvents. The crude extract, after suitable concentra tion procedures, was resolved into three toxic fractions by column and thin layer chromatography. The infrared spectra of the toxic fractions (I > Ic, Id) are shown in Fig. 2. The similarity between the spectra of the toxic fractions and that of phthalate-like compounds i s readily apparent. To emphasize this point, we have shown the spectrum of a tygon-tubing extract in Fig. 2, as well as the non-toxic fraction (IHe) which i s usually discarded during the extraction procedure. Note that the tygon extract and fraction I I I have a strong absorption peak at 7.8-7.9 μ (arrows) that i s not present in the toxic fractions, I » Ic> Id- The remainder of the spectra are very similar. The presence of the phthalate compounds in a l l fractions was verified by mass spectroscopy. Fragments with the diagnostic values of 279, 167, and 149 generated by phthalates, particularly dim ethyl hexyl phthalate (20), were readily obtained. Further mass spectroscopic analyses, performed by Dr. D. Brent of the Burroughs-We11come Laboratories, revealed the presence of addi tional fragments with m/e values > 700. It i s presumed that these were generated by the G. breve toxin (unpublished results). We cite this example to i l l u s t r a t e the fact that these man-made additions w i l l add to the technical problems encountered in the study of natural products suspected of having a specific physio logical effect. a

C

a

Diversity and Distribution of Biodynamic Compounds A l l phyla have representative organisms as sources of bio dynamic compounds. Because of the pyramidal distribution of organisms i n the sea, the phytoplankton» zooplankton, and smaller


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Compounds


Frequency (cm ')

λ(ρ) Proceedings of the First International Conference on Toxic Dinoflagellate Blooms

Figure 2. Infrared spectra of G . breve toxins and tygon tubing extract. Upper abscissa, frequency (cm' ); lower abscissa, wave length (μ = mi crons); ordinate, absorbance. I = toxic fraction I ; h — toxic fraction l J = toxic fraction I ; III = nontoxic fraction III ; T — tygon tubing extract (21). 1

A

c;

D

D

C

A

C

(ex)



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invertebrates not only occur in larger numbers, but have a higher incidence of speciation and thus may yield a greater variety of bioactive compounds. In other words, they have evolved into specific ecological niches which form a balanced community of organisms. A second feature which affects the distribution of biodynamic compounds i s the seasonal succession of algal blooms, which follow c y c l i c a l hydrographie changes in the coastal environment (e.g. changes in s a l i n i t y during the summer, enrichment of coastal waters from land run-off following seasonal rains, etc.). It has been suggested that seasonal factors play a role in the outbreak of red tides or the incidence of similar phytop1ankton-1inked toxicities (22). For example, a study of the monthly incidence of ciguatoxin poisonings during 1966-1968 showed that there was a peak of the incidence of toxicity during the summer months of 1968, particularly June-July (6). The level of toxicity was low during the winter months and began to r i s e during the spring. As discussed earlier, the increased toxicity was due to a parallel increase in the growth of algae. In a study on the incidence of hemolytic agents concentrated by oysters, which were f i e l d collected from 17 stations along the coast of North Carolina, we found a 2 to a 5-fold increase in the hemolytic t i t e r during the months of September and October, coincident with increase in phytoplankton blooms in this region (23). The hemolysin was completely absent during the winter months. The incidence of hemolysin appearance was taken to be an indication that algae containing hemolytic components were being concentrated by the oysters. A similar assay was used to monitor an incipient redtide outbreak in Florida (24). The seasonal occurrence of red tides in the coastal environment has been well documented (22,25). For example, Hartwell (25) recently described the hydrographie factors which may set the stage for the red-tide outbreaks of the dinoflagellate Gonyaulax tamarensis. Until recently, these organisms were rarely seen in the western gulf of Maine. However, during September 1972, a bloom was seen in the coastal waters off New Hampshire. As many as 4600 yg of toxin/100 g meat of the soft-shelled clam had been accumulated at that time. A second smaller bloom occurred i n early June of 1974 and a major one recurred in August 1974. Evidence was presented to show that shifts of a variety of hydrographie factors such as s a l i n i t y , temperature, water currents, level of nutrients, and dissolved oxygen favored the outbreak of red tides during the summer months of those years. Steidinger (26) also examined the factors which may influence the incidence of red tides, but from a more ecological point of view. According to her studies, there are at least 3 elements common to red-tide outbreaks : (a) the rate of increase in population size, (b) the presence of favorable hydrographie factors and (c) the maintenance of dinoflagellate populations in discrete "patches" by other hydrologie or météorologie forces. The


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interplay between these factors sets the stage for a red-tide outbreak. How such an interplay i s brought about i s not known at present, but these investigations do show that i t i s a combination of biological and physical forces that w i l l bring about an expansion or bloom in specific phytoplankton populations. Were i t not for the liberation of potent toxins into the marine environment, the presence of these algal blooms would have gone undetected.


Detection and Identification of Biodynamic Compounds It i s unreasonable to expect that every biodynamic compound w i l l exert dramatic and widespread actions such as the mass fish mortalities which accompany the outbreak of red tides. It would also be an unsatisfactory state of affairs to rely on such ecological disasters before we proceed to identify the toxic principle involved. One's a c t i v i t i e s would be reduced to a "follow-up strategy: await a red tide, gather sufficient material, and then discover i t s mode of action. Whatever the socio-economic implications of this course of action, not only are red tides sporadic and d i f f i c u l t to predict, toxins are produced at extremely low concentrations (i.e. at a few micrograms/liter). It i s only because they are specific and potent that they do not escape our attention. Table II shows the relative toxicity of a variety of marine toxins (1). Even when one considers lethality as the index of activity, compounds such as tetrodotoxin, saxitoxin, and Gymnodinium breve toxin are 11

Table II Relative Toxicities of Selected Marine Biotoxins Source

Toxin

Palytoxin Saxitoxin Tetrodotoxin Sea snake venom Ciguatoxin Prymnesin Holothurin A Ostracitoxin

a

Zoanthid (Palythoa sp) Dinoflagellate (G. catenella) Puffer fish (S. rubripes) Sea snake (L. semifasciata) Moray eel (G. javanicus) Golden alga (P_. parvum) Sea cucumber (A. agassizi) Boxfish (0. lengitinosus)

a

Lethal dose (yg/kg) 0.15 3.4 8.0 130 500 1,400 10,000 200,000

b

See (1) for references; L D or minimum lethal dose in mice From "Marine Pharmacognosy" with permission of Academic press. 5 0



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effective at very low concentrations. It has been reported that these toxins inhibit specific physiological a c t i v i t i e s (i.e. neuronal transmission, active transport, hemolysis, etc.) at very low concentrations (y 10-8-10-14 g/L). Yet lethality i s almost the universal bioassay used (1,3,27), and i n many cases i t i s the only one. Most often, i t i s used with laboratory animals rather than organisms derived from the estuarine environment (7). Since marine animals communicate with each other through the medium which surrounds them, a biologically active compound must exert i t s i n i t i a l stimulatory or noxious effect at the c e l l membrane . This barrier need not be at the g i l l s or at the outer integument but may reside at a specific locus within the animal, i.e. the nervous system, kidney, hepatopancreas, etc. The biological a c t i v i t i e s of marine bioactive compounds should thus be examined in terms of effects on membrane-dependent a c t i v i t i e s . This would include assays based on electrophysiological measurements, transport mechanisms, as well as the binding to specific membrane sites which govern the activity of membrane-bound enzymes. In addition, an a f f i n i t y for membrane components may result in the disruption of membrane structure. An example of this interaction i s seen in the hemolytic activity of toxins from the euryhaline alga Prymnesium parvum (28). A variety of experimental approaches for determining the activity of compounds on excitable membranes (e.g. the giant axon of the squid, neuromuscular junctions, etc.) are presently available (3). For example, methods using the voltage-clamp technique and recording microelectrodes have defined the specific effects of marine toxins on the ionic conductances of excitable cells (see [3] and [29] for review). The frog skin preparation in which one relates changes i n the short c i r c u i t current to the active transport of ions has also been used to measure the activity of toxins from Gymnodinium breve (30). Another useful approach employs the frog nerve-muscle preparation to study the effect of biotoxins on the release and activity of neurotransmitters. It has proved valuable in the analysis of toxins derived from dinoflagellates and blue-green algae (3). It i s clear from the above that the study of biodynamic compounds i s in essence based on the structure-function relationship which they have with biological membranes. This statement implies that the activity of these compounds i s dependent on the existence of membrane sites bearing a unique and specific a f f i n i t y to effector compounds. This approach i s analogous to one based on the receptor concept developed for studies on drug and hormone act ion. According to this concept, a drug i s active because of i t s interaction with a specific cellular component to which i t binds. Subsequent events or a series of events are then triggered by this interaction (32). For example, a specific enzyme may be activated or inhibited, a transmitter substance may be released or a second chemical effector produced. Toxins



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which are highly reactive must have such a specific interaction by virtue of their chemical structure. Their relative potency is i n effect a measure of their a f f i n i t y to a specific membrane component. This situation i s thought to explain the activity of tetrodotoxin. It has been shown to have a unique a f f i n i t y for the sodium channels i n the giant axon of the squid or lobster. The inward movement of sodium i s prevented and although the movement of potassium i s unaffected, the conduction of e l e c t r i c a l pulse along the nerve axon i s blocked (see [.1,3] for r e f s . ) . A comparison between saxitoxin and tetrodotoxin shows that differences in physico-chemical properties may account for variations i n their physiological activity as discussed by Doig et^ a l (1). For example, both toxins possess an N-alkylsubstituted guanidinium ion, (H N)2C=NH , as part of their structure. Such ions are thought be act as current carriers and possible substitutes for sodium ions. However, the bulkiness of the remainder of the molecule offers a steric hindrance which impedes the passage of the guanidinium ion, and thus blocks the movement of sodium ions through "channels" i n the c e l l membrane. Henderson and coworkers (31) recently examined the binding properties of both toxins at the sodium channels of nerve membrane preparations i n the presence and absence of a series of monovalent, divalent, and trivalent cations. They provide evidence to show that the sodium channel possesses a negatively charged site with an apparent pKa between 5 and 6. It i s at this site that both tetrodotoxin and saxitoxin w i l l bind and block the channel with the guanidinium ion portion of the molecule. Divalent and trivalent cations reversibly compete with the toxins for this site as do some of the monovalent ions. This elegant approach i s of necessity possible only with toxins of known chemical structure. The extent to which our understanding of membrane function w i l l be advanced by a parallel understanding of the physico-chemical attributes of marine biotoxins i s considerable. +

2

Concluding Remarks Marine organisms are linked to each other through a dynamic network of chemical interactions. Bioactive compounds are the elements of this network and as such serve to establish and maintain the physiological and ecological balance of the communities in the coastal environment. It i s thought that the s p e c i f i c i t y and potency of their action i s an expression of their a f f i n i t y to specific membrane sites. It i s only through a mult idisciplinary approach that we w i l l gain an understanding of the marine environment. We w i l l have at our disposal compounds to be used as probes of membrane function in both research and medicine.


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Acknowledgements This work was supported in part by a Food and Drug Administration Grant to GMP (FD 00120) and a Research Career Development Award to DFM (1 K04 GM 4269) from NIGMS.


Abstract Although our knowledge of the exact chemical structures of bioactive compounds derived from marine sources is limited, it is of value to consider to what extent they originate as by-products of metabolism, constitutents of the organisms that produce them, or as chemically altered compounds introduced by man into the environment. Bioactive compounds were considered in terms of the biological activity they possess, toxicity being one example. The physiological effects of compounds such as prostaglandins and sex attractants were discussed to emphasize the role of bioactive compounds which is not necessarily detrimental to other organisms in the ocean. The structure-function relationship of bioactive compounds was examined at some length and their value as probes of biological activity in research and medicine was evaluated. Literature 1.

2. 3.

4. 5. 6.

7.

8.

9. 10. 11.

Cited

Doig, M.T., Martin, D.F. and Padilla, G.M. In: "Marine Pharmacognosy" (Eds. Martin, D.F. and Padilla, G.M.), pp. 1-35, Academic Press, New York, 1973. Müller, D.G., Jaenicke, L., Donike, M. and Akinobi, T. Science (1971) 171, 815. Sasner, J.J., Jr. In: "Marine Pharmacognosy" (Eds. Martin, D.F. and Padilla, G.M.), pp. 127-177, Academic Press, New York, 1973. Pedersen, Μ., Senger, P. and Fries, L. Phytochemistry (1974) 13, 2273. Benson, A.A. and Lee, R.F. Scientific American (1975) 232, 77. Banner, A.H. In: "Bioactive Compounds from the Sea" (Eds. Humm, H.J. and Lane, CE.), pp. 15-36, Marcel Dekker, New York, 1974. Vemberg, F.J. and Vernberg, W.B. (Eds) "Pollution and Physiology of Marine Organisms", 492 pp., Academic Press, New York, 1974. Halstead, B.W. In: "Drugs from the Sea" (Ed. Fruedenthal, H.D.), pp. 229-239, Marine Technology Society, Washington, D.C., 1968. Weinheimer, A.J. and Spraggins, R.L. Tetrahedron Letter (1969) No. 59, 5185. Sims, J.J., Lin, G.H.Y. and Wing, R.M. Tetrahedron Letter (1974) No. 39, 3487. Gonzalez, A.G., Darias, J., Martin, J.D. and Pascual, C. Tetrahedron (1973) 29, 1605.


36. 12. 13. 14. 15.

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17. 18. 19. 20. 21.

22. 23. 24. 25.

26.

27. 28. 29. 30. 31. 32.

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Okaichi, T. and Hashimoto, Y. Bull. Jap. Soc. Sci. Fish. (1962) 28, 930. Yasumoto, T., Watanabe, T. and Hashimoto, Y. Bull. Jap. Soc. Sci. Fish. (1964) 30, 357. Baker, J.T. and Duke, C.C. Aust. J. Chem. (1973) 26, 2153. Mueller, G.P. and Rees, D.A. In: "Drugs from the Sea" (Ed. Fruedenthal, H.D.), pp. 214-255, Marine Technology Society, Washington, D.C., 1968. Weinheimer, A.J., Metzner, E.R. and Mole, M.L., Jr. Tetrahedron (1973) 29, 3135. Kern, W.R. In: "Marine Pharmacognosy" (Eds. Martin, D.F. and Padilla, G.M.), pp. 37-84, Academic Press, New York, 1973. Seba, D.B. and Corcoran, E.F. Science (1973) 191, 925. Ogner, G. and Schnitzer, M. Science (1970) 170, 317. Mayer, F.L., Jr., Sailing, D.L. and Johnson, J.L. Nature (1972) 238, 411. Padilla, G.M., Kim, Y.S. and Martin, D.F. Proc. 1st Int. Conf. on Toxic Dinoflagellate Blooms, Massachusetts Science and Technology Foundation, Wakefield, Mass., pp. 299-308, 1975. Prakash, Α., Medcof, J.C. and Tennant, A.D. Bulletin 177, Fisheries Research Board of Canada, Ottawa, 1971. Padilla, G.M. and Bragg, R.J. Proc. 4th Ann. Conf. Marine Technology Society, Washington, D.C., pp. 193-204, 1968. Martin, D.F., Martin, B.B. and Padilla, G.M. Environ. Letters (1972) 2, 239. Hartwell, A.D. Proc. 1st Int. Conf. on Toxic Dinoflagellate Blooms, Massachusetts Science and Technology Foundation, Wakefield, Mass., pp. 47-68, 1975. Steidinger, K.A. Proc. 1st Int. Conf. on Toxic Dinoflagellate Blooms, Massachusetts Science and Technology Foundation, Wakefield, Mass., pp. 153-162, 1975. Humm, H.J. and Lane, C.E. (Eds.) "Bioactive Compounds from the Sea", 215 pp., Marcel Dekker, New York, 1974. Martin, D.F. and Padilla, G.M. Biochim. Biophys. Acta (1971) 241, 213. Narahashi, T. Fed. Proc. (1972) 31, 1124. Kim, Y.S., Mandel, L.J., Westerfield, M., Padilla, G.M. and Moore, J.W. Environ. Letters (1975) (In press). Henderson, R., Ritchie, J.M. and Strichartz, G.R. Proc. Nat. Acad. Sci. USA (1974) 71, 3936. Cuatrecasas, P. Ann. Rev. Biochem. (1974) 43, 169.


Marine Chemistry in the Coastal Environment

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