Biotransfer/accumulation of toxins produced by dinoflagellate Prorocentrum concavum to domino damsel (Dascyllus trimaculatus) fish
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Biotransfer/accumulatiou of toxins produced by dinoflagellate Prorocentrum concavum to domino damsel (Dascyllus trimaculatus) fish
Kosa, Maha Bahjat, M.S.
The University of Arizona, 1991
UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106
BIOTRANSFER/ACCUMULATION OF TOXINS PRODUCED BY
DINOFLAGELLATE Prorocentrum concavum TO DOMINO DAMSEL
(Dascvllus trimaculatust FISH.
by
MAHA BAHJAT KOSA
A Thesis Submitted to the Faculty of the
DEPARTMENT OF NUTRITION AND FOOD SCIENCE
In Partial Fulfillment of the Requirements For the Degree of
MASTER OF SCIENCE
WITH A MAJOR IN FOOD SCIENCE
In the Graduate College
THE UNIVERSITY OF ARIZONA
1991 2
STATEMENT BY AUTHOR
This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University library to be made available to borrowers under rules of the library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarships. In all other instances, however, permission must be obtained from the author.
SIGNED:
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
Dr. Douglas L.Park Assoc. Professor of Nutrition and Food Science. 3
DEDICATIONS
To my family for their love, encouragement and great support in which without them would have not been able to achieve my goals 4
ACKNOWLEDGMENT
My greatest thanks and appreciation are extended to Dr. Douglas L. Park for all his technical advice, assistance, moral support and continuous encouragement throughout my degree program.
I would also like to thank Mr. Sam Rua for his technical support and assistance throughout the experiment. Special thanks are extended to Mrs. Lillian Juranovic, Mrs.
Lili Hao, Mr. Scott Rigsby and Ms. Raquel Gribble for all the assistance. Further thanks go to the University of Arizona, Department of Nutrition and Food Science and to the members of the committee Dr. Charles Gerba and Dr. Donald Lightener. 5
TABLE OF CONTENTS
Page
LIST OF ILLUSTRATIONS 9
LIST OF TABLES 11
ABSTRACT 13
1. INTRODUCTION 14
2. LITERATURE REVIEW 16
a. Occurrence 16
b. Source 17
c. Types of Fish Implicated in Ciguatera Poisoning 21
d. Toxin Accumulation in Fish 23
e. Chemical Properties of Toxins 25
f. Pharmacology 26
g. Analytical Methodology 30
h. Clinical Symptoms 34
i. Medical Treatment 36
3. MATERIALS AND METHODS 38
a. Culturing of the Dinoflagellate 38 6
b. Large Scale Culturing Operation 46
c. Morphological Characteristics of Prorocentrum concavum 48
d. Harvesting Operation 50
e. Cell Lyophilization 51
f. Preparation of Fish Diet 52
g. Proximate Analysis of Fish Diet 52
h. Exposure of Fish to Toxins Produced by Dinoflagellate 54
i. Fish Feeding Study 56
j. Fish Euthanasia 57
k. Determination of Toxin Potential . 57
1. Determination of Toxicity in Fish 59
m. Toxicity Testing Techniques 60
n. Determination of Ciguatoxin and Related Polyether Compounds 61
o. Okadaic Acid Analysis 65
4. RESULTS AND DISCUSSION 66
a. Large Scale Culturing of Dinoflagellates 66
b. Morphological Characteristics of Prorocentrum concavum 69
c. Harvesting of Prorocentrum concavum Cells 69
d. Fish Diets 69 7
e. Proximate Analysis 76
f. Behavioral and Toxic Effects of Prorocentrum concavum Diets on Fish 79
g. Toxicity Testing (Brine Shrimp Assay) 86
h. Okadaic Acid Analysis 93
i. Stick Test Assay (S-EIA) 100
j. Comparative Toxicity 103
5. CONCLUSION AND FUTURE WORK 105
APPENDICES:
APPENDIX A: Effect of extract of standard control diet on brine shrimp (Artemia sp.) mortality 106
APPENDIX B: Effect of extract of 50/50 Standard control/ dinoflagellate cell fProrocentrum concavum') diet on brine shrimp (Artemia sp.) mortality 107
APPENDIX C: Effect of extract of 100% Dinoflagellate cell fProrocentrum concavum^ diet on brine shrimp (Artemia sp.) mortality 108
APPENDIX D: Effect of okadaic acid on brine shrimp (Artemia sp.) mortality 109
APPENDIX E: Effect of fish tissue extract for fish fed standard control diet on brine shrimp (Artemia sp.) mortality 110
APPENDIX F: Effect of fish tissue extract for fish fed 50/50 Standard Control/dinoflagellate cell diet on brine shrimp (Artemia sp.) mortality Ill 8
APPENDIX G: Effect of fish tissue extract for fish fed 100% dinoflagellate cell diet on brine shrimp (Artemia sp.) mortality 112
APPENDIX H: Effect of fish tissue extract for fish that died as a result of feeding on 50/50 standard control dinoflagellate cell diet on brine shrimp (Artemia sp.) mortality 113
APPENDIX I: Effect of fish tissue extract for fish that died as a result of feeding on 100% dinoflagellate cell diet on brine shrimp (Artemia sp.) mortality 114
APPENDIX J: Effect of extracts of aquaria water taken during the feeding study (day 19) on brine shrimp (Artemia sp.) mortality 115
APPENDIX K: Effect of extracts of aquaria water taken at the end of feeding study on brine shrimp (Artemia sp.) mortality 116
REFERENCES 117 9
LIST OF ILLUSTRATIONS
Figure Page
1 Transmission of ciguatera toxins to humans through the marine food chain 18
2 Structure of okadaic acid 27
3 Procedure for large-scale culturing of Prorocentrum concavum 45
4 Large scale culturing of Prorocentrum concavum using 20 liter carboys 47
5 Large scale culturing of Prorocentrum concavum using 20 liter plastic bags 49
6 Diagram of Aquaria/diet designation 55
7 Diagram of Stick-Enzyme Immunoassay (S-EIA) 63
8 Prorocentrum concavum cell 70
9 Attachment of Prorocentrum concavum cells by mucus 71
10 Daughter Prorocentrum concavum cell emerging from parent cell 72
11 Daughter and skeleton of Prorocentrum concavum cell 73
12 Toxicity of 50/50 Standard control/dinoflagellate cell (Prorocentrum concavum) diet extract to brine shrimp fArtemia sp.) at 8 hours 87
13 Toxicity of 100% dinoflagellate cell (Prorocentrum concavum) to brine shrimp fArtemia sp.) at 8 hours 88
14 Toxicity of okadaic acid to brine shrimp fArtemia sp.) at 8 hours 89 10
15 HPLC chromatogram of Prorocentrum concavum diets at mobile phase 80 + 20 (ACN + H20) 95
16 HPLC chromatogram of standard control diet at mobile phase 80 + 20 (ACN + H20) 96
17 HPLC chromatogram of fish tissue extract at mobile phase 80 + 20 (ACN + H20) 97
18 HPLC chromatogram of fish tissue extract at mobile phase 70 + 30 (ACN + H20) 98
19 HPLC chromatogram of fish tissue extract at mobile phase 75 + 25 (ACN + H20) 99 11
LIST OF TABLES
Tables Page
1 Modified K enriched seawater medium composition 39
2 Enriched seawater medium "enriched f/2" primary stock solution for trace elements 40
3 Trace metals working stock solution for enriched seawater media 41
4 Vitamin primary and working stock solution used in enriched seawater media 42
5 Ingredients composition of the standard control diet 53
6 Composition of the substrate solution for (S-EIA) assay 64
7 Comparison of Prorocentrum concavum dry cell weight (g/L) cultured in different large scale containers 68
8 Comparison of Prorocentrum concavum dry cell weight yield cultured in 2.8 liter flasks using different methods of harvesting 74
9 Composition of diets containing Prorocentrum concavum cells 75
10 Proximate analysis of diets used in fish feeding experiment 77
11 Average weight loss (g/fish) for all the fish treatment groups 80
12 Effect of Prorocentrum concavum diets on Domino damsel fish (Dascyllus trimaculatusl mortality 83 12
13 Comparison of the number and percentage of fish mortality with respect to diet and aquaria position 84
14 Quality of aquaria water tested during the feeding study (Day 19) 91
15 Quality of aquaria water tested at the end of the feeding study 92
16 High performance liquid chromatographic analysis of fish diets and fish tissue extracts for okadaic acid 94
17 Toxic response of fish tissue samples to antibodies specific to ciguatoxin and other polyether compounds 101
18 Toxic response of fish homogenate samples to antibodies specific to ciguatoxins and other polyether compounds 102
19 Comparative toxicity of fish diets containing Prorocentrum concavum and extracts of fish exposed to these diets 104 13
ABSTRACT
Toxic dinoflagellate Prorocentrum concavum cells (DC) implicated in ciguatera poisoning were lyophilized and mixed with a standard fish food and fed to Domino damsel fish fDascvllus trimaculatus'i. Damsel fish fed 50/50-SC/DC and 100% DC diets exhibited physical and behavioral changes as well as death. Feed refusal was apparent among the fish fed 100% DC diet. Toxicity of the control group could be attributed to previous exposure of the fish to other polyether compounds in its natural habitat or even other chemicals. Fish extracts of both control and treatment groups were toxic when tested on the Stick-enzyme immunoassay for ciguatoxin. Okadaic acid was detected in
P. concavum. but no okadaic acid vras found in any of the fish tissue extracts. Further studies are needed to determine the transfer of toxin to the fish through diet before any conclusion is made. 14
INTRODUCTION
Ciguatera is a spanish term which refers to poisoning upon eating toxic marine fish (Lange, 1987; Wither, 1982). This illness occurs mainly in Hawaii, the Caribbean and the South Pacific Islands (Halstead, 1988). Eventhough this phenomenon has existed for centuries, only recently the source of the toxin has been identified and studied due to the limited amount of toxin available. The origin of toxin is believed to be from dinoflagellates. Prorocentrum concavum is one of the many dinoflagellate implicated in ciguatera poisoning and has been found to produce okadaic acid a polyether toxin isolated from barracuda implicated with ciguatera poisoning (Gamboa and Park, 1989).
Other dinoflagellates found to be toxic are Gambierdiscus toxicus. Prorocentrum lima. Prorocentrum mexicanum. Gonvaulax polvedra. Coolia monotis and Osteoropsis ovata (Carlson, 1984; Tindall, 1985). All these dinoflagellates are single celled, photosynthetic motile algae that attach to macroalgae and dead coral surfaces. These dinoflagellates have been found to produce polyether toxins. Some of these toxins are fat soluble like ciguatoxin, scaritoxin and okadaic acid and others are water soluble like maitotoxin, one of the most potent ciguateric toxins (Dickey, 1990; Yasumoto, 1971 and
1973). It is believed that 50,000 cases of ciguatera poisoning occurs annually worldwide
(Ragelis, 1984).
Ciguatera poisoning is one of the most common seafood poisoning of non bacterial origin that occurs in the United states and its territories (Doorenbos, 1984). 15
Humans get exposed to the toxin through the marine food chain theory: Herbivorous fish are believed to feed on toxic dinoflagellates and carnivorous fish in turn feed on toxic herbivorous fish thereby accumulating more toxin(s) (Randall, 1958). Ciguateric fish accumulate toxins in their bodies and in higher amounts in their viscera for a long time without being affected. Barracuda, snapper, ambeijack, parrotfish and mackerel are few of the 400 species of fish implicated in ciguatera poisoning. This illness involves variety of symptoms ranging from mild gastrointestinal to severe neurological problems that tend to stay and reoccur for a long time (Bagnis, 1979).
The occurrence of variable symptoms is attributed to the presence of more than one toxin (Hokama,1985). Few drugs have been found to treat the neurological symptoms but no one drug has been found yet to treat all the variable symptoms of ciguatera. This phenomenon has caused a major economic loss to the endemic areas where local fish is banned from consumption, in addition to the cost of health treatment
(Ragelis, 1984). Many assays have been developed to detect ciguatera toxins but the most sensitive and specific is an immunochemical assay developed to screen toxic fish and protect the consumer.
This study has been undertaken to evaluate the potential transfer of toxins produced by Prorocentrum concavum to Domino damsel fish (Dascvllus trimaculatust. a dinoflagellate and fish respectively implicated in ciguatera poisoning outbreaks. 16
CHAPTER 1
LITERATURE REVIEW
Occurrence of Ciguatera Poisoning:
Ciguatera poisoning, an illness associated primarly with the tropics, occurs in humans upon the consumption of fish that have harbored toxins in their bodies. This phenomenon has been noticed in the tropical regions for a long time. Captain James
Cook recorded an illness with his soldiers upon eating what probably was a red bass in the South Pacific. His description matches what is called today ciguatera poisoning.
Other recordings of fish poisoning are found in history but most of them are not accurate. The term ciguatera originated from the Caribbean referring to intoxication upon eating snails (Turbo pica) called cigua (Lange, 1987; Ragelis, 1984; Wither, 1982).
The current term of ciguatera refers to poisoning as a result of ingesting toxic marine fish
(Hokama, 1986; Ragelis, 1984). It is believed that 50,000 cases of ciguatera poisoning occurs every year worldwide. This illness exists in the tropical and subtropical coastal regions of the world, where it is primarly confined to the islands of the tropical regions
(Banner, 1976). This disease occurs throughout the Caribbean Islands, Florida, Gulf of
Mexico, Hawaii, all the archipelago in the tropical Pacific, on the Great Barrier Reef of
Australia, in Japan and across the Indian Ocean to Madagascar (Grant, 1984). There are also reports of ciguatera poisoning in the Mediteranean Sea off the coast of Israel 17
(Eisenkraft, 1988), in the Red Sea of the Coast of Egypt (E.S. Abd-Alla, Personal
Communication) and in Venezuela (Doorenbos, 1984).
Source of Ciguatera Toxins:
The origin of ciguatera toxins is not exactly known; however, is suspected to be produced by dinoflagellates and passed up to humans through the marine food chain
(Figure 1). The idea of fish feeding on toxic dinoflagellates was proposed in a theory by Randall (1958). He concluded that herbivorous fish feed on benthic dinoflagellates and accumulate the toxin and in turn carnivorous fish feed on toxic herbivorous fish and become poisonous. The earliest record in history with this theory dates back to the
1800's, when a British physician wrote that West Indian fish became poisonous as a result of eating toxic benthic dinoflagellates (Chisholm, 1808). A similar belief was held among the fishermen and native islanders of the Pacific like Hawaii (Banner, 1959) and the Gilbert Islands (Cooper, 1964).
Stomach contents of surgeon fish (herbivorous sp) were found to contain ciguatoxin-like substances which led to conclusion that these fish became toxic through their diets (Yasumoto, 1971). In another study, where the dinoflagellate Gambierdiscus toxicus was isolated from the gut of surgeon fish, the relationship between dinoflagellates and fish toxicity was supported (Hokama, 1987). These fish were tested for toxicity using a Stick-enzyme immunosorbent assay (S-EIA). Eighty seven (87)% of them were found to be toxic while 98% contained G.toxicus in the gut content. In an early study TOXIC CARNIVORE
HERBIVORE INGESTS TOXIC DINOFLAGELLATES
TOXIN PRODUCING s DINOFLAGELLATES
TOXIC HERBIVORE
Figure 1: Transmission of ciguatera toxins to humans through the marine food chain. 19 conducted in the Palmyra and Line Islands, algae was present in the gut contents of 106 poisonous fish tested (Dawson, 1955). In 1967, a lipid soluble toxin was isolated by
Scheuer (1967) from Moray eel liver and was named ciguatoxin. Five years later,
Yasumoto (1971) isolated a new toxin from surgeon fish in addition to ciguatoxin. The toxin was more water soluble and was named maitotoxin after a local Tahitan name
"maito" for surgeon fish. Later in 1974, another toxin was isolated from parrot fish fScarus gibbusl. a major cause of ciguatera poisoning in the Gambier Island. The toxin, named scaritoxin, was found to be lipid soluble (Bagnis, 1974).
After careful study of the parrot fish diet, it was concluded that these fish feed only on microalgae and fine detritus. A major discovery by Yasumoto (1977) identified a dinoflagellate from the Gambier Island to be the source of ciguatera toxins. Samples of detritus studied were discovered to contain toxins. This finding led to the identification of dinoflagellate Gambierdiscus toxicus named after the Gambier Island.
This dinoflagellate in its natural habitat is photosynthetic, single celled and attaches to macroalgae unless disturbed. This organism was believed to be the most likely cause of ciguatera (Yasumoto, 1977).
Further studies showed that G. toxicus collected from its natural habitat produced ciguatoxin and larger amounts of maitotoxin. The ciguatoxin from the moray eel liver was compared to the ciguatoxin from £J. toxicus. and were found to have similar chromatographic and pharmacologic properties (Yasumoto, 1977). In addition, they found that the maitotoxin from the dinoflagellate was similar to the one isolated from the 20 surgeon fish (Yasumoto, 1971 and 1979). Therefore, it was concluded that Q. toxicus was the source of ciguatoxin and maitotoxin in reef fishes. When 0. toxicus was cultured in the laboratory, only maitotoxin was produced which is one of the most potent of ciguatera toxins (Yasumoto, 1980). The reason ciguatoxin is not produced in cultured dinoflagellates is attributed to the absence of factors that enhance toxin production found in nature but not provided in culture (Hokama, 1986).
Other toxins implicated with ciguatera poisoning are okadiac acid and brevetoxin.
Okadiac acid has been isolated from a South Pacific dinoflagellate Prorocentrum lima originally thought to be scaritoxin (Yasumoto, 1978 and 1982). P. lima was also found to produce ciguatoxin (Yasumoto, 1978). Recently, okadiac acid was isolated from a
Caribbean dinoflagellate Prorocentrum concavum and it was suggested that okadiac acid might have a larger role in ciguatera poisoning than originally thought (Dickey, 1990).
Juranovic (1989) isolated okadiac acid from unialgal culture of G.toxicus and
P.concavum. Juranovic (1989) reported in her study toxic isolates from P.lima which was later identified as P.concavum.
Other dinoflagellates have been isolated and tested to be toxic such as
Prorocentrum mexicanum. Prorocentrum rhathvmun. Gvmnodinium sanpuineum and
Gonvaulax polvedra (Carlson, 1984; Tindall, 1985). For an in depth understanding of the dinoflagellates implicated in ciguatera poisoning refer to a review prepared by
Juranovic and Park (1991). All these benthic dinoflagellates are single celled organism, photosynthetic and attach to macroalgae and coral surfaces. These dinoflagellates secrete 21 large amounts of mucus which enables the cells to attach to each other and form strands
(Besada, 1982). The macroalgae are believed to provide nutrients and shelter to the
dinoflagellates (Bomber, 1985). Extracts of macroalgae added to the media for culturing
these dinoflagellates in the laboratory have showed enhancement of growth (Carlson,
1984; Bomber, 1985).
Types of Fish Implicated in Ciguatera Poisoning:
Most ciguatoxic fish are bottom reef dwellers. They are categorized into
herbivorous, omnivorous and carnivorous fish (Halstead, 1978). Ciguatoxic fish are
restricted to species that either feed on microalgae and detritus or carnivorous fish that
feed on toxic herbivorous and omnivorous Fish (Halstead, 1988). More than 400 species of fish world wide are thought to be implicated with ciguatera poisoning outbreaks
(Halstead, 1965). Among the most common fish that accumulate ciguateric toxins are ambeijack, barracuda, snappers, mackarel, grouper and moray eels. Other fish are damsel, mullet, bonefish, seahorse, angel fish and sword fish (Halstead, 1978). Not all
the fish of one species are found to be toxic everywhere. Throughout the Pacific ocean area moray eels, red bass, snappers and ambeijack are the fish most commonly associated with ciguatera. In Florida, barracuda is the most poisonous fish which resulted in its ban from consumption (Lange, 1987).
Relationship between fish size and toxicity has been studied in the Line Island by
Hessel and Banner. In 1959 and 1960, Banner examined 437 random samples of red 22 snappers (L.bohari in the Line Island and concluded that fish weighing less than 1 kg had less toxicity (25%) compared to the fish larger than 5 kg which showed 100% toxicity
(Banner, 1963). In 1960, Hessel came to the same conclusion. He examined 190 samples of red snappers in the Line Island and reported that fish weighing less than 1 kg had 18% toxicity compared to 65% toxicity in fish weighing more than 2.8 kg. Higher toxicity among bigger fish has been documented from outbreaks where people have consumed large carnivorous fish (Hokama, 1986). Ciguatoxin-like substances have been isolated from the carnivorous fish, Spanish mackerel, in Queensland, Australia (Endean,
1983). Later findings by the same researcher showed that ciguatoxin was detected in fish viscera of small barracuda fish (less than 3 kg) caught in the same area. This suggested that Spanish mackerel could feed on small size toxic barracuda and accumulate higher amounts of toxins.
Damsel Fish (Dascvllus trimaculatus):
Dascvllus trimaculatus are one of the many kinds of fish implicated in ciguatera poisoning (Halstead, 1978). These fish are too small to be consumed by man but can contribute to the toxicity of carnivorous fish diet (Halstead, 1988). D. trimaculatus fish belong to the family of the Pomacentridae and are found in the tropical and subtropical areas of the world. There are many genera and species of the Pomacentridae family in the Indo-Pacific area. In the Western Indian Ocean, the Pomacentridae family have five common genera which are Abudefdef. Amphiprion. Chromis. Dascvllus and Pomacentrus 23 (Bock, 1978). The Pomacentidae fish are mostly small, laterally compressed with bright
distinguished colors.
Young Dascvllus trimaculatus have a velvet black color with three sharp white
spots. One spot on the head and one spot on each side of the body below the dorsal fin.
The adult fish color changes into pinkish brown with faded white spots (Bock, 1978).
D. trimaculatus fish are omnivorous and feed at mid level level in the reef ecosystem
(Allen, 1972; Halstead, 1988). These fish are confined to coral reefs and are found
around rocky areas which they use as a shelter. The young D. trimaculatus are found
to be associated with anemones (Radianthum oritteri and Stoichactis gigantum) (Allen,
1972). They are also seen around small coral heads often Pacillopora (Allen, 1972;
Bock, 1978). In comparison, the adults are not associated with anemone or corals and
they swim freely in a small groups of around 50 fish near rocky areas and reefs (Allen,
1972). However, the adults often take refuge in the anemones when they are threatened.
^ The D. trimaculatus are opportunistic, colonize, exploit reefs and lagoonal habitats and
are known for their strong territorial behavior (Bock, 1978).
Toxin Accumulation in Fish:
Fish exposed to toxins in their environment accumulate the toxins in their bodies.
Heavy metals such as mercury have been shown to accumulate in the fish flesh and in
higher amounts in their viscera. Research evaluating the mercury content of marine fish
near Venezuela detected almost two times the amount of mercury in fish liver (0.361
Hgfg) than in the flesh 0.190 ng/g (Shrestha, 1988). Another study conducted in Baja 24
California, Mexico on the mercury in fresh water fish, reported that an average mercury concentration in the viscera of 0.05 jtcg/g (Galindo et al, 1988).
Ciguatera toxins have been shown to accumulate in the flesh and in higher amounts in the viscera. Helfrich detected 50 times more toxin in the liver than the flesh of red snapper in the Palmyra Island (Helfrich, 1963). Yasumoto (1969) detected 100 times more ciguatoxin in the liver as in the flesh of Q. javanicus. Another study reported by Endean (1984) on the barracuda, detected 7 times more ciguatoxin in the viscera than the flesh. Sometimes even when the flesh did not contain detectable levels of toxins, the liver contained 100 times more toxin as it was found in moray eel
(Scheuer, 1969). However, this phenomena was not observed in the current study when testing fish that were exposed to diets containing the toxic dinoflagellate P.concavum.
Ciguateric fish are believed to store toxins in their bodies without being affected by it (Banner, 1960). The current study found that when the marine fish,
D.trimaculatus. were fed toxic dinoflagellates, the fish showed physical and behavioral changes. Inducing toxicity in fish has been reported in a study feeding 10 g of toxic red snapper to non-toxic surgeon fish every other day for 18 months (Banner, 1963). The surgeon fish were found to be toxic when they were fed to mongooses. However, the authors assumed that the control fish were not toxic eventhough they did not test the control fish for toxicity. Feeding marine fish control diet were found to be toxic by the author of this document and it is attributed to the fish being exposed to the toxin in its natural habitats. Toxic fish are believed to retain their toxicity over a long period of 25 time. A study reported by Banner (1965) showed no detectable significant decrease in toxicity when toxic red snappers were maintained on non-toxic Hawaiian skipjack tuna and herring for 30 months. This study suggests that the fish store the toxin in their bodies indefinitely.
Chemical Properties of the Toxins:
The process of isolating and purifying ciguatera toxins has been time consuming and laborous due to limited amounts of the toxin even in the most toxic fish. One metric ton of moray eel viscera yielded only 1 /xg of toxin (Scheuer, 1969). This toxin was named ciguatoxin and was found to be soluble in organic solvents (methanol, ethanol, 2- propanol and acetone) (Hokama, 1986). Its partial structure was determined (Scheuer,
1969) and had an LDJ0 of 0.45 /ig/kg (i.p) in mice. The empirical formula was determined to be C^H^NO^ or C^H^NO^ by CI plasma desorption mass spectrometry. Ciguatoxin is lipophilic, light yellow viscous oil, heat stable with molecular weight of 1,111.7 +, 0.3 daltons. Ciguatoxin isolated from moray eel and dinoflagellate G. toxicus had similar chromatographic and pharmacologic properties.
Maitotoxin, one of the most potent of ciguatera toxins, was isolated from surgeon fish
(Yasumoto, 1971). It has an LD50 of 0.2/xg/kg (i.p) in mice. This toxin was reported to be a metabolite of £. toxicus based on similar chromatographic and pharmacological properties (Yasumoto, 1971 and 1979). Maitotoxin is water soluble and believed to be a precursor of ciguatoxin. 26
Scaritoxin, another polyether toxin implicated in ciguatera poisoning, was isolated from parrotfish by (Yasumoto, 1973). It has an LDM of 30 ftg/kg (i.p) in mice and was postulated to be a metabolite of ciguatoxin. It produces ciguatoxin-like physiological symptoms, but is chromatographically different. Scaritoxin is lipid soluble and its molecular structure has not been established.
Okadaic acid, a polyether toxin similar to ciguatoxin, was isolated from dinoflagellate P. lima (Figure 2) and its molecular formula was determined to be
C44H68013 (Murakami, 1982). This toxin has been also implicated in diarrhetic shellfish poisoning (Murata, 1982) and was also isolated from two sponges Halichondria okadiac and H.melanodocia (Nakajima, 1981; Yasumoto, 1980). Okadiac acid was isolated from barracuda implicated in ciguatera poisoning (Gamboa and Park, 1985). Recent work by
(Dickey, 1990) isolated okadiac acid from another dinoflagellate P. concavum. Its LDj0 was determined to be 210 /^g/kg (i.p) in mice and confirmed its molecular formula proposed by Murakami (Dickey, 1990). Hokama (1988) reported similar biological and
TLC characteristics of okadaic acid to ciguatoxin. Not all the ciguatera toxins have been identified.
Pharmacology:
Many of the ciguatera toxins (ciguatoxin, maitotoxin, brevitoxin and okadaic acid) appear to have the same ionophoretic effects on( Na+, Ca2+, K+) permeability through the cell membrane (Hokama, 1986). Early studies on the mode of action of ciguatoxin HO OH OH
Figure 2: Structure of okadaic acid.
PO 28 concluded that it had anticholinesterase activity (Li, 1965). This conclusion was based on in vitro experiments with rabbit intestinal segment in which an inhibition of human and bovine erythrocyte cholinesterase was observed. Later studies done by Ogura (1967) disproved the anticholinesterase activity of ciguatoxin when no electroencephalograph responses of the anticholinesterase agent was detected. Ciguatoxin was found to exert its effect on many sites.
Ciguatoxin effect on the nervous system caused a tetrodotoxin-sensitive increase in sodium-ion permeability and depolarization of the resting membrane (Rayner, 1972).
Ciguatoxin has a maked effect on the respiratory and the cardiovascular systems. The respiratory arrest result from depression of the central respiratory center in the brain
(Cheng, 1969). On the cardiovascular system ciguatoxin had a biphasic action in an anaesthsized rat. First, it caused bradychardia and hypotension then was followed by tachychardia and hypertension (Li, 1965).
The effects of ciguatoxin on smooth muscle are complex. On the guinea pig tissues, ciguatoxin enhances the inhibition of the taenia caecum (Miyahara, 1985) and causes excitation of the vas deferens (Ohizumi, 1981; Shibata, 1981). These effects are associated with norepinephrine release. In contrast ciguatoxin induces sustained contraction of the guinea pig ileum (Lewis, 1984). Similar effects of excitation and inhibition are observed in other tissue. Ciguatoxin contracts the thoracic aorta strip of the rabbit (Legrand, 1982) and relaxes the spontaneously contracting rat jejunum (Miller,
1984). Many of these effects of ciguatoxin can be best explained by the depolarization 29 of nerve terminals and the release of neurotransmitters.
Maitotoxin effects on respiratory and cardiac systems of anaesthetized cats have been studied by (Legrand, 1982). Hyperventilation, hypertension and tachycardia were observed and followed by slight bradychardia when the cat was induced with sublethal dose. The effects of maitotoxin in vitro were studied by Miyahara (1979). He observed a biphasic response on guinea pig atria followed by a progressive decrease of the rate and force of contractions. Maitotoxin was found to strongly inhibit the Na+ - K+ ATP-ase from microsomes of cats and human kidneys (Bergmann, 1982). Other studies done on rat pheochromocytoma cell lines, found an increase in Ca2+ in- flux and Ca2+ dependent release of epinephrine and dopamine (Takahachi; 1982, 1983). Similar actions were observed in the guinea pig isolated ileum, taenia caecum and vas deferens in the rabbit thoracic aorta (Ohizumi, 1983).
Okadiac acid (OA), a polyether compound isolated from P.lima and P.concavum. was found to be indistinguishable from ciguatoxin chromatographically. OA also causes sodium ion permeability across the membrane like ciguatoxin. A smooth muscle contraction in the absence of Ca2+ was found to be triggered by OA (Ozaki, 1987;
Shibata, 1985). The mode of action of OA was compared to another polyether compound
Calyculin isolated from a marine sponge (Ozaki, 1987) and was found to activate sodium channels and contractile elements to induce smooth muscle contraction. Lately, okadiac acid was also found to induce tumors which bind to their own receptors present in particulate and cytosol fractions. The mode of action of these compounds is not yet fully 30
understood (Fujiki, 1988).
Scan toxin, another toxin isolated from toxic fish, has a pharmacological actions
similar to ciguatoxin. Scaritoxin caused a depolarizing action on excitable membrane
(Rayner, 1977). On the cardiovascular system, scaritoxin had a biphasic action on a rat
atria similar to ciguatoxin effects (Legrand, 1979). In the pentobarbital anaesthetized cats, scaritoxin evoked respiratory and cardiovascular depression similar to those of
ciguatoxin. In the guinea-pig atria, scaritoxin caused a strong increase in potency of the
acetylcholine negative inotropic and chronotropic effects (Rentier, 1980). Data about
scaritoxin are still limited but is thought to be a metabolite of ciguatoxin.
Analytical Methodology:
Many biological assays have been used to detect ciguatoxins in fish, but none of
the early assays were quantitative, specific or sensitive to ciguatoxins. Toxic fish cannot
be distinguished from non-toxic fish on the basis of smell, taste or appearance. As a result detection methods have been established by the islanders in the endemic areas.
Natives of these places would feed the fish viscera to a pet cat, dog or turtle and observe for a change in behavior or even death. Some people would look for a change in color of silver coin placed inside the fish after it is cooked (Lewis, 1986). Others would rub the fish viscera on the inside tissue of their mouth and watch for numbness. Only the latter of the above methods was specific. Banner (1960) tested 37 species of animals and concluded only five were sensitive to fish feeding tests. The cats were rejected because 31
they would regurgitate the toxic fish. The rats were eliminated due to their high
tolerance. The turtles and the crayfish were rejected because of difficulty in quantifying
symptoms. The mongoose was chosen to be the animal for standardized screening tests.
Further studies by (Banner, 1961) resulted in the development of a mouse assay
which was based on injecting mice intraperitoneally (i.p) with toxic fish extract and were
observed for 24 hours. The results are expressed in mouse unit where one mouse unit
is equal to the amount of toxin that kills a 20 g mouse in 24 hours (World Health
Organization, 1984; Hoffman, 1983). Symptoms included lower rectal temperature, excessive salivation, difficulty breathing, lacrimation and death. The major draw back
to the mouse assay is it utilizes large amounts of the toxin in addition to the cost of
maintaining the animals and the time it takes to run the assay.
A new sensitive assay (Brine Shrimp) was developed to detect ciguatera toxins
(Granade, 1976). Extracts from toxic fish caused toxicity to brine shrimp similar to
toxicity obtained from the mouse assay (Granade, 1976). This test has been used for the
bioassay of fungal toxins, dinoilagellate toxins, morphine like analgesic, anaesthetic and insecticides. This assay is based on exposing shrimp larvae to toxic fish extracts and observing for mortality. Brine shrimp assay is economical, sensitive and good for toxicity screening. This bioassay was used by Juranovic (1989) and found it to be a good test for screening ciguatoxins.
Recently a bioassay using mosquitos was developed (Bagnis, 1987; Chungue,
1984; Pompon, 1987). This procedure involves injecting ciguatoxin in mosquitos 32
intrathoracically and toxicity is expressed by LDJ0. Good correlations between this assay
and the cat and the mouse assay have been observed. The advantages of this assay it is
rapid, simple and only requires small amounts of fish extracts. Other assays developed
used guinea pig ileum, guinea-pig atrium, isolated frog nerve fiber, crayfish nerve cord,
human and mouse hemolytic tests, Chicken embryo and Salmonella/microsomal assay
(Benoit, 1986; Dickey, 1982; Escalona de Motta, 1986; Endean, 1986; Miller, 1986;
Miyahara, 1970; Miyahara, 1984 ; Juranovic, 1989).
Reasearchers (Gamboa & Park,1985 ; Sawyer, 1984) monitored mouse
temperature depression following (i.p) injection of toxic fish or dinoflagellate extracts as
a measurment of ciguatoxin toxicity. A pronounced temperature decline was observed
following an (i.p) injection of G.toxicus methanol extract (Juranovic, 1989). McMillan
(1983) developed a dose response curve from a series of mouse assays and concluded that
the mouse assay was the most reliable and preferred to cats and mongooses.
It was not until the late 1970's the most quantitative, specific and sensitive assays
were developed to detect toxins directly from the fish (Hokama, 1977). A
radioimmunoassay (RIA) developed, was based on sheep polyclonal antibody coupled to
1-125. This assay was successful in distinguishing toxic and non-toxic fish to protect the
consumer. Good correlation was achieved when comparing the RIA, mouse and in vitro
guinea-pig atria in a series of studies (Kimura, 1982). Other studies detected ciguatoxin
in 93% of the fish tissue implicated in ciguatera poisoning (Kimura, 1982). The
disadvantages to this assay were only fish bigger than 9 kg could be tested, high cost, 33 time and cross reactivity with other polyether toxins.
In 1983, an enzyme-immunoassay (EIA) was developed and instead of 1-125 a horseradish peroxidase was coupled to the polyclonal antibody (Hokama, 1983).
Comparison of clinically diagnosed toxic with non-toxic consumed fish demonstrated a statistically significant difference between the two groups (Hokama, 1984). This test was feasible, easy to run, screen any size fish and can be tested on viscera as well as fish flesh. It proved to be sensitve, specific, simple and highly useful in screening fish for cosumption but cross reactivity with other polyether toxins was still observed (Hokama,
1983).
An improved version of the (EIA) called stick-enzyme immunoassay (S-EIA) was developed and proved to be very successful (Hokama, 1985). A coated Bamboo stick with correction fluid is inserted in a fish flesh, fixed in methanol, rinsed in a series of buffer washes and finally placed in the substrate. Color developed in the substrate is compared to a color chart. This test was able to detect toxins from fish tissues as well as fish extracts and had the ability of performing it in the field since it used no equipment. In addition, it is rapid, specific and sensitive to polyether toxins. The disadvantage of this test was false positive results were still obtained ; However, no false negative results were observed (Hokama, 1987). It is believed that polyether compounds such as brevetoxin, maitotoxin and okadaic acid cross react with ciguatoxin which may have accounted to the false positive reported in the RIA, EIA and S-EIA tests (Hokama,
1977; Kimura, 1982; Kimura, 1982a; Hokama, 1985). 34
The latest version of the immunoassays is the simplified solid-phase immunobead assay (S-PIA) (Hokama, 1989). A more specific monoclonal antibody (M-Ab) and a coated bamboo paddle is used. This test is much simpler and easier than the S-EIA to use in the laboratory as well as the field. Coated Bamboo paddles exposed to fish toxins from flesh or extract are fixed in methanol and immersed into suspension of latex bead and monoclonal antibodies specific to ciguatoxin. When this test was used in conjunction with the S-EIA test on 160 toxic fish implicated in ciguatera poisoning in Hawaii, there was a 100% agreement (Hokama, 1990). Efforts for improving this aassay are in progress (D.L.Park, Personal Communication).
Clinical Symptoms in Humans:
Ciguatera poisoning in humans involves a variety of symptoms. These symptoms range from being mild (such as diarrhea) to severe ones like Vertigo. The variable symptoms displayed by people suggests the presence of multiple toxins in the various kinds of fish consumed (Withers, 1982; Hokama, 1985). Most symptoms observed among people intoxicated with ciguatera poisoning are gastrointestinal, neurological and cardiovascular (Bagnis, 1979; Grant, 1984; Lange, 1987; Ragelis, 1984; Withers, 1982).
The gastrointestinal symptoms involve diarrhea, vomiting, abdominal cramps and nausea
(Bagnis, 1979; Ragelis, 1984; Withers 1982). These symptoms appear within 10 minutes to 12 hours and may last for up to 7 days but they are self limiting.
Gastrointestinal symptoms are accompanied by neurological symptoms which 35 involve paresthesia, temperature reversal, dysthesia, motor incoordination, vertigo and loss of equilibrium (Bagnis, 1979; Gillispie, 1986; Hokama, 1986; Withers, 1982). The neurological symptoms tend to stay for months or years depending on the severity of toxicity in the consumed fish (Bagnis, 1984, Withers, 1982). Paresthesia and temperature reversal sensations are the major distinctive symptoms of ciguatera (Gillispie, 1986;
Ragelis 1984). It is the neurological symptoms that differentiates ciguatera poisoning from other food poisoning. Gastrointestinal and neurological symptoms are common in patients who have consumed herbivorous fish like surgeon fish. Initial exposure to ciguatera poisoning do not cause immunity instead sensitization occurs (Bagnis, 1979;
Lange, 1987; Ragelis, 1984). Neurological symptoms sometimes reoccur under stress or when consuming alcohol or even eating non-toxic fish (Gilispie, 1986; Lange, 1987;
Raegelis, 1984).
Cardiovascular symptoms involve hypotension, bradychardia, and tachycardia
(Bagins, 1979; Hokama, 1986; Ragelis, 1984). In contrast cardiovascular symptoms occur in addition to neurological and gastrointestinal symptoms when ingesting carnivorous fish like snappers and grouper (Bagnis, 1979; Hokama, 1986: Gillispie,
1986). Symptoms are more severe among natives of endemic areas rather than tourists due to the repeated exposure (Lange, 1987). Mortality can occur due to dehydration or cardiac arrest but is very rare (Bagnis, 1979; Hokama, 1986; Lange, 1987; Ragelis,
1984; Withers, 1982). For an in depth review of the ciguatera symptoms refer to
Juranovic and Park (1991) and Juranovic (1989). Medical Treatment:
Despite the fact that ciguatera poisoning outbreaks have existed for centuries, no complete treatment has been found for this illness yet. Various remedies have been used by the natives of the endemic areas. These treatments mainly involve the ingestion of fruits or aqueous extracts of plant leaves. A very popular treatment in the South Pacific is ingestion of roasted leaves and bark of the monpakoi plant froumefortea arrgenteal
Linneaus (Hiyama, 1943). One of the best known treatments in the Gilbertese islands in the South Pacific is the fruit of (te non), the Indian Mulberry (Morinda critrifolia^
Linneaus. This treatment involves consumption of a fruit mixture where unripe fruits from this tree are crushed with ripe ones and then mixed with coconut juice (Cooper,
1964).
Since ciguatera poisoning induces a variety of complex symptoms, finding one treatment to relieve all the symptoms has not been possible yet. Recently some treatments; however, have shown improvements in partially reducing symptoms displayed by ciguatera illness. For severe vomiting and diarrhea, intravenous fluids and electrolytes should be repleted to prevent dehydration which could lead to death
(Johnson, 1983; Withers, 1982). Artificial respiration and oxygen is used in the case of severe cardiac distress (Withers, 1982; Yasumoto, 1984), Electrolytes and atropine have been used for hypotension and bradycardia (Tantall, 1980). Atropine and calcium have shown protective action against ciguatoxin nerve cell membrane (Broadbent, 1987).
Dopamine infusion has been used for severe hypotension (Anthony, 1986). Induced 37 vomiting is encouraged within hours of intoxication to rid the body of the toxin (Johnson,
1983). Other therapies with inconclusive results have been used like calcium gluconate, procaine amide, vitamins (B6> BI2) and vitamin C (Gillispie, 1986; Halstead, 1988).
One successful drug in treating ciguatera poisoning is protamide. This drug has been used in the Bahama Island and 15 other countries but it is not approved in the U.S.
(Ragelis, 1984). Another successful treatment was found in Queensland, Australia where a patient suffering from itching, numbness of the feet, malaise and lethargy after hours of consuming fish was completely cured with (25 mg) of Amitriptyline (Bowman, 1984).
In another case of two people suffering from ciguatera symptoms in the U.S., this drug helped relieve most of their symptoms except for temperature reversal and puritus,
(Calvert, 198.7). Amitriptilyne acts as a membrane stabilizer by blocking sodium channels in excitable membrane and also has an anticholingeric actions (Miller, 1985).
The most recent treatment was proven successful in the Marshall Islands (Palafox,
1988). Twenty four patients with acute ciguatera poisoning improved dramatically when given 250 mL of 20% intravenous mannitol. All the patients experienced less neurologic and muscular dysfunction and the gastrointestinal symptoms disappeared slowly. Palafox proposes the use of mannitol for initial use in patients with significant ciguatera symptoms even though the mechanism of action of mannitol is not yet known. 38
CHAPTER 2
MATERIALS AND METHODS
Culturing of the Dinoflagellates:
Organism:
Single celled microscopic dinoflagellate Prorocentrum concavum used in this study, was provided by L. Provasoli and R.R.L. Guillard, Bigelow Laboratory for Ocean
Sciences, McKnown Point, West Brothbay Harbor, Maine 04575.
Media:
Enriched seawater modified K medium was used to culture the dinoflagellates.
Modified K medium is based on Keller and Guillard's K medium (Keller and Guillard,
1985). The composition of the medium, vitamins and stock solutions are shown in
Tables 1 through 4. In this study a macroalgal extract was added to the media instead of Tris and silicate (J.W. Bomber, Personal Communication), Preparation of the macroalgal extract will be discussed later. An antibiotic solution of 0.1 % penicillin/streptomycin (2:1) was added to the medium. (Durand Clement 1987; Keller and Guillard, 1985) The pH of the media was adjusted to 7.8-8.2 using sterile (3N)
HCL and (1.5M) Na2 C03 solutions as necessary. Table 1: Modified K enriched seawater medium composition
Compound Concentration fmM)
NaNo3 8.83 X 10 '
2 NaH2P04 H20 1.00 X 10"
Vitamins
Thiamin • HC1 3.00 X 104
Biotin 2.10 XKT6
7 B12 3.70 X 10'
Trace Metals
FeEDTA 1.17X10'2
MnCl2 9.00 X 10^
s ZnSo4 8.00 X 10
5 CoS04 8.00 X 10"
5 Na2Mo04 3.00 X 10"
EDTA (Naj) 1.00 X 101
NH«C1 5.00 X 10"2
5 Na2Se03 1.00 X 10"
(J.W. Bomber, Personal Communication; Keller and Guillard, 1985;
Juranovic, 1989) 40
Table 2: Enriched seawater medium "enriched f/2" primary stock solution for trace elements.
Salt Primary Stock (w/v) *
CuSo4 5H20 0.98
ZnSo4 • 7H20 2.20
or ZnCl2 1.05
CoCl2 6H20 1.00
MnCl2 • 4H20 18.00
Na2Mo04 2H20 0.63
* w/v = n grams of salt diluted to 100 ml with distilled water
(Guillard, 1975; Juranovic, 1989) 41
Table 3 : Trace metals working stock solution for enriched seawater media
Compound Amount Ferric Sequestrene " 5 g Cu primary stock 1 ml Zn primary stock 1 ml Co primary stock 1 ml Mn primary stock 1 ml Mo primary stock 1 ml Distilled water up to 1 liter
* Dyestuffs & Chemical Divisions, CIBA-GEIGY Corporation, Greensboro, N.C 27419.
(Guillard, 1975; Juranovic, 1989) 42
Table 4 : Vitamin primary and working stock solution used in enriched seawater media
Vitamin Primary Stock Working Stock Biotin 0.1 mg/ ml 1.0 ml
B12 1.00 mg/ml 0.1 ml Thiamin • HC1 20.0 mg Distilled water up to 100 ml
(Guillard, 1975; Juranovic, 1989) 43
Media Preparation:
The media used to grow dinoflagellates was natural seawater obtained from a natural seawater well located in Puerto Penasco, Mexico. The seawater was stored in
55 gallon plastic drums at 4°C until use. Water was siphoned into four and six liter flasks and diluted with de-ionized distilled water to 32 7„, salinity. Media was prepared in two and four liter polycarbonate bottles. Salinity was determined by using ATAGO dual scale hand refractometer (S/MILL, 32-10 Honmachi, Itabashiku, Tokyo 173, Japan).
All the nutrients except the vitamin solution were added to the seawater and the pH of the media was adjusted to 4.0 with (3N) HCL. The media was autoclaved at 121°C (15 psi) for 20 minutes per liter of seawater in the container. Sterile media was stored at
4°C prior to use. Before inoculating the cultures, vitamin and antibiotic solutions were added aseptically to the medium and the pH was adjusted to 7.8-8.2 with sterile sodium carbonate. Macroalgal extract, trace metal solution, antibiotics, hydrochloric acid, and sodium carbonate were all stored at 4°C. Stock vitamin solutions were stored at 0° C; all other solutions were stored at room temperature. All glassware used in culturing operation was acid washed with 10% nitric acid, rinsed with de-ionized distilled water and sterilized by autoclaving at 121°C (15 psi) for 20 minutes.
Macroalgal Extract Solution:
Brown macroalgae (Sargasum sp.) was collected off the Southern California coast and stored in plastic bags at 4°C. The macroalgae was cut into small pieces and mixed 44 with de-ionized distilled water in a 1:3 ratio, respectively. The mixture was blended until a homogenous solution was obtained. The mixture was centrifuged (14,703 g) three times, the liquid extract collected and the residue discarded. The extract was autoclaved to achieve sterility. In order to remove any precipitated solids as a result of the autoclaving operation, the extract was centrifuged once again. The sterile extract was stored at 4°C until use. To obtain the equivalent dry weight of the extract, 1 ml of the extract was dried in quadruplicate at 110°C for 4 hours and weighed after cooling in a desiccator to room temperature. A 40 mg equivalent dry weight of macroalgal extract was added per liter of modified K medium (J.W. Bomber, Personal Communication).
Vitamin Stock Solution:
Twenty (20 mg) of Thiamin.HCL in was mixed with (1 ml) of Biotin, (0.1 ml) of vitamin B12 and dissolved in deionized distilled water to a total volume of 100 ml. The solution was sterilized using 0.45 ^m filter disc connected to a syringe. The sterile solution was aseptically dispensed into sterile vials (4ml/vial) and stored at 0° C until use.
Culture Maintenance:
Unialgal culturing was accomplished using tubes/flasks ranging from 10ml-2.8L.
Master cultures were kept in 10 ml glass tubes. Mass culturing was performed in 2.8L fernbach flasks, 20 L carboys and 20L plastic bags. The scale up procedure used is shown in Figure 3. All cultures were maintained in enriched natural seawater K medium 45
10 mi tubes I 125 ml flask I 250 ml flask
vV 500 ml flask
1000 ml flask
>v 2800 ml fernbach Harvest (every 25 days)
20 liter plastic bags 20 liter carboy I I Harvest Harvest (every 25 days) (every 25 days)
Figure 3: Procedure for large - scale culturing of Prorocentnim concavum. 46 at pH 7.8 and 32 °!00 salinity as described earlier. General culture conditions were
24-27° C temperature and alternate light/dark cycle of 14/10 hours. Lighting was provided by four 40 watt cool white fluorescence lamps (General Electric shop lite).
Illumination was at 45 microeinstein m"2 sec"1. Four lights were used for every incubator or vat. Culture transfers were performed at intervals of 15-21 days and harvesting of large scale cultures was performed every 21-25 days.
Large Scale Culturing Operation:
Large scale culturing operations were accomplished using 2.8L fernbach flasks, 20L carboys and 20L plastic bags suspended in a 300L water bath vat.
Fernbach Flasks:
Cultures grown in 1000 ml flasks were aseptically transferred into 2.8L fernbach flasks containing one liter of K medium. Inoculated fernbach flasks were kept in incubators at 24-27°C with alternate lighting as described previously. Each flask was turned 180 degrees every two days to insure even exposure to the light, Transfers were performed every 21 days and harvesting was performed every 21-25 days.
Carboys:
One or two 2.8L fernbach flasks were inoculated into each carboy containing two liters of K media. Due to the size of the carboys, transfers were performed in the 47
Figure 4: Large-scale culturing of Prorocentrum concavum using 20 liter carboys. 48 incubator. Cultures were gently aerated with filtered air through 0.45 /im filter disc connected to an aquarium pump (Second Nature, Willinger Bros, INC. Wright Way,
Okland, NJ 07136) through teflon plastic tubing (Figure 4). The same criteria for temperature and lighting was used for the carboy as for the fembach flasks. Two liters of K medium were added to each carboy weekly after the first inoculum.
Plastic Bags:
A three hundred gallon round fiber glass vat (custom made) was partially filled with water maintained at 24-27°C. A water heater and cooling system were used to control the water temperature. Six 20L disposable plastic bags (Shamrock Foods Co,
1900 Ruthrauf Rd, Tucson, Arizona) were suspended in the vat using a metal hanger made to hook to the bag cap (Figure 5). Cultures were aerated as described for the carboys. Illumination was provided by 4 cool fluorescent lights suspended with chains from the top of the vat. Two liters of K medium were added to each bag every week after the 1st inoculation. The same criteria applied for harvesting as for the fernbach and carboys.
Morphological Characteristics of Prorocentrum concavum.
In the current study morphological characteristics of P.concavum were studied using the scanning electron microscopy (SEM) (Dalenberg and Park, 1990). Cultures were grown in 1000 ml flasks and maintained in the same manner as mentioned earlier. 49
Figure 5: Large scale culturing of Prorocentrum concavum using 20 liter plastic bags. 50
Culture samples were taken at an interval of 3 days for 21 days. Cultures were processed using a basic aldehyde fixation procedure, followed by usual mounting and coating processes to prepare samples for scanning electron microscopy.
Harvesting Operation:
Cell Count Technique:
Cells were scraped off the sides and the bottom of the flasks/bags with a rubber spatula and combined all in plastic container. Sampling was done in quintuplet and cells were counted under a bright field microscope (Cooke, Troughton and Sims LTD, At
York, England) using a hemacytometer (Hausser Scientific, Blue Bell, PA 19422). The number of cells per cubic mm was determined as follows:
# cells/cubic mm = # of Cells counted x 5 x dilution # of cubic mm in chambers
Average cell density (cells/ml) was obtained through calculations.
Cell Harvesting Operation:
During the first phase of the study, harvesting was accomplished using a plankton net. Cell cultures were retained in the net by pouring the medium through the net. The medium filtrate was decontaminated with 10% bleach and discarded. The cell pellet was scraped off the net with a rubber spatula and concentrated using a centrifuge (14,703 g).
In the later phases of the study, harvesting was accomplished by passing the culture 51
through a continuous flow cream clarifier (New World's Standard Series, DE LEVAL.
No 619). Cell cultures were poured in the supply can and the flow of the culture was
controlled. Culture then passed through the regulatory cover and into the bowl where
separation occurred due to the centrifugal force. Cell pellets accumulated on the inside
of the centrifugal bowl and the liquid medium passed out where it was decontaminated
and discarded. The cell pellet was scraped off the bowl with a rubber spatula,
transferred with a small amount of liquid left inside the bowl to centrifuge bottles. Final
centrifugation (14,703 g) was used to concentrate the cells. Cell pellets were stored at
4°C until lyophilization.
Cell Lyophilization:
Cell pellets obtained from different harvests were combined and dried using a
Lyph-Lock 18 liter freeze drier system (Labconco Corporation, Kansas City, MO
64132). The cell pellet was shell frozen in a fast freeze dry flask by rolling the flask in a dry ice/acetone bath. The flasks were then connected to the freeze drier when the
temperature reached -40°C and the vacuum was equal to (7 n Hg). Upon completion of
the freeze drying operation, the vacuum was removed, the flasks disconnected and the cells scraped off. Dry cells were weighed and stored in jars in a desiccator until preparation of diets or analysis.
The number of dinoflagellate cells/g was determined by:
# cells / g = total # cells total dry cell wt (g) 52 Preparation of Fish Diet:
Three diets were prepared for this study: Standard Control SC diet, 50/50
Standard Control/Dinoflagellate cell 50/50-SC/DC diet, and 100% Dinoflagellate cell
100% DC diet. The SC diet was obtained from the Environmental Research laboratory,
University of Arizona, Tucson, AZ. The fish diet was prepared by mixing the ingredients together and pelleting using a California Pellet Machine (California Pellet Machine,
Inc.). The ingredients of the SC diet is shown in (Table 5). The standard control/dinoflagellate cells 50/50-SC/DC diet was prepared by mixing equal weights of the standard control diet and dry dinoflagellate cells. The mixture was freeze dried as described earlier and stored at room temperature in a desiccator until use. The 100% dinoflagellate cell diet consisted of pure freeze dried Prorocentrum concavum cells.
Proximate Analysis of Fish Diets:
Samples of the SC, 50/50-SC/DC and 100% DC diets were analyzed for percent (%) moisture, fat, protein, ash and acid digested fiber. The analyses were performed in duplicates according to the AOAC methodology sections (930.15 , 954.02, 954.01,
942.05, 989.03), respectively (AOAC 1990). The analysis was conducted at the Food
Analysis Laboratory, Department of Nutrition and Food Science, University of Arizona,
(Courtesy of Dr.Charles Weber).
Samples of the three diets were also tested for toxicity using the brine shrimp and stick- enzyme linked immunosorbent assay S-EIA. Samples of the diets were also tested Table 5: Ingredients composition of the standard control diet.
Ingredients Amount
Wheat 48.8 %
Soybean oil meal 27.0 %
Anchovy meal 10.0 %
Gluten 5.2 %
Fish Solubles 4.0 %
Tuna oil 4.0 %
Poultry Vitamin Pre-mix 0.55 %
Trace Mineral mix 0.20 %
NaCl 0.20 %
Ascorbic Acid 0.05 %
(Environmental Research Laboratory, University of Arizona) 54
for okadaic acid (OA) by the HPLC method reported by Lee (1980).
Exposure of Fish to Toxins Produced by Dinoflagellate:
Domino damsel (Dascvllus trimaculatus) fish were exposed to diets containing varied mounts of Prorocentrum concavum cells and/or SC diets as previously described.
Establishment of Aquaria:
Five 20 gallon aquaria were purchased from a local pet store. Four aquaria were placed on wooden shelves supported by blocks. The fifth aquarium was placed on a counter nearby (Figure 6). Each tank used an under gravel filter system and air stones were suspended in the lift tubes inserted into the filter. Artificial seawater at 327 salinity was added to each tank followed by crushed coral to a height about 2 inches.
0.15 g of ammonium chloride was added and the air pump was connected. The aquaria were allowed to subside for one day. A mixture of bacterial strain 25 /*! of
(Nitrosomonas and Nitrobacter) (Cycle, Rolf C.Hagen Corp, Mansfield, Ma. 02048) was added the following day and the ammonia level were allowed to settle for one week.
Two non toxic plastic plants (Second Nature, Willinger Bros, Inc. Wright way,
Oakland, NJ, 07136) were placed in each aquarium to provide shelter for the fish.
Additional filtration [Aqua Clear 200 Dual Filtration, Rolf C. Hagen (U.S. A) Corp, 50
Hampden Road, P.O Box # 634, Mansfield, MA 02048] was mounted on the back of each aquaria to maintain the cleanliness of the water. 1 2
L
5
3 4
Aquaria Diet 1 & 4 100% Dinoflagellate cell. 2 & 3 50/50 Standard control/dinoflagellate cell. 5 Standard control. Ul Ul 56
Domino damsel fish (Dascyllus trimaculatust were purchased from a pet store in
Mesa, AZ (Salty Dog Pet Shop, Home Depot Center, 1320 S. Country Club Dr, Mesa,
AZ. 85210). Initially 15 fish were placed in each aquarium. The fish in each tank were fed lg of SC diet daily for two months before commencing with the dinoflagellate feeding experiment.
Fish Feeding Study:
The position of the aquaria containing the fish to be exposed to the various diet combinations were selected randomally. Position of each aquarium/diet is presented in
(Figure 6). A total of 27 fish were exposed to each toxin containing diet. The control aquarium contained 14 fish. Fish in each aquarium were weighed (wet weight) by transferring to a beaker containing aquarium water. The fish were returned to the tank one more day before the feeding study started to minimize the stress on the fish. As the feeding study started, each aquaria received 0.5g/day of the appropriate diet. The diets were broken into smaller pieces with a spatula and small amount was given at a time to insure total consumption and avoid accumulation of the food in the tank.
The experiment was designed to continue for 30 days; However, due to the feed refusal exhibited by the fish receiving the 100% DC diet, the feeding regime was changed and, as a result took longer than 30 days. These fish were switched to SC diet for three days. Feeding the 100% DC diet was resumed for three days. Feeding refusal occurred again; therefore, the fish were put on SC diet for six days. The fish were then 57 given SC diet which contained concentrated extract of 100% DC added to it. Finally they were given the 50/50-SC/DC diet. At the end of the study all the fish were maintained in the aquaria for one day without feeding to insure the removal of any residual diet from their gastrointestinal system. Fish from each tank were weighed (wet weight) and euthanized.
Fish Euthanasia:
Euthanasia was done according to (Public Health Service Policy on Humane Care and the Use of Laboratory Animals). Fish were euthanized by decapitation to minimize pain and suffering. The viscera were removed by cutting the lower part of the body and removing with pointed forceps. All the viscera of fish from one tank were combined and stored in a plastic bag at -80° C until analysis. The same procedure was used for the head and the flesh portions of the fish.
Determination of Toxin Potential:
All the fish diets, aquaria water and the fish samples exposed to toxic dinoflagellate diets and/or Standard control diet, were extracted and tested for toxicity.
Diets;
One (1) g portions of SC diet, 50/50-SC/DC diet and the 100% DC diet were weighed, ground to a homogenous form and transferred to centrifuge bottles. An 80% methanol/water solution was added and the cells were lysed using sequential sonication 58
(Branson model #2200, Eagle Rd., Danbury, CT. 06810-1961) for 30, 20, and 10 minutes. After each sonication, cells were centrifuged (14,703 g) for 10 minutes. This procedure was repeated two more times. The supernatant was collected and filtered through Whatman #2 filter paper and extra thick fiber glass filter paper (Gelman Sciences
600 South Wayner Rd.t Ann Arbor, MI 48106) to remove any cell residue left. The cell pellet was discarded. A roto-evaporator was used to remove methanol at 65°C.
Excess water left in the sample was removed by heating. The concentrate was taken to a known volume with absolute methanol and stored at 4°C until used for toxicity testing.
Aquaria Water:
Aquaria were tested for water quality during and at the end of the feeding study.
Water was tested for the presence of nitrate an end product of toxic fish waste which accumulates in the water and affects the water quality. Water was also tested for ammonia and nitrite levels. Testing for ammonia, nitrite and nitrate was accomplished using a Sea Test Kit (Aquarium systems, 8141 Tyler Blvd, Mentor, Ohio 44060). Water was also tested for toxicity during and at the end of the feeding study by taking 500 ml water samples from each aquarium. The samples were extracted with the same volume of hexane for 5 minutes. The hexane layer was retained. Hexane was evaporated using the roto-evaporator at 65°C . The remaining residue was dissolved in 50 ml absolute methanol. The samples were evaporated to dryness and the residue was redissolved in
15 ml absolute methanol and stored at 4°C until ready for testing. 59 Determination of Toxicity in Fish:
Toxin extraction from fish was accomplished according to the McMillan method
(McMillan, 1980) as modified by the Food and Drug Administration (Douglas Park,
Personal Communication). This procedure involves three major steps: Extraction,
Purification, and Partition.
Extraction:
Fish tissues (flesh, head, and viscera) were defrosted and autoclaved at 121° C
(15 psi) for 10 minutes. Test portions from fish feeding on the diet were combined and homogenized using a Biohomogenizer (Biospec Products, Inc. P.O Box 722 Bartesvill,
OK 74005). The samples were mixed with acetone (1:1; w/v) ratio using a wrist action shaker (Burrel Corporation, Pittsburgh, PA U.S.A.) for 5 minutes and centrifuged
(14,703 g) for 15 minutes. The acetone extraction step was repeated 3 times and all extracts were combined.
Purification:
Acetone extracts were held overnight in a freezer at -18°C and the precipitate filtered using a cold Buchnner funnel and Whatman #\ Filter paper. Acetone was removed with a roto-evaporator at a 45°C. Methanol was added at a ratio 2:1. The mixture was washed 3 times with hexane (1:1 ratio) and hexane portion discarded.
Residual methanol was removed using a roto-evaporator at 64°C. 60
Partition:
Partially purified extracts were transferred to a separatory funnel, mixed with chloroform (1:1; w/v) ratio and allowed to partition. The chloroform layer was collected.
Partition step was repeated two times and all extracts were combined. Chloroform was
removed on a steam bath and the residue weighed. Extracts were stored in vials in a
freezer at 0°C. For toxicity testing, samples were taken in minimal volumes of chloroform.
Toxicity Testing Technique:
Brine Shrimp Assay:
Sample extracts to be tested were taken in known volume of solvent, diluted as necessary and added to borosilicate glass tubes (12x75 mm). Solvents were evaporated to dryness under nitrogen on a heat block. Shrimp eggs were incubated for 24 hours at
31°C in artificial seawater prepared by adding 15 g of sea salt (Aquarium Systems, 8141
Tyler Boulevard, Mentor, Ohio 44060) per 500 ml deionized distilled water. Hatched larvae were separated from the eggs by using light. Brine shrimp solution was diluted in order to obtain 15-20 shrimp/ml. One (1) ml of brine shrimp solution was added to each tube containing previously dried test portions or controls. Samples were evaluated in quintuplet. All tubes were incubated at 31°C and surviving larvae were determined by counting the number of dead shrimp/tube at 4, 8, 16, 24, and 48 hours. After 48 hours surviving larvae were killed with heat and the total number of shrimp/tube was 61
counted to determine the percent mortality. Positive and negative controls were included
in every test.
Determination of Ciguatoxin and Related Polyether Compounds:
ELISA Assay:
A stick-enzyme immunosorbent assay (S-EIA) based on the enzyme linked
immunosorbent assay (ELISA) developed by researchers at the University of Hawaii
(Hokama, 1977, 1979, 1983, 1985, 1987) was used to determine toxicity due to
ciguatoxin and other polyether compounds in fish tissues and fish extracts. All reagents and materials necessary to perform the test were provided by Dr. Hokama (University of Hawaii at Manoa, John A. Burns School of Medicine, Department of Pathology, 1960
East-West Road, Honolulu, Hawaii 96822). Proper function of the test was confirmed
by positive control obtained from ciguatoxic fish and fresh substrate prepared every time
the test was done. Preparation of the substrate, buffers and other solutions is discussed
later. Test portions were analyzed in triplicate plus a blank stick.
Bamboo sticks coated with correction fluid (Pentel Correction Pen, Pentel Co.,
LTD, Code-ZLM 1 -W) were inserted in fish tissues or dipped in fish extract and allowed to dry in tubes for 15-30 minutes. The sticks were then fixed in methanol for
1 second and allowed to dry for 5-10 minutes. Sticks were washed gently in Tris buffer
B (0.05M) wash one for 5 seconds and excess solution was blotted on a tissue. The 62 sticks were then dipped in the tube containing the antibody for 1 minute and excess fluid drained inside the tube. Later the sticks were washed gently in two washings of Tris buffer B wash 2 and 3, respectively, for 15 seconds each. The excess solution after buffer wash 3 was blotted on a tissue. Finally every stick was immersed in a tube containing 0.3 ml substrate which contained Tris buffer B, H202, and 4-chlorol-naphthol and shaken for 10 seconds. After incubating for 10 minutes, the sticks were removed and the substrate color was compared to the color chart. Diagram of the S-EIA test is shown in (Figure 7) . The color chart was rated from clear (zero) to dark purple (5).
Ratings of 0 - 1.2 was considered nontoxic, 1.3 - 1.9 borderline, and 2-5 was considered toxic. Analyses were conducted in triplicate and results were averaged.
Samples were also read at 405 and 540 nm wavelength using an ELISA reader (Titertek
Multiskan MCC/340).
Substrate Solution:
In a 25 ml volumetric flask added 5 ml of (3.0% H2O2) and 25 (A of (0.02%) 4
Chloro-l-naphthol. Added Tris buffer B to make 25 ml. The solution was transferred to a capped flask covered with aluminum foil and was shaken for 5 minutes in an action hand wrist shaker (Burrel Corporation, Pittsburgh, PA). The solution was filtered in the dark using Whatman #1 filter paper. The filtrate was transferred to dark flask covered with aluminum foil. Coated bamboo stick 1 second 5 seconds
blot excess buffer
Oa-U w drain methanol buffer wash 1 Mono-Ab Ab inside tube
10 minutes 15 seconds 15 seconds
blot excess buffer
W substrate buffer wash 3 buffer wash
Figure 7: Diagram of the Stick-Enzyme Immunoassay (S-EIA). 64
Table 6: Composition of the substrate solution for the Stick-Enzyme Immunoassay.
Compound Amount
4 Chloro-l-naphthol (0.02%)" 25 (xl
Hydrogen peroxide (3.0%)b 5 ml
Tris buffer B c 20 ml
Substrate total volume prepared was 25 ml.
* 0.075 g 4 chloro-l-naphthol dissolved in 1 ml absolute methanol.
b 1 ml (30%) H202 diluted into 9 ml of deionized-distilled water.
e 7.5 g Trizma (0.05M) dissolved in 1000 L deionized-distilled water. 65 Okadaic Acid Analysis:
High Performance Liquid Chromatography (HPLC) Procedure:
Samples of standard control diet, 50/50 standard control/dinoflagellate cell diet,
100% dinoflagellate cell diet and extracts from fish tissues (flesh, head, viscera) were sent to Dr. Jean-Marc Fremy (CNEVA, Laboratoire Central d'Hygiene Aliamentaire, 43, rue de Danzig, f-75015 Paris, France) for okadaic acid (OA) analysis. The method is based on (Lee, 1980) HPLC method for determination of fatty acids, using 9- diazomethylanthracene (ADAM) as a fluorescent and ultra-violet label.
Samples were evaporated to dryness and redissolved in 500 pi of chloroform.
This solution was esterified to 100 p1 of 0.1 % ADAM reagent for derivatization. The derivatized isolate was purified using silica gel column (SEP PAK) conditioned with hexane:chloroform, washed with hexane:chloroform followed by chloroform.
Derivatized okadaic acid was eluted with chloroform:methanol (95+5). The isolate was dissolved in 100 #tl methanol and 10 p 1 were injected into the instrument. The analyses were performed using a LICHROCART 250-4 HPLC and a Cartridge Supersphere 100
RP-18 (MERCK) column (5 jiM pore size and 250 mM length). Eluting solvents were acetonitrile:water (80:20), (70:30) and (75:25) at a rate of 1 ml/minute. Spectrometric detection was performed using an F 1000, MERCK fluorometer (365 nm excitation; 412 nm emission) Proper function of the system was confirmed by analyzing a reagent blank,
OA standard and addition of OA standard to samples. 66
CHAPTER 3
RESULTS AND DISCUSSION
Large Scale Culturing of Dinoflagellates:
Prorocentrum concavum cultures grown in K media exhibited excellent growth and high cell density. The decision to use K media was based on previous studies in this laboratory (Juranovic, 1989). P. concavum yielded higher cell growth in K media than the L20-1 media. The addition of a macroalgal extract to the K media has been shown to increase the dinoflagellate growth (Carlson, 1984; Bomber, 1985). The macroalgae/dinoflagellate association in nature suggests that the macroalgae are beneficial to the dinoflagellate by providing them with vitamins, chelating agents and nutrients. In the current study, P.concavum cells attached to the containers, in particular the bottom, in addition to attaching to each other and forming strands (Figure 9).
P. concavum cells had a tan-brownish color and under a light microscope , cells were motile and had an oval shape with double cell wall. Cells grown in 2.8 liter fernbach flasks were turned every two days to assure even exposure to the light. In comparison, cells grown in 20 liter carboys and plastic bags were in suspension continuously due to the aeration which created gentle movement of the cells. The use of carboys and plastic bags for mass culturing provided larger surface area for the organism to attach and grow. Also, the addition of 2 liter media each week after the first 67 inoculum, provided more nutrients for the cells which resulted in higher growth and cell
mass. Comparison of the three kinds of containers used for culturing, showed that the carboys yielded the highest dry cell weight (Table 7).
A similar yield for G. toxicus (0.29 g/L) was reported by Withers (1981). The
highest number of cells/g obtained in our study for P. concavum culture was 1,2 X 108 cells/g. Bomber (1985) reported 8.2 x 103 cells/g, 6.9 x 103 cells/g, and 5.1 x 103 cells/g for £. toxicus, H. gibbesii. and P.capitalus respectively (Bomber, 1985).
Antibiotics were added to the culture to minimize the bacterial contamination observed in P.concavum by Juranovic (1989). The presence of low levels of bacteria in the unialgal cultures did not appear to adversely affect the dinoflagellates. Carlson (1984); however, reported bacteria containing P. concavum cultures enhanced the dinoflagellate growth. It was suggested that bacteria aided in macroalgal metabolism which was beneficial for the growth of P. concavum.
There were advantages and disadvantages observed in the use of different containers for culturing. The major disadvantage of using glass fernbach flasks and carboys was the time required for cleaning. The plastic bags had the advantage of being disposable; however, they would inflate occasionally and come up on top of the water.
When this occurred it was necessary to remove the air by pressing on the bags and allowing the air to escape through the aerator tubes. All cultures contained some dead cells which had whitish color and the cells looked empty with no cell material under the microscope. 68
Table 7: Comparison of Prorocentrum concavum dry cell weight (g/L) cultured in different
large scale containers.
Culture Liters Dry cell Dry cell weight g/L Vessel weight (g) 14.2 3.81 0.27 2.8 L fernbach flask 21.6 4.77 0.22
22.6 5.86 0.26 41.3 10.49 0.25 Average 0.25
7.28 2.31 0.32 20 L carboy 9.29 3.30 0.35 22.4 3.38 0.15 19.4 6.02 0.31 23.0 8.32 0.36 Average 0.30
29.0 6.76 0.23 20 L plastic bags 44.6 8.60 0.19 31.4 9.82 0.31 42.6 10.66 0,25 Average 0.24 69 Morphological Characteristics of Prorocentrum concavum:
Prorocentrum concavum cells observed using the scanning electron microscopy appeared to have openings covering the entire outer surface of the cell (Figure 8) (Faust,
1990). Mucus layer appeared to attach the cells together (Figure 9). Cells reproduced by opening their shells where a new (daughter) cell came out (Figure 10). No doubling of cell number was observed during reproduction. The younger cells also appeared to have openings on their surface which increased in number with age. Dead cells appeared as an empty shell (Figure 11).
Harvesting of Prorocentrum concavum:
Harvesting P. concavum cultures using the continuous flow centrifugation cream clarifier proved to be much more efficient than the plankton net. The advantages of the cream clarifier are higher cell pellet recovery, decrease harvest time by over 50% and minimal use of the centrifuge. There also appeared to be increased recovery of the cells; for example, harvesting about 22 liter of culture media grown in 2.8 L fernbach flasks in a cream clarifier yielded 0.26g/L dry cell weight compared to 0.22 g/L using the plankton net. Comparison of the two harvesting methods is shown in (Table 8).
Fish Diets:
The three diets used in this study had different textures due to the difference in composition. The Standard control SC diet had a hard texture and the pellet size was too Figure 8: Prorocentrum concavum cell. Figure 9: Attachment of Prorocentrum concavum cells by mucus. Figure 10: Daughter Prorocentrum concavum cell emerging from parent cell. 73
Figure 11: Daughter and skeleton of Prorocentrum concavum cell. 74
Table 8: Comparison of Prorocentrum concavum dry cell weight yield cultured in 2.8L
flask using different methods of harvesting.
Harvest Total No. Total No. No. Cells/ml Dry cell Dry cell Method Liters Cells weight * (g) weight g/L Cream 22.6 3.8 x 108 1.69 x 104 5.86 0.26 clarifier
Plankton 21.6 3.02 x 108 1.39 x 104 4.77 0.22 net
Cells weighed after being freeze dried. Table 9: Composition of diets containing Prorocentrum concavum cells.
Diet Total No. cells Dry cell weight (g) No. cells/g
50/50-SC/DC' 1.18x10® 40.11g 2.94 x 107
100% DC b 3.03 x 10® 35.66g 8.5 x 107
" 50/50 Standard control/dinoflagellate cell diet b 100% Dinoflagellate cell diet 76 large for this particular fish species (Domino damsel). It was, therefore necessary to break it to smaller pieces before feeding. The 50/50-SC/DC dinoflagellate cell diet was easier to prepare for feeding due to the presence of dinoflagellates. The 100% DC diet was very flaky. In fact, some of the 100% DC diet became a powder-like consistency when it was broken into smaller pieces. That also happened with the 50/50-SC/DC diet but to a lesser extent. When this occurred the powder-like diet was mixed with few drops of water, cut into small pieces and allowed to dry before feeding to the fish. The
P.concavum composition of the 100% DC and the 50/50-SC/DC diets is presented in
(Table 9).
Proximate Analysis:
The proximate analysis conducted on the diets used in this study showed that the
SC diet was more nutritionally complete than the 50/50-SC/DC and the 100% DC diets.
Since the objectives of the current study was to determine the biotransfer of toxins from the diet to the fish, nutritional quality of the diet was not of concern. Formulated fish diets have certain specifications for nutrients for growth and quality of feed. Small amounts of moisture in the feed are required to make it more palatable while minimizing microbial growth and spoilage (Lovell, 1989). The 100% DC diet had slightly lower amount of moisture than the SC and the 50/50-SC/DC diet (Table 10). The amount of fat in the 100% DC and the 50/50-SC/DC diets was much lower than the SC diet. Fat percentage in a fish diet higher than about 8% affects the water quality especially if the Table 10: Proximate Analysis of diets used in fish feeding experiment.
Diet % Moisture % Fat % Protein % Ash % ADF4
SCb 4.07 ± 0 7.81 ± 0.08 34.53 ± 0.39 6.40 ± 0.02 3.55 ± 0.13
50/50-SC/DCc 4.33 ±0.05 1.06 + 0.14 24.21+ 0.20 25.45 ±0.21 8.65 ± 0.06
100% DCd 3.34 ±0 1.16 ± 0.05 13.58 ±0.06 50.06 ±0.01 12.50 ±0.37
* Acid digested fiber b Standard control diet c 50/50 Standard control/dinoflagellate cell diet d 100% Dinoflagellate cell diet 78 feed is not consumed immediately (Dan Parker, Personal Communication). The SC diet had about 35% protein which is within the range recommended (25-50%) for fish growth. The 50/50-SC/DC diet had 24% protein which is optimum for fish growth. On the other hand, the 100% DC diet had much lower percentage of protein. Protein is used as an efficient source of energy by fish unlike land animals which rely mainly on carbohydrates as an energy source (Weatherley, 1986). Mineral requirement for fish should not exceed 10% since higher amounts interfere with the nutrients metabolism
(Dan Parker, Personal Communication). The SC diet had a sufficient amount of minerals
(about 6%) whereas the 50/50-SC/DC and 100% DC diets had 4-8 times higher amounts than the SC diet (Table 10). This may have affected the metabolism of the fish on these diets. Fiber is not a critical factor in fish diet; however, small amounts are needed for digestion. The SC diet had 3.5% fiber whereas the 50/50-SC/DC and the 100% DC diet had much higher amounts.
Fish Acclamation:
The Dascvlus trimaculatus fish when first were brought and placed in the aquaria, exhibited a shy and frightened behavior. These fish would become alarmed and hide when anyone approached the aquaria. This behavior persisted until the person would leave. The fish were small about 1 inch in length. The acclamation period was about one month during which a total of 7 fish died. The remaining 68 fish adjusted well to their new environment. Also, the shy and fearful behavior was no longer apparent and 79
they grew and increased in size. Eventually the fish became aggressive toward each
other and developed their own territories and individual dominance. During the
adjustment period, the fish fed well on the custom made fish diet but apparently the
amount given to them daily (lg/tank) was more than they would consume. Occasionally
the fish were observed picking the food from the gravel; however, most of the fish went
after the food when it was floating or when it was coming down.
Behavioral and Toxic Effects of P.concavum Diets on Fish:
During the period of the feeding study, all the fish were watched for any change in feeding patterns and behavior. They were watched daily during and following the time of feeding. The control fish did not exhibit any change in their feeding pattern or
behavior. All the fish survived and there was no change in swimming patterns or appearance among this group. Their normal aggressive behavior was observed
throughout the study. The wet weight of the fish taken before and after the feeding study indicated an average weight loss of (0.24g/fish) eventhough it appeared they were feeding well (Table 11). Many reasons could be attributed to the weight loss. One reason could be that higher amounts of protein were needed for growth. Smaller fish require higher amounts of protein than larger fish due to the rapid increase in size in the early stage
(Lovell, 1989; Weatherely, 1986). Another possible reason would be that in their natural habitat these fish feed on plant as well as animal sources since they are omnivorous. That means their protein and fat intake could be higher than what they were given 35%. In 80 Table 11 : Average weight loss (g/fish) for all the fish treatment groups.
Diet No. Fish Fish Avg weight Avg wt loss Weight (g) g/fish g/fish sc- (s)b 14 26.00 1.86 0.24
(d) 0 - - -
(t) 14 29.40 2.1 -
50/50-SC/DC c (s) 9 16.58 1.8 0.6 (d) 4 5.46 1.4 1.0 (0 13 31.30 2.4 —
50/50-SC/DC (s) 6 15.39 2.6 0 (d) 8 8.04 1.0 1.3 32.10 2.3 (0 14 —
100% DC d (s) 10 19.98 2.0 0 (d) 4 3.85 1.0 1.0 (0 14 28.00 2.0 —
100% DC (s) 12 28.77 2.4 0 (d) 1 0.70 0.7 1.6
(t) 13 30.20 2.3 - " Standard control diet.
b s - survived; d - died; t - total. c 50/50 Standard control/dinoflagellate cell diet.
d 100% Dinoflagellate cell diet. 81
this study they were only fed one kind of diet. Although we tried to simulate the natural
habitat, these fish could have been stressed in this environment. All these factors could
have contributed to the fish loosing weight during the feeding study.
The fish group receiving the 50/50-SC/DC diet did not respond well to this diet.
Since the Dascvllus fish were found to feed only on floating food or while it was going
down, small amounts of feed were given at a time to insure total consumption and avoid
feed loses. As a result it took about 4-5 hours daily to feed this group of fish. This
treatment group ate about half of the food given to them. After 9 days of exposure to the
50/50-SC/DC diet, their appearance changed as well as their behavior. The fish developed whitish color spots and streaks on their bodies. Fish mortality was observed.
More aggressive patterns became apparent and the fish bit the fins of other fish. In addition, the 3 characteristic sharp white color spots for this species started fading away.
Changes in swimming pattern were also observed.
The affected fish would swim sideways with no balance. They would isolate
themselves from the rest and stay in the back of the tank in one location. Usually they
would become immobile, land on a tree or on the gravel and appear dead. Attempts to capture these fish with a net would cause them to move very slowly in a big circle and land on the gravel head down. Some of these fish were observed being sucked to the filter. When the filter was turned off, the fish were still alive but could not swim. Some of the affected fish which were immobile, had difficulty breathing. Other fish would stay in one location and flap their fins faster and more often than normal. All the fish that 82 exhibited the change of appearance and behavior usually would not feed, were picked on by the other fish and would die within 1-2 days. This is a possible way how the affected
fish in nature falls a prey to the other fish.
Twelve fish out of 27 total fish in this group died (Table 12). Among this group of fish, there was higher mortality in the aquarium situated on the bottom row (Figure
6, Table 13). It is not apparent why such a difference in mortality occurred since the fish in both aquaria received the same diet. All of the fish that died weighed less than 2.5 grams. This showed a higher affinity of the toxin for the smaller fish. The high fish mortality observed over a short period of time is attributed to acute toxicity. The wet weight of the fish taken before and after feeding indicates a higher weight loss in the fish that died than the fish that survived in both aquaria. The fish that died earlier lost an average of (1.15 g/fish). On the other hand, the fish that survived lost only (0.3 g/fish)
(Table 11). It is not apparent why the fish that died lost more weight than the surviving fish. One reason could be that since their diet was low in protein and fat which suppressed their growth. High minerals and fiber also affect fish growth by interfering with their metabolism and prevents the absorption of the nutrients available.
The fish group receiving the 100% DC diet started refusing to feed after four days of feeding. At feeding time they would approach the food, but once they knew it was the same food they would not respond to feeding. Therefore it took around 8 hours a day to feed them. Immediately upon switching to the SC diet normal feeding patterns resumed. To see if the feed refusal was in general or was it only to toxic food, the fish 83
Table 12: Effect of Prorocentrum concavum diets on Domino damsel fish fPascyllus
trimaculatus) mortality.
Diet Total No. fish/tank No. Dead fish % Mortality
SC» 14 0 0
50/50-SC/DCb 27 12 44
100% DCc 27 5 18
" Standard control diet b 50/50 Standard control/Dinoflagellate cell diet c 100% Dinoflagellate cell diet 84
Table 13: Comparison of the number and percentage offish mortality with respect to
aquaria location and diet.
Location Diet Total No. Fish %
Fish / Aquarium mortality / Aquarium Mortality
Top 100% DC 0 13 1 8
Top 50/50-SC/DC b 13 4 31
Bottom 50/50-SC/DC 14 8 57
Bottom 100% DC 14 4 29
Side SC c 14 0 0
8 100% Dinoflagellate cell diet b 50/50 Standard control/Dinoflagellate cell diet c Standard control diet 85
were fed the 100% DC and SC diet for 3 days intervals. Feed refusal was observed with
the 100% DC diet. To determine whether the fish feeding refusal was due to the algae
itself or the toxin, a 100% DC extract was added to the SC diet. When the fish were given the algal extract SC diet mixture, total refusal was observed again. This step verified the fish refusal to the food was due to the presence of the compounds in the
toxic algal extract rather than the algae itself.
The observations of the present study are the first report of this phenomenon with
finfish. Khalil (1989) reported a similar observation when oysters were exposed to
Gonyaulax monilata. This same treatment group was fed the 50/50-SC/DC and SC diets.
The fish had a greater affinity for the SC diet. Mortality was observed in this treatment group too. Prior to death, the affected fish had the same change in behavior patterns and appearance as mentioned previously. The fish had no balance and as a result would swim upside down. They would isolate themselves and stay in the back of the tank without movement and would be attacked by the other fish. Their fins were bitten and whitish color spots developed on their bodies.
In this treatment group, the fish that survived did not show any weight loss whereas the fish that died lost an average of (1.3 g/fish) (Table 11). The 100% DC diet treatment group had only 18% mortality (Table 12) whereas the 50/50-SC/DC diet treatment group exhibited 44% mortality. Aquaria position may have had the same effect on fish mortality observed in the 50/50- SC/DC treatment group since fish in the lower shelf aquaria had a higher percentage of mortality (Table 13). 86 Toxicity Testing:
Toxicity of Fish Diets to Brine Shrimp:
The SC diet showed no toxicity to brine shrimp (Appendix A). Both the 50/50-
SC/DC and 100% DC diets caused high toxicity to brine shrimp (Figure 12, 13). The
50/50-SC/DC diet caused 91% mortality to brine shrimp at 8 hours with a dose equivalent to 2.94 x 10® cells (Appendix B). The 100% DC diet caused 98% mortality at 8 hours with a dose representing 8.5 x 10s cells (Appendix C). Dose response curve for the 50/50-SC/DC, 100% DC diets and okadaic acid have been established (Figure 12,
13, 14 ; Appendix D). Control solutions were included for each test performed.
Aquaria Water:
Water samples tested for ammonia, nitrite and nitrate during the feeding study were within the acceptable levels except for the nitrate (Table 14). Ammonia is a toxic waste product of fish therefore level <0.1 ppm is required. All the aquaria water tested had an acceptable level of ammonia. Nitrites, which are produced from ammonia by bacterial action, are not as hazardous but are still toxic. All the aquaria water tested had undetectable levels of nitrite. Nitrates are the product of bacterial action on nitrite and eventually accumulate in the aquarium unless the water is changed on a regular bases.
The acceptable level of nitrate in the water is > 20 <40 ppm. The nitrate levels for three aquaria were within the acceptable range. Control aquarium; however, had (>40 <70 ppm) nitrate and one aquarium of the fish receiving the 50/50-SC/DC diet had % Mortality
4 8 16 24 48 Time (hours)
HH 1,47 x 109cells/g IH 2.94 x lO'cells/g
Figure 12: Toxicity of 50/50 Standard Control/Dlnoflagel late Cell fProrooentrum concavumi diet extract to brine shrimp fArtemia sp.) at 8 hours.
-jCD % Mortality
HBIP^ 16 Time (hours)
Figure 13: Toxlcliy ol 100£ dlnoflagel late oeU fProrocentrum concavum) to brine shrimp fArtemia sp.) at 8 hours. % Mortality 1 00
10 24 Time (hours)
Figure 14: Toxlcily of okadalc acid to brine shrimp fArtemia sp.) at 8 hours. 90
(>70<100 ppm) nitrate (Table 14). Water samples tested at the end of the feeding study had acceptable levels of ammonia, nitrite and nitrates (Table 15). Aquaria water tested for toxicity on brine shrimp during and after the end of the study showed no toxicity (Appendix J and K). Therefore no further testing was performed on the water.
Fish Tissue Samples:
Samples of flesh from fish receiving the SC diet representing 0.02g caused 48% mortality of brine shrimp at 16 hours. The fish heads and the viscera with a dose of
O.Olg and 0.006g, respectively, caused no toxicity to brine shrimp (Appendix E). The toxicity observed in the flesh portion could be due to accumulation of compounds occurring naturally in the fish environment before they were brought to our laboratory.
Similarly, flesh samples representing 0.015g from fish fed the 50/50-SC/DC diet caused
50% mortality to brine shrimp at 16 hours (Appendix F). The head and the viscera representing 0.005g and 0.003g, respectively, did not cause any mortality to the brine shrimp. Tissue extracts from fish fed 100% DC diet caused only 10% mortality to brine shrimp at 16 hours. The fish flesh, head and viscera were all of equal toxicity
(Appendix G). The fish that died as a result of feeding on the 50/50-SC/DC diet showed no toxicity to brine shrimp (Appendix H). Flesh extract representing 0.015g from fish that died as a result of the 100% DC diet caused 32% mortality to brine shrimp at 16 hours. The head and viscera caused no toxicity (Appendix I). Apparently, samples representing 0.015 - 0.031g of flesh caused 50% mortality to brine shrimp. Samples that 91
Table 14: Quality of aquaria water tested during the feeding study (day 19).
Diet Ammonia Nitrite Nitrate
(ppm) (ppm) (ppm)
SC» < 0.1 < 0.2 > 40 < 70
100% DC b < 0.1 < 0.2 > 20 < 40
50/50-SC/DC c < 0.1 < 0.2 > 20 < 40
50/50-SC/DC < 0.1 < 0.2 > 70 < 100
100% DC < 0.1 < 0.2 > 20 < 40
" Standard control diet b 100% Dinoflagellate cell diet c 50/50 Standard control/dinoflagellate cell diet 92
Table IS: Quality of aquaria water tested at the end of the feeding study.
Diet Ammonia Nitrite Nitrate
(ppm) (ppm) (ppm)
sc- < 0.2 < 0.2 >10 <20
100% DC b < 0.2 < 0.2 >10 <20
50/50-SC/DC c < 0.2 < 0.2 >10 <20
50/50-SC/DC < 0.2 < 0.2 >10 <20
100% DC < 0.2 < 0.2 >10 <20
* Standard control diet b 100% Dinoflagellate cell diet e 50/50 Standard control/dinoflagellate cell diet 93
represented less than 0.015g did not exhibit any toxicity. The same non-toxic phenomena
was observed with the head and viscera that did not cause any toxicity. Due to the
limited amount of samples tested, it was not possible to concentrate the samples enough
to get a dose response similar to the flesh samples.
Okadaic Acid Analysis:
Results of high performance liquid chromatographic (HPLC) analyses for OA
using the 80:20 (ACN:H20) mobile phase are presented in Table 16. OA was detected in the 100% DC and the 50/50-SC/DC diet at RT=14.45 min. Eight (8) ng of Standard
(OA) was detected at RT= 14.46 min (Figure 15). No OA was detected in the SC diet
(Figure 16). The dotted line and arrow points to where the (OA) would have been
detected if it was present in the sample. OA was detected at RT= 14.44 min in the OA spiked SC diet (Figure 16). No OA was detected in the fish tissue extracts, instead interferences were observed at RT=13.7 min (Figure 17). To avoid interferences the fish
tissue extracts were reanalyzed using a 70:30 mobile phase (Figure 18). No OA was present and the arrow refers to where the OA would have been detected. Standard OA
(8ng) was detected at RT=28.7 min. Finally the fish tissue extracts were analyzed with a 75:25 mobile phase (Figure 19). Again no OA was detected in the fish tissue samples and the Standard OA (8ng) was detected at RT= 18.57 min. This test did not show transfer of toxins (OA) produced by P concavum to the fish at a (24:1) feed to tissue ratio. Table 16 : High performance liquid chromatographic analysis of fish diets and fish tissue extracts for okadaic acid.
Sample Diet OA (figlg)
SC SC ND' 50/50-SC/DC 50/50-SC/DC 219
100% DC 100% DC 487
Fish flesh SC ND b Fish head SC ND b Fish viscera SC ND d
Fish flesh 50/50-SC/DC ND b Fish head 50/50-SC/DC ND b Fish viscera 50/50-SC/DC ND c
Fish flesh 100% DC ND c Fish head 100% DC ND c Fish viscera 100% DC ND d
" < 1 fig/g b < 5 /xg/g
0 < 10 fig/g d < 20 [xg/g V 8 ng okadaic acid 50/50-SC/DC diet 100 % DC diet
Figure 15: HPLC Chromatogram of Prorocentrum concavum diets at mobile phase 80 + 20 (ACN + H 0). 2 us ui 9 C 6 •€ T* . 1'
a
« I II I I I I I M IM 1 II I I I I I I • • r c •' o- r- i * t * * » t»• 111 * i i i i »i i i i »»i _ rl II <4 tf* 1* k' 6- Vi 1 *4 •• r»i r i 8 ng okadaic acid standard control diet standard control diet spiked with 2 ng OA
Figure 16: HPLC Chromatogram of standard control diet at mobile phase 80 + 20 (ACN + H20). XO o\ 8 ng okadaic acid 0.12 mg fish viscera 0.6 mg fish viscera
Figure 17: HPLC Chromatogram of fish viscera extract at mobile phase 80 + 20 (ACN + H20). 8 ng okadaic acid 0.3 mg fish viscera 0.4 mg fish flesh
Figure 18: HPLC Chromatogram of fish tissue extract at mobile phase 70 + 30 (ACN + H20). & m n9
©
ILiL 4LO © «iaX
1 J
i i l l «ii i it i• i i n i i i an* t M i nn r • ri - U 14 *9 ,r* n ux — r* *« 8 ng okadaic acid 0.17 mg fish head 0.77 mg fish flesh
Figure 19: HPLC Chromatogram of fish tissue extract at mobile phase 75 + 25 (ACN + H20). u> ID 100
Stick-Enzyme Linked Immunosorbent Assay (S-EIA):
The fish tissue samples (flesh and viscera) were tested in duplicates using stick- enzyme linked immunosorbent assay (S-EIA) as shown in Table 17. All fish flesh samples from the different treatment groups were shown to be positive. However, viscera samples exhibited a wide range of toxic responses. Viscera of fish from the control aquaria were negative. Fish receiving the 50/50-SC/DC diet exhibited borderline toxicity. This was apparent for surviving as well as fish that died before completing the feeding study. On the other hand the viscera of fish group that received the 100% DC diet and died before the end of the experiment exhibited positive toxicity whereas surviving fish had borderline toxicity. Due to the limited amount of test material, not all the individual fish were tested. For viscera samples, viscera from each treatment group were combined and tested. The heads were not tested with this system since it was not possible for the stick to penetrate a bony structure.
Fish homogenate samples were also tested in duplicates. All the flesh and the head homogenate samples from all the treatment groups tested positive (Table 18). A positive toxic response was also observed for fish viscera from different treatment groups except for the control fish viscera. There was a general agreement when comparing the results of the S-EIA test on the whole fish tissue and homogenate fish samples. A borderline response observed in the viscera and head tissues compared to positive response in the homogenate sample is possibly due to homogenization which allowed for extraction and homogenous distribution of toxins in the samples. The stick test assay is sensitive, fast 101
Table 17: Toxic response of fish tissue samples to antibodies specific to ciguatoxins and other related polyether compounds."
Diet Sample S-EIA Results Average 1st run b 2nd run b SC flesh + c + + head NT d NT NT
viscera - - -
50/50-SC/DC flesh -f + +. (died) head NT NT NT viscera ± + +.
50/50-SC/DC flesh ± + ±_ head NT NT NT
viscera +. - +_
100% DC flesh + + + (died) head NT NT NT viscera + + +
100% DC flesh + + + head NT NT NT
viscera ± - +
0 Method of Hokama (1987) b Mean of two analyses c (2.0 - 5.0) - positive (+); (1.3 - 1.9) - borderline (±); (0.0 - 1.2) - negative (-) d NT - Not tested 102 Table 18: Toxic response of fish homogenate samples to antibodies specific to ciguatoxins and other related polyether compounds."
Diet Sample S-EIA Mean Score S-EIA Avg score 1st runb 2nd run b SC flesh + c + + head + + + viscera
50/50-SC/DC flesh + + + (died) head + + + viscera + + +
50/50-SC/DC flesh + + + head + + +
viscera + +. +
100% DC flesh + + + (died) head + + + viscera + +
100% DC flesh + + + head + + + viscera + + + " Method of Hokama (1987) b Mean of two analyses 0 (2.0 - 5.0) - positive (+); (1.3 - 1.9) - borderline (+) ; (0.0 - 1.2) - negative (-) 103
and specific to ciguatoxin and other polyether toxins.
Comparative Toxicity:
Okadaic acid, a polyether compound associated with ciguatera poisoning was detected in high amounts in the 100% DC and the 50/50-SC/DC diets (Table 19). No
OA was detected in the fish tissue extracts at a detection level of 20 fig OA/g fish flesh.
This shows that there was no transfer of okadaic acid from the feed to the fish at a feed
to tissue ratio of (24:1). However, most of the fish tissue extracts were very toxic when tested on the stick test assay (S-EIA). The toxicity obtained from this assay could be attributed to the presence of other polyether compounds. This fish in its natural habitat could have accumulated polyether compounds other than okadaic acid that were not detected by the HPLC method.
Brine shrimp results were not in full agreement with the S-EIA test and the reason could be a factor of specificity. The brine shrimp assay is only a screening test whereas the S-EIA test is sensitive and specific to ciguatoxin and other polyether compounds.
Most of the fish viscera and head extracts from different treatment groups showed no toxicity to brine shrimp but were very toxic to stick test assay S-EIA. The reason could be that there is very small amounts of toxins in these tissues that were detected by the stick test since it is more sensitive to polyether compounds. 104 Table 19: Comparative toxicity of fish diets containing Prorocentrum concavum and extracts of fish exposed to these diets. Treatment Sample Brine shrimp S-EIA" S-EIA • OA analysisb toxicity tissue homogenate pglg SC diet c + d + ND 0
50/50-SC/DC diet + + + +' + + 219 100% DC diet + + + + + + 487
SC flesh + + + + + ND 8 head - NT + ND
viscera - - - ND
50/50 flesh - + + ND
(died) head - NT + ND
viscera - + + ND
50/50 flesh + + + + ± NT
head - NT + NT
viscera - + NT
100 % DC flesh + + + + + ND
(died) head - NT + ND
viscera - + + ND
100% DC flesh - + + NT
head - NT + NT
viscera - +_ + NT a Mean of two analyses d (1.3-1.9) borderline (+); (0.0-1.2) negative (-). b HPLC analysis (Lee, 1980) c Not detected c No Toxicity ' + + + + >90% toxicity; + + + 40-50% toxicity d (2.0-5.0) positive(+); 8 Not tested 105 CONCLUSION
1 - Unialgal Prorocentrum concavum cultures produced 487 fig of okadaic acid/g of
dry cell weight.
2 - Exposure of Damsel fish fPascvllus trimaculatus) to diets containing P.concavum
showed modified behavior and mortality.
3 - No okadaic acid was detected in fish tissue extracts of fish exposed to diets
containing P.concavum.
4 - There was no transfer of okadaic acid from the diets containing P.concavum to
the Domino damsel fish at a feed to tissue rate of 24:1.
5 - Fish receiving 100% P.concavum diets exhibited feed refusal.
6 - Toxicity exhibited by the fish tissue extracts could be attributed to other
polyether toxins implicated in ciguatera poisoning or even other
compounds present in the fish natural habitat.
FUTURE WORK
Further studies are needed to perform the feeding study for a longer period of time using fish that are raised in the aquarium instead of wild fish to avoid previous exposure to toxic compounds. The fish diet should contain higher protein with no more than 10-25% dinoflagellate cell content. In addition the control and the dinoflagellate diet should be nutritionally equal. 106
APPENDIX A
EFFECT OF EXTRACT OF STANDARD CONTROL SC DIET ON BRINE
SHRIMP (Artemia sp.) MORTALITY.
% Mortality3
Time Doseb
(Hour) 10 5 1 0.1 0.01 0.001
4 3 3 0 0 1 1
8 4 4 1 2 2 2
16 6 5 1 2 2 4
24 7 9 1 2 2 5
48 6 10 0 3 6 10 a % Mortality = % Mortality dosed shrimp-% Mortality control shrimp. b Dose represent fig of SC diet c Mean control mortality = 15 % 107
APPENDIX B
EFFECT OF EXTRACT OF 50/50 STANDARD CONTROL/
DINOFLAGELLATE CELL (Prorocentrum concavum) DIET ON BRINE
SHRIMP (Artemia sp.) MORTALITY.
% Mortality*
Time Dose'
(Hour) 2.94 x 106 1.47 x 10s 2.94 x 105 2.94 x 104 2.94 x 103 2.94 x 102
4 60 63 59 1 1 3
8 91 94 84 37 3 4
16 98 98 98 93 8 4
24 98 98 98 96 8 4
48 96 98 96 96 15 12
* % Mortality = % Mortality dosed shrimp - % Mortality control shrimp b Dose represent # of cells/g APPENDIX C
EFFECT OF EXTRACT OF 100% DINOFLAGELLATE CELL (Prorocentrum
concavumt DIET ON BRINE SHRIMP (Anemia sp.) MORTALITY.
% Mortality"
Time Doseb
(Hour) 8.5 x 106 4.25 x 106 8.5 x 10s 8.5 x 104 8.5 x 103 8.5 x 102
4 63 56 66 3 0 0
8 98 98 98 28 0 0
16 98 98 98 92 1 0
24 98 98 98 94 4 0
48 96 96 96 96 15 3
• % Mortality = % Mortality dosed shrimp - % Mortality control shrimp b Dose represent # cells/g 109
APPENDIX D
EFFECT OF OKADAIC ACID ON BRINE SHRIMP fArtemia sp.)
MORTALITY.
% Mortality'
Time Doseb
(Hour) 100 50 10 1 0.1 0.01
4 5 0 1 2 0 2
8 7 2 1 5 1 2
16 30 9 10 0 0 0
24 57 32 36 0 0 0
48 59 58 60 0 0 0
' % Mortality= % Mortality dosed shrimp- % Mortality of control shrimp
b Dose represent ng of okadaic acid 110
APPENDIX E
EFFECT OF FISH TISSUE EXTRACT FOR FISH FED
STANDARD CONTROL DIET ON BRINE SHRIMP fArtemia sp.)
MORTALITY.
% Mortality'
Doseb
Time Flesh Head Viscera
(Hour) 0.02 0.01 0.006
4 38 2 0
8 49 4 0
16 48 3 0
24 47 3 0
48 42 0 0
" % Mortality = % Mortality dosed shrimp - % Mortality control shrimp b Dose represent g of fish tissue Ill
APPENDIX F
EFFECT OF FISH TISSUES EXTRACT FOR FISH FED 50/50 STANDARD
CONTROL/DINOFLAGELLATE CELL DIET ON BRINE SHRIMP
(Anemia sp.) MORTALITY.
% Mortality"
Doseb
Time Flesh Flesh Head Viscera Viscera
(Hour) 0.015 0.0075 0.005 0.003 0.006
4 50 1 0 4 0
8 50 0 0 4 0
16 50 3 0 5 0
24 48 I 0 0 0
48 43 2 0 0 0
" % Mortality = % Mortality dosed shrimp - % Mortality control shrimp b Represent g of fish tissue 112
APPENDIX G
EFFECT OF FISH TISSUES EXTRACT FOR FISH FED 100%
DINOFLAGELLATE CELL DIET ON BRINE SHRIMP (Artemia sp.)
MORTALITY.
% Mortality'
Dose b
Time Flesh Head Viscera Viscera
(Hour) 0.007 0.0035 0.0025 0.005
4 6 7 0 10
8 9 7 2 13
16 10 6 13 17
24 12 5 12 20
48 7 0 7 23
' % Mortality = % Mortality dosed shrimp - % Mortality control shrimp b Dose represent g of fish tissue 113
APPENDIX H
EFFECT OF FISH TISSUE EXTRACT FOR FISH THAT DIED AS A
RESULT OF FEEDING ON 50/50 STANDARD CONTROL/
DINOFLAGELLATE CELL DIET ON BRINE SHRIMP (Artemia sp.)
MORTALITY.
% Mortality"
Doseb
Time Flesh Head Head Viscera Viscera
(Hour) 0.0025 0.0045 0.009 0.002 0.004
4 4 4 4 1 0
8 4 4 6 3 0
16 8 4 7 4 0
24 13 2 7 5 0
48 8 0 5 0 0
• % Mortality = % Mortality dosed shrimp - % Mortality control shrimp b Dose represent g of fish tissue 114
APPENDIX I
EFFECT OF FISH TISSUE EXTRACT FOR FISH THAT DIED AS A
RESULT OF FEEDING ON 100% DINOFLAGELLATE CELL DIET ON
BRINE SHRIMP fArtemia sp.) MORTALITY.
% Mortality *
Doseb
Time Flesh Flesh Head Head Viscera Viscera
(Hour) 0.015 0.031 0.0035 0.007 0.003 0.006
4 25 38 4 0 0 0
8 28 57 4 5 1 0
16 32 56 4 6 0 1
24 31 61 6 4 0 0
48 26 65 1 5 0 0
• % Mortality = % Mortality dosed shrimp - % Mortality control shrimp b Dose represent g of fish tissue 115
APPENDIX J
EFEECT OF EXTRACTS OF AQUARIA WATER TAKEN DURING THE
FEEDING STUDY (DAY 19) ON BRINE SHRIMP (Artemia sp.) MORTALITY.
% Mortality"
Time Dose b
(Hour) Control 1 2 3 4
4 4 5 3 1 9
8 6 7 3 6 9
16 4 5 1 4 7
24 4 5 1 5 7
48 0 2 0 4 4
* % Mortality = % Mortality dosed shrimp - % Mortality control shrimp b Dose represent 8.3 ml aquaria water 116
APPENDIX K
EFFECT OF EXTRACTS OF AQUARIA WATER TAKEN AT THE END
OF FEEDING STUDY ON BRINE SHRIMP fArtemia sp.) MORTALITY.
% Mortality"
Time Doseb
(Hour) Control 1 2 3 4
4 8 1 4 1 4
8 8 1 6 1 5
16 6 0 4 0 3
24 6 0 5 0 3
48 0 1 0 0 2
' % Mortality = % Mortality dosed shrimp - % Mortality control shrimp
b Dose represent 8.3 ml aquaria water 117
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