Seafood Toxins - ACS Publications - American Chemical Society

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Paralytic Shellfish P o i s o n i n g A n Emerging Perspective CLARICE M. YENTSCH

Downloaded by CORNELL UNIV on August 10, 2016 | http://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch002

Bigelow Laboratory for Ocean Sciences, W. Boothbay Harbor, ME 04575 Dinoflagellates are rapidly gaining a reputation for being the "nasties" in the marine environment. The slate now includes organisms causing Paralytic Shellfish Poisoning (PSP), Neurotoxic Poisoning (NSP), Ciguatera Poisoning, and more recently described Diarrhetic Shellfish Poisoning (DSP). Problems with toxic d i n o f l a g e l l a t e s are indeed global (1)• I w i l l confine my comments to PSP, the well-documented human a f f l i c t i o n r e s u l t i n g from the consumption of s h e l l f i s h containing d i n o f l a gellate-derived toxins, namely saxitoxin and related compounds. Biological oceanographers have traditionally wrestled with sampling strategies. Inappropriate sampling has hindered our understanding of organisms with patchy d i s t r i b u t i o n (e.g. dinof l a g e l l a t e s ) . High technology developments with regard to flow cytometry/sorting o f f e r promise to increase our understanding of the c e l l biology, and remote sensing offers promise to increase our understanding of the oceanography (Figure 1). Despite these advancements, immediate gains i n an appreciation of the biogeographical d i s t r i b u t i o n w i l l only be possible once a sound simple global monitoring program i s effected. In an attempt to achieve a conceptual balance, l e t us consider the c e l l biology, the biogeographical d i s t r i b u t i o n , and the oceanography. They are a l l i n t e r r e l a t e d and thus an increase of knowledge i n one area strengthens our interpretation of another area. C e l l Biology Surprisingly, most of the d e f i n i t i v e c e l l biology on toxic dinof l a g e l l a t e s has yet to be observed, understood and described. What was once thought to be the major causative genus, Gonyaulax, i s now known to contain a wide variety of s i m i l a r s t r a i n s / varieties/species (2). Although the taxonomy of these organisms i s s t i l l i n a state of uncertainty, d e f i n i t e biochemical differences are obvious — primarily toxin content (3). While i n i t i a l l y saxitoxin was considered a single toxin causing s h e l l f i s h poisoning, i t i s now 0097-6156/84/0262-0009$06.00/0 © 1984 American Chemical Society

Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

S E A F O O D TOXINS


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clear that there are suites of toxins, that these suites vary from one geographic region/organism to another, and that these toxin suites vary with l i f e stage of the producing d i n o f l a g e l l a t e and are altered i n molecular structure within the s h e l l f i s h which acts as a vector of the toxins (4). In short, the s i t u a t i o n once interpreted as a very simple one, i s highly complex. Toxigenesis, biochemistry, physiology and c e l l cycle analysis are b a s i c a l l y s t i l l unknowns. Flow cytometers/sorters o f f e r promise as a research tool here. Flow cytometry combines revolutionary l a s e r , electronics and optics technology (Figure 2). One i s able to analyze c e l l s at very rapid rates (greater than 2000 c e l l s per second) on a c e l l b y - c e l l basis. Feasible measurements include: autofluorescence of the chlorophyll pigments and accessory pigments, as well as induced fluorescence, such as adding hydrogen peroxide, which results i n toxin fluorescence (5); and uptake of various dyes s p e c i f i c for DNA (6-7) (therefore d i r e c t growth rate estimates are possible), RNA and protein (therefore n u t r i t i o n a l concentration estimates are possible). L i p i d stains, pH s t a i n s , membrane potential stains and immunofluorescent antibody preparations are reagents which provide additional information. Another part of this instrumentation i s a charging c o l l a r and d e f l e c t i o n plates. B a s i c a l l y the c e l l s , i n single f i l e , are surrounded by a sheath of saline which can be charged i f the c e l l s going through the charging c o l l a r are of i n t e r e s t . Charged c e l l s are then sorted into d i f f e r e n t containers by the d e f l e c t i o n plates. C e l l s are viable after this process, and can be used for establishing cultures or subsequent EM investigation and biochemical experimentation. A study of interest to the PSP problem i s photoadaptation. We know that phytoplankton organisms grow i n what i s often termed an unbalanced environment. That i s , l i g h t and nutrients, both basic requirements, do not occur i n the same location. Holligan et a l . (8) show potential new growth graphically (Figure 3). The c e l l s of toxic d i n o f l a g e l l a t e s , as well as others, seem to "prefer" a low l i g h t environment, just at the interface of the low-light-high-nutrient zone. Thus, there are masses of dinof l a g e l l a t e s at subsurface maxima near f r o n t a l boundary layers (Figure 4). To continue to grow, c e l l s must adapt to the low l i g h t environment. I t i s hypothesized that this i s accomplished by an increase i n pigmentation per c e l l . In the laboratory, increased pigmentation has been measured i n bulk analyses. We are now able to measure such phenomena on an i n d i v i d u a l c e l l basis (Figure 5). In these laboratory experiments, we have grown the c e l l s i n various l i g h t i n t e n s i t i e s . Chlorophyll indeed does increase on a per c e l l basis. Upon microscopic observation i t i s clear that there i s organelle photomorphogenesis, that i s the chloroplasts per se actually change from the spaghetti-shaped to very t i g h t l y packed more ovoid-shaped e n t i t i e s (9; Figure 6). Whether or not t h i s occurs i n the natural environment has yet to be demonstrated. Biogeography On the other side of the balance i s the biogeography. Distribut i o n patterns were once thought to be simple, but are now


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Paralytic Shellfish Poisoning

SATEUITES

Β Q UJ

80pg/100g) f o r years 1972 t o 1982. Bottom p a n e l i s " s t a r t d a t e s " i n d i c a t i n g when i n i t i a l l e v e l s over q u a r a n t i n e a r e w i t n e s s e d . These dates f a l l i n c a t e g o r i e s 1) t r a n s i t i o n from mixed t o s t r a t i f i e d water column (May) and v i c e v e r s a (Sept.) and 2) d u r i n g summer m e t e o r o l o g i c a l events.


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4) creation of a microclimate i n shallow ponds, embayments and estuaries, appropriate for encystment and/or d i n o f l a g e l l a t e d u p l i cation. In our region, the l a t t e r i s generally associated with the spring r i s e i n t o x i c i t y i n nearshore areas coincident with excystment of the dinoflagellates (J.W. Hurst, pers. comm.) while the f i r s t three are generally associated with summer and autumn t o x i c i t i e s , which can r e s u l t i n very dangerous toxin l e v e l s . Currently, s h e l l f i s h toxin pattern data and/or species enumeration patterns are coalescing with physical oceanographic data hopefully leading to an environmental predictive model which may be applied to broad geographic regions throughout the Gulf of Maine.


Human Impact The past decade has been characterized by the human concern of science for a better environment. Coincidently, there has been a surge of media presentations reporting or implying spreading of toxic red t i d e s . I t i s no wonder that the public has been provoked into asking numerous relevant questions. The questions can be reduced to two. Although the questions appear related, they are not i d e n t i c a l . B a s i c a l l y 1) Are red tides caused by pollution? and 2) Do human a c t i v i t i e s serve to aggravate red tide problems? Perhaps we need reminding that the regions of the world's oceans most severely affected by toxic red tides have waters which are r e l a t i v e l y p r i s t i n e . In the U.S., Alaska and Maine coastal areas are severely affected by organisms leading to P a r a l y t i c S h e l l f i s h Poisoning, yet each state has populations of approximately one m i l l i o n , and there i s l i t t l e i n d u s t r i a l development along coastlines of over 3,000 miles for Maine and 7,500 miles for Alaska. In each case the rocky coast protrudes into the cold waters with f i n g e r - l i k e projections and i s l a n d systems, and each region has a shelf-break several miles offshore which sets up physical conditions which encourage generation of massive subsurface populations of toxic d i n o f l a g e l l a t e s . These populations p e r s i s t throughout the time of water s t r a t i f i c a t i o n , commonly mid-April to mid-October i n these l a t i t u d e s . I t i s now hypothesized that when a proper sequence of meteorological events, such as persistent offshore winds, drive the surface waters offshore, these subsurface waters are forced inshore over the clam f l a t s and mussel beds. Hence the s h e l l f i s h feed on the toxic d i n o f l a g e l lates contained i n this water mass and thus the s h e l l f i s h consumed by humans may be t o x i c . Red tides are indeed a natural phenomenon and have been known to exist p r i o r to recorded h i s t o r y . There are noted accounts i n the Bible as well as Indian legend. D i n o f l a gellates have flourished for over 600 m i l l i o n years as compared with human 20 m i l l i o n year venture on Earth. The assessment that red tides are a natural phenomenon, however, does not absolve humans from concern. Some data suggest an apparent spread i n geographic distribution, increase i n i n t e n s i t y , and extension i n duration. Note the use here of the word "apparent." An argument could be posed from our recent data (Table I) against t h i s . While we do not argue that there i s an apparent decline, these data serve to demonstrate several f a l l a cies i n using scant and inconsistent data. F i r s t , we assume data are collected i n a regular manner. While Maine can boast an


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SEAFOOD TOXINS Table I. S h e l l f i s h Toxin Content. Numbers of Marine S h e l l f i s h Samples Which Meet Stated C r i t e r i a total number of samples


1979 1980 1981 1982 1983 Original Marine

500ug/ 100g

% of >2000yg/ t o t a l 100g

% of >5000yg/ t o t a l lOOg

% of total

0.6 2 136 2983 4.6 11 0.4 4191 330 0.7 2.8 31 7.9 119 0.2 4572 158 9 34 0.7 3.5 0 2257 55 2.4 0 0 2701 0.5 63 15 2.3 data courtesy of J.W. Hurst and P. Hoyt, Maine Dept. Resources.

-

-

outstanding monitoring program, sampling frequency and spacing i s not constant. Both are increased during times when toxin levels are high, thus biasing the data sets. There i s great value i n current toxin data analysis and I would strongly encourage researchers i n regions where large data sets exist to see whether or not there has been an increase i n high toxin l e v e l scores. From the scant b i t s of analyzed data, there i s no way to ascertain whether there i s an actual global increase or whether this impression i s generated by increased human awareness and observational powers which have been fine-tuned. In some l o c a l regions where long-term phytoplankton studies have been conducted (e.g. Oslofjord; Norway, Seto Island, Japan; and S p l i t , Yugoslavia) investigators have found correspondence i n increased p o l l u t i o n and increased d i n o f l a g e l l a t e blooms. Correspondence, however, does not necessarily r e f l e c t a cause-and-effeet r e l a tionship. In some cases, the type of p o l l u t i o n ( i . e . adding increased organics and nutrients) would be predicted to enrich the environment to encourage increased phytoplankton growth, yet we do not understand why dinoflagellates outcompete other plankton. A recent research thrust concerns the benthic resting cysts of toxic d i n o f l a g e l l a t e s (the so-called dormant "over-wintering" stage i n the l i f e history common i n our sediments from October to A p r i l ) (17, 27-34). Benthic monitoring of cysts, possibly with corers, could t e l l the investigator h i s t o r i c a l l y whether the species has been i n the region and possibly for how long. B a s i c a l l y , benthic cysts reseed blooms on a seasonal b a s i s . This has serious implications to human a c t i v i t i e s . The possible effects may be manifested i n a wide range of marine a c t i v i t i e s . In p a r t i c u l a r , those engaged i n projects such as the seeding of s h e l l f i s h beds, s h e l l f i s h culture, and marine dredging operations should be altered to possible dangers. Microscopic cysts are e a s i l y carried with dredged-up sediment and may be transported f a r from the source after dumping at sea. Caution should be exercised when s h e l l f i s h are being transferred from one geographical region to another. Cysts may be buried and l o s t , or by contrast, numbers of motile c e l l s s u f f i c i e n t f o r a bloom condition can result from merely one cyst i n less than one month (assuming one doubling per day, a rate e a s i l y documented i n culture and f i e l d conditions). Once introduced into a new area, cysts may d i r e c t l y contaminate


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s h e l l f i s h and they may establish d i n o f l a g e l l a t e populations.


The Future:

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a more permanent l o c a l

toxic

Global Monitoring

While early work claimed that PSP i s confined to the temperate l a t i t u d e s , reports from Venezuela, Malaysia, the P h i l l i p i n e s and Mexico lead us now to believe that oceanographic conditions, not some feature associated with l a t i t u d e , are d i c t a t i n g the onset. There i s a s t r i k i n g overlap of areas affected by toxic d i n o f l a g e l lates of the genus Gonyaulax and regions of high t i d a l d i s s i p a t i o n (35; see Figure 10). This hypothesis needs to be tested using rigorous s t a t i s t i c a l procedures. A proper impact statement of human a c t i v i t i e s and r e s u l t i n g informed decisions w i l l need to await national and global information bases with data collected i n a controlled uniform manner so that intercomparisons are v a l i d . In my mind, t h i s i s the singular most important e f f o r t necessary for future understanding and prediction of toxic d i n o f l a g e l l a t e s . While remote sensing, a t o o l useful for the study of oceanographic features y i e l d i n g synoptic data sets, and flow cytometry and sorting, a tool useful for the study of c e l l u l a r biology, are promising high technology developments, the major understanding w i l l result from far simpler approaches. We believe that s h e l l f i s h toxin data can be used as a chemical tracer i n the marine environment (13). The signal integrator i n the water column can be the ubiquitous and common mussel (Figure 11) and the signal integrator i n the benthic layer can be the scallop (Figure 12). Both cases can y i e l d important data on seasonal and year-to-year v a r i a t i o n . It i s d i f f i c u l t to conceive of a more useful continuous sampling device than the common mussel. I t works well i n a variety of climatic conditions as well as extreme weather conditions. I t i s nearly indestruct i b l e , and the signal i s uniquely integrated over time, r e l i e v i n g the investigator from concern about " f i l t e r i n g diurnal migration, t i d a l v a r i a t i o n , advection fluctuations and the l i k e from the data. I believe that we need to be r e a l i s t s . At the time of this w r i t i n g , there i s no clear evidence to indicate whether there i s an unnatural spreading i n d i s t r i b u t i o n of the causative organisms, increased duration of the seasonal occurrence or increased intens i t y of the t o x i f i c a t i o n . To respond to any claims of apparent spreading a sound global d i n o f l a g e l l a t e monitoring e f f o r t should be immediately established. Figure 13 i s a reasonable approach. The equipment i s not costly. I t involves: a small pump ( L i t t l e Giant at ~$100), garden hose (30 meters for -$30), a small generator (Honda 6K Watt at -$400) and a small grab (-$400). For less than $1,000 one can be t o t a l l y operational from a row boat. Additional gear can be added as affordable to follow temperature, s a l i n i t y , pigments, nutrients and bioluminescence i n the water column (Figure 14). For a global monitoring program to be e f f e c t i v e , I believe that there must be monitoring of the environment, the oceanography, the motile c e l l s i n the water, the benthic resting cysts i n the sediments (which can be undertaken year-round), and the toxin i n the s h e l l f i s h . The program must be consistently embarked 1 1



SEAFOOD TOXINS

F i g u r e 10. Major t i d a l d i s s i p a t i o n areas of the w o r l d , the g r e a t e r the number, the g r e a t e r t h e energy i n v o l v e d . After M i l l e r (23).

= Signal

producer

= Signal

Signal

recorder/integrator

F i g u r e 11. U s i n g t o x i n m o l e c u l e s as water column t r a c e r s of presence of t o x i n p r o d u c i n g d i n o f l a g e l l a t e s i n m o t i l e form. The common m u s s e l , found w o r l d - w i d e , i s used as the s i g n a l r e c o r d e r / integrator. = Signal producer

= Signal

= Signal

recorder/integrator

F i g u r e 12. U s i n g t o x i n m o l e c u l e s as b e n t h i c t r a c e r s of presence of t o x i n p r o d u c i n g d i n o f l a g e l l a t e s i n c y s t form. The s c a l l o p i s used as t h e s i g n a l r e c o r d e r / i n t e g r a t o r .


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Hydrography

a

Cyst Monitoring Phytoplankton Monitoring


Toxin Monitoring Humon Consumer

^ ^

-

Harvester

a

Morket

*

-

Environmental Monitoring

Shellfish

Dinoflagellates Diatoms

light temperature salinity nutrients trace metals runoff winds

F i g u r e 13. Suggested m o n i t o r i n g program d e s i g n which i n c l u d e s t o x i n m o n i t o r i n g i n s h e l l f i s h , p h y t o p l a n k t o n m o n i t o r i n g ^ and hydrography and e n v i r o n m e n t a l m o n i t o r i n g .

F i g u r e 14. Water column a n a l y s i s i n v o l v i n g pump p r o f i l i n g system. In the system p i c t u r e d , c o n d u c t i v i t y (an index of s a l i n i t y ) and temperature are p r o f i l e d . The f l u o r o m e t e r measures i_n v i v o f l u o rescence from c h l o r o p h y l l (from a l l p h y t o p l a n k t o n ) and d i s c r e t e samples are taken f o r c e l l counts and n u t r i e n t c h e m i s t r y . P h o t o d e t e c t o r s can be employed f o r the measurement o f b i o l u m i nescence. L i g h t measurements range from S e c c h i d i s c r e a d i n g s t o more s o p h i s t i c a t e d transmissometry and s p e c t r a l r a d i o m e t r y instruments.


SEAFOOD TOXINS

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on g l o b a l l y , thus most hopefully sponsored by World Health Organization (WHO), Food and Agriculture Organization (FAO), International Union of Pure and Applied Chemists (IUPAC) or similar non-partisan i n t e r e s t groups, and must be long term — that i s , no less than 10 years i n i t i a l l y . The program must not fluctuate with funding sentiments: a case i n point i s the C a l i f o r n i a s i t u a t i o n which i n 1980 proved to be disasterous (19). Public awareness does need enlightenment. Assay methods do need s i g n i f i c a n t improvement. Yet, I f e e l that no thrust w i l l be as important i n providing leaps i n our understanding as w i l l a sound global monitoring program. I believe that i t i s the respons i b i l i t y of government, and c o l l e c t i v e governments concerned with human welfare and the maintenance of sound f i s h e r i e s , to pursue such programs. Acknowledgments I thank W.M. Balch, P.M. Holligan, J.W. Hurst, F.C. Mague and C.S. Yentsch f o r rewarding discussions. P. Boisvert prepared the manuscript and J . R o l l i n s prepared the i l l u s t r a t i o n s . Partial funding was provided through FDA 223-77-2314, NIH 5-RO1-ES0132903, NMFS/NEFC NA-82-FA-C-00043 and the State of Maine. This i s Bigelow Laboratory f o r Ocean Sciences contribution number 84002.

Literature Cited 1. Baden, D. G. Intnl. Rev. Cytol. 1983, 82, 99-150. 2. Taylor, F. J . R. In "Toxic Dinoflagellate Blooms"; Taylor, D. L . ; Seliger, H. H . , Eds.; Elsevier North Holland: New York, 1979; pp. 47-56. 3. Steidinger, K. A. In "Prog. Phycol. Res."; Round, F . ; Chapman, Eds.; Elsevier: New York, 1983; Vol. 2, pp. 147-188. 4. Shimizu, Y . ; Yoshioka, M. Science 1981, 212, 547-49. 5. Yentsch, C. M. Toxicon, 1981, 19, 611-21. 6. Yentsch, C. M.; Mague, F. C.; Horan, P. K.; Muirhead, K. J . exp. mar. Biol. Ecol. 1983, 67, 175-83. 7. Karentz, D. J . Protozool. 1983, 30, 581-8. 8. Holligan, P. M.; Balch, W. M.; Yentsch, C. M. submitted. 9. Yentsch, C. M.; Cucci, T. L.; Glover, H. E . ; Phinney, D. A . ; Selvin, R. submitted, Mar. Biol. 10. White, A. W. Can. Tech. Rept. Fish. Aquat. Sci. 1982, 1064, 12 pp. 11. Carreto, J . I . ; Lasta, M. L . ; Negri, R.; Benavides, H. Instituto Nacional de Investigacion y Desarroclo Pesquero 1981, Argentina, 52 pp. 12. Davison, P.; Yentsch, C. M. Technical Report 1982, Instituto Nacional de Pesca, Montevideo, Uruguay. 13. Hurst, J . W.; Yentsch, C. M. Can. J . Fish. Aquat. Sci. 1981, 38, 152-6. 14. Lembreye, G. V. Apartado Anales del Instituto de al Patagonia 1981, Magallanes, Chile, 12, 273-89. 15. Okaichi, T. Rept. Fac. Agriculture 1982, Kagawa Univ., Japan, 239 pp. 16. Sampayo, M. A. de; Cobecadas, G. Instituto Nacional de Investigacao dao Pescas 1980, Alges-Praia, Lisbon, Portugal, 25 pp.


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23

Prakash, A . ; Medcof, J . C.; Tennant, A. D. Bull. Fish. Res. Bd. Can. 1971, 177, 87 pp. 18. Saunders, S.; Sample, T . ; Matsuda, R. Metro Rept. Water Pollution Control Dept. Seattle, WA, 31 pp. 19. Sharpe, C. A. 1982, California Dept. Health Services, Sanitary Engineering Section, 75 pp. 20. Tangen, K. In "Toxic Dinoflagellate Blooms"; Taylor, D. L.; Seliger, H. H . , Eds.; Elsevier North Holland: New York, 1979; 179-82. 21. White, A. W. Rept. to ICES Advisory Comm. on Marine Pollution 1980, 11 pp. 22. Simpson, J . H . ; Bowers, D. Deep-Sea Res. 1981, 28A, 727-38. 23. Miller, G. R. J . Geophys. Res. 1966, 2485-89. 24. Pingree, R. D.; Pugh, P. R.; Holligan, P. M.; Forster, G. R. Nature London 1975, 258, 672-77. 25. Holligan, P. M.; Harbour, D. S. J . Mar. Biol. Assoc. U.K. 1976, 1075-93. 26. Yentsch, C. S. In "Remote Sensing Applications in Marine Science and Technology"; Cracknell, A. P . , E d . ; D. Reidel Publ. Co., 263-97. 27. Steidinger, K. A. Int. Conf. Toxic Dinoflagellate Blooms; LoCicero, V . , Ed.; Mass. Sci. Tech. Fdn.: Wakefield, MA, 1975, 153-62. 28. Dale, B. In "Toxic Dinoflagellate Blooms"; Taylor, D. L.; Seliger, H. H . , Eds.; Elsevier North Holland: New York, 1979; 443-52. 29. Dale, B.; Yentsch, C. M.; Hurst, J . W. Science 1978, 201, 1223-5. 30. Anderson, D.; Wall, D. J . Phycol. 1978, 14, 224-34. 31. Yentsch, C. M.; Lewis, C. M.; Yentsch, C. S. Bioscience 1980, 30, 251-4. 32. White, A. W.; Lewis, C. M. Can. J . Fish. Aquat. Sci. 1982, 39, 1185-94. 33. Thayer, P. E . ; Hurst, J . W.; Lewis, C. M . ; Selvin, R.; Yentsch, C. M. Can. J. Fish. Aquat. Sci. 1983, 40, 1308-14. 34. Selvin, R. C . ; Lewis, C. M.; Yentsch, C. M.; Hurst, J . W. Toxicon, in press. 35. Yentsch, C. M.; Holligan, P. M.; Balch, W. M . ; Tvirbutas In "Tidal Mixing and Plankton Dynamics"; Bowman, M.; Yentsch, C. M.; Peterson, R., Eds.; Springer Verlag: New York, in press. 36. Esaias, W. In "Primary Productivity in the Sea"; Falkowski, P. G . , Ed.; Plenum Press: New York, 1980; 321-37. RECEIVED February 6, 1984


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