/AA PH0000009 RADIATSOM RESISTANCE OF PARALYTIC SHELLFISH POISON (PSP) TOKliS
A Thesis Pressntsd to Faculty of the College of Home Economics University of the Philippines DllSman
In Partial Fulfillment of the requirements for the Degree of Master of Science In Food Science
By EDITH M. SAN JUAK April, 2000 CERTIFICATE OF APPROVAL
The thesis attached hereto entitled "Radiation Resistance of Paralytic Shellfish Poison (PSP) Toxins" prepared by Edith M. San Juan in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN FOOD SCIENCE is hereby accepted.
P. ACEVEDO Adviser
/
ALU h MARIA PjATRIClXvWANZA, Ph.D. fANDA M. DELA RQl Ph.D. Critic PaneNUIember
Accepted as partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN FOOD SCIENCE.
FLOR CRISANfrA jffp$ALVEZ, Ph. D. Dean, College of Home Economics ABSTRACT
Radiation resistance of paralytic shellfish poison (PSP) toxins, obtained from Pyrodinium bahamense var. compression in shellstocks of green mussels, was determined by subjecting the semi-purified toxin extract as wel! as the shelistocks of green mussels to high doses of ionizing radiation of 5, 10,15 and 20 kGy. The concentration of the PSP toxin** was determined by the Standard Mouse Bioassay (SMB) method. The radiation resistance of the toxins was determined by plotting the PSP toxin concentration versus applied dose in a semilog paper. The DID value or decimal reduction dose was obtained from the straight tine which is the dose required to reduce the toxicity level by 90%. The effects of irradiation on the quality of green musseis In terms of its physico- chemical, microbiological and sensory attributes were also conducted. The effect of irradiation on the fatty acid components of green mussels was determined by gas chromatography.
Radiation resistance of the PSP toxins was determined to be lower in samples with initially high toxicity level as compared with samples with initially low toxicity level. The Dio values of samples with initially high PSP level were 28.5 kGy in shelistocks of green mussels and 17.5 kGy in the semi-purified toxin extract. When the PSP level was low initially, the Dio values were as high as 57.5 and 43.5 kGy In shelistocks of green mussels for the two trials, and 43.0 kGy in semi-purified toxin extract.
The microbial load of the irradiated musseis was remarkably reduced. No difference in color and odor characteristics were observed in the mussel samples subjected to varying doses of ionizing radiation. There was darkening in the color of mussel meat and its juice. The concentration of the fatty acid components in the fresh green mussels were considerably higher as compared with those present in the irradiated mussels, though some volatile fatty acids were detected as a result of irradiation. iii ACKNOWLEDGMENTS
I wish to express my sincere appreciation and deep gratitude to a lot of people who assisted me in undertaking and completing this study.
I wish to thank Prof. T.P. Acevedo, my thesis adviser, for her expert guidance, Invaluable suggestions, and unwavering support and encouragement throughout the duration of this study. I would also like to extend my gratitude to my critic, Dr. A.M. Dela Rosa and my panel member Dr. Ma. P.V. Azanza, for their constructive criticisms and invaiuable suggestions and Insights.
I would like to thank the following institutions for their support:
Department of Agriculture-Bureau of Agricultural Research Fisheries Sector Program / Manpower Development Program for Fisheries (DA-BAR FSP/MDPF) Scholarship Program for the study grant given;
National Food Authority-Food Development Center for their invaluable support;
Philippine Nuclear Research Institute for the accommodation In their facilities, especially to Ms. Cafoatfln and her staff, Luvy, Haydee, Yeng, Jun, Frank and Mang Jun; to Ms. Zeny de Guzman and Zena of the chemistry section; and to Ate Josle;
Iv Red Tide Laboratory at the Marine Science Institute, University of the Philippines, Dillman, for their unselfish accommodation, especially to Dr. Rhodora V. Azanza and her staff, Ltta, LIHbeth, Arlynne and Tess;
Bataan Red Tide Testing Center of the Department of Science and Technology, especially to Ms. Sally Roman and her staff, for the accommodation In their testing laboratory;
Ride Tide Task Force of the Bureau of Fisheries and Aquatic Resources, especially to Ms. Zeny Abuso, Mr. Lite Gonzatos, Miriam and Jay, and Josle for the training on mouse bloassay method;
College of Home Economics Food Pilot Plant for the us® of their facilities, to Belie, Abigail and their staff;
My friends at the College of Home Economics, Elsa, Mary Ann, Hubert, Elvle, Ate Fe, Emy, Ariene, Ate Linda Myrna, Lotls, Blanca, Beth, Mang Neme, and Mang Lino, for their many acts of kindness and heart- warming thoughts;
My offlcemates at FDC, to Dr. Alicia O. Lustre for her permission to go on study leave; to Glenda, Joy, Pearl, Olive and Luvy for their technical assistance In the fatty add analysis; to Meyen, Fe, Amy, Vivian, Myrna, Ariene, Badette, Beth and Malou for their eagerness In the sensory evaluation of the mussel samples; to Jeje and her staff at the R&D Section, and to everyone for their encouragement and moral support; My parents, tola, sisters, brother and in-iaws for their understanding, support and encouragement;
My loving husband Joey and children Cocoy, Karen and Tin-Tin for the Inspiration and to whom this work Is dedicated;
And God Almighty who made everything possible.
vi TABLE OF CONTENTS
Page
Title Page „ i Certificate of Approval ii Abstract iii Acknowledgements Iv Table of Contents vii List of Tables x List of Figures xiii List of Appendices xvi
INTRODUCTION 1
REVIEW OF LITERATURE
Mussels 4 Description of Mussels 4 Mussel Distribution 4 Habitat 6 Feeding Habit 6 Reproduction 6 Culture of Mussels 7 Methods of Mussel Farming 7 Mussel Production 8 Red Tide and Paralytic Shellfish Poisoning . 8 Red Tide 8 Causative Organism 10 Mechanism of Red Tide 11 Outbreaks of Paralytic Shellfish Poisoning (PSP) 17 Paralytic Shellfish Poison (PSP) 20 Chemical Structure of Saxltoxin 22 Mode of Action of Saxitoxin 25 Toxicology/Pat hop hysiology 27
vii Clinical Picture 27 Economic Implications 29 Standard Mouse Bioassay for Paralytic Shellfish Toxins. . . 32 Selection of Animals 33 Storage of PSP Toxins 34 Food Irradiation 34 The Process of Food Irradiation 34 Ionizing Radiation 35 Mechanism of Food irradiation 36 Factors Affecting Radiolytic Products 38 Effects of Irradiation on Food 38 Effects of Irradiation on Microorganisms 40 Activity in Biological Systems 41 Radiation Resistance (D10 Value) of Organisms 43 Factors Affecting Dio Values 48 Bacterial Toxins 52 Irradiation as a Food Process 53 Dose and Dosimetry 54 Food Irradiation Costs 54
METHODOLOGY
Raw Materials 56 Preparation of Samples for Irradiation 57 Preparation of Samples for Toxin Extraction 61 Determination of PSP Toxin Concentration by Standard Mouse Bioassay Method 62 Irradiation of Samples 62 Shellstocks of Green Mussels (Sample A) 62 Semi-Purified Toxin Extract 63 Phase 1. Radiation Resistance of Paralytic Shellfish Poison (PSP) Toxins 64 Radiation Resistance of PSP Toxins at High Toxicity Level . . .64 Radiation Resistance of PSP Toxins at Low Toxicity Level ... 64 Determination of Decimal Reduction Dose (Dio Value) 64 Phase 2. Effects of ionizing Radiation on the Quality Characteristics of Shellstocks of Green Mussels 65 Effects on Shellstocks of Green Mussels Subjected to Different Doses of ionizing Radiation 65 Effects on Shellstocks of Green Mussels Irradiated at the Dio Value 67
VIII Phase 3. Changes in the Fatty Acid Profile of irradiated Shellsfocks of Green Mussels 69 Effects on Sheilstocks Exposed to Different Doses of Different Doses of Ionizing Radiation 69 Effects on Sheilstocks of Green Mussels Irradiated at the D10 Value 69 Identification of Fatty Acids 69
RESULTS AND DISCUSSION
Phase 1. Radiation Resistance of Paralytic Shellfish Poison (PSP) Toxins 71 Radiation Resistance of PSP Toxins at High Toxlcity Level. . . 71 Radiation Resistance of PSP Toxins at Low Toxicity Level ... 76 Phase 2. Effects of Ionizing Radiation on the Quality Characteristics of Sheilstocks of Green Mussels 86 Effects on Sheilstocks of Green Mussels Subjected to Different Doses of Ionizing Radiation 85 Effects on Sheilstocks of Green Mussels Irradiated at the D10 Value 91 Phase 3. Changes in the Fatty Acid Profile of Irradiated Shellstocks of Green Mussels 93 Effects on Shellstocks of Green Mussels Subjected to Different Doses of Ionizing Radiation 95 Effects on Sheilstocks of Green Mussels Irradiated at the Value 106
SUMMARY AND RECOMMENDATIONS 109
REFERENCES 112
APPENDICES 121
Ix LIST OF TABLES
Table Page 1 Mussel production and value of production (BFAR.1998; Bureau of Agricultural Statistics, 1998) 9
2 Composition of paralytic shellfish toxins in organisms of tropical waters (Yasumoto et a/., 1984) 24
3 Symptoms of paralytic shellfish poison (PSP) (Pastor 4 Approximate lethal doses for various organisms (Urbain, 1986) 42 5 D10 values of bacteria (Urbain, 1986) 47 6 Effect of oxygen on Dto values of four strains of Satnonetta (Urbain, 1986) 50 7 Change of D10 value of Streptococcus faecium A21 In phosphate buffer with temperature (Urbain, 1986) 51 8 Concentration of PSP toxins from shellstocks of green mussels (or Sample A) with high initial toxin level 72 9 Concentration of PSP toxins from semi-purtfied toxin extract (or Sample B) obtained in mussels with high initial toxin level 74 10 Concentration of PSP toxins from shellstocks of green mussels (or Sample A) with low initial toxin level (Trial 1). .77 11 Concentration of PSP toxins from semi-purified toxin extract (or Sample B) obtained in mussels with low initial toxin level (TriaM) 79 Table Page 12 Concentration of PSP toxins from shelistocks of green mussels (or Sample A) with low initial toxin level (Trial 2) . . 81 13 Concentration of PSP toxins from semi-purified toxin extract (or Sample B) obtained in mussels with low initial toxin level (Trial 2) 83 14 Chemical characteristics of frozen irradiated and unirradiated shellstocks of green mussels 86 15 Color and odor characteristics of frozen irradiated and unirradiated shelistocks of green mussels 88 16 Microbiological characteristics of frozen irradiated and unirradiated shelistocks of green mussels 90 17 Effects of irradiation at the D10 value of 28.5 kGy on the quality characteristics of frozen shelistocks of green mussels with high initial toxin level 91 18 Tentatively identified fatty acids and their concentrations in frozen irradiated shellstocks of green mussels with high Initial toxin level 97 19 Tentatively identified fatty acids and their concentrations in frozen irradiated shellstocks of green mussels with low Initial toxin level (Trial 1) 101 20 Tentatively identified fatty acids and their concentrations in frozen irradiated shellstocks of green mussels with low initial toxin level (Trial 2) 104 21 Tentatively identified fatty acids and their concentrations in frozen shellstocks of green mussels irradiated at the D-m value of 28.5 kGy in samples with high initial toxin level 106 XI Table Page 22 Somer's Table on death time and mouse unit relations for paralytic shellfish poison 126 23 Scoring system for the intensity of odor descriptors . ... 136 xil LIST OF FIGURES Figure Page 1 Map of the Philippines showing the farming areas of mussels and oysters (PCARD, 1977) 5 2 Pyrodinium cells types (Taylor and Fukuyo, 1989) 12 3 Hydrographic parameters and Pyrodinium bahamense population in Manila Bay from January 1992 to December 1994 (Bajaras and Re fox, 1996) 16 4 Horizontal distribution of Pyrodinium cells in Manila Bay during the 1992 blooms (Bajaras and Relox, 1996) .... 19 5 Horizontal distribution of Pyrodinium cells in Manila Bay during the 1993 blooms (Bajaras and Relox, 1996) .... 19 6 Horizontal distribution of Pyrodinium ceils in Manila Bay during the 1994 blooms (Bajaras and Relox, 1996) .... 19 7 Toxins associated with PSP. Several (de-NEO, de-GTX I/IV) have not been reported in the literature, but are postulated to occur based on presence of the other (Taylor, 1988) 23 8 Schematic diagram of survival curves 45 9 Relationship of the surviving spores of Ctostridium botufinum 33A with pH. Spores suspended in buffer solution and irradiated at -50°C with 9kGy (Urbain, 1986) 49 10 Change with irradiation temperature of D10 values of Spores of Clostridium botulinum 33A in ground beef (Urbain, 1986) 49 xiii Page 11 Arrangem ent of packed shellstocks of green m ussels in a styroporebox (11a, top view; 11b, front view). The packs are arranged from top to bottom as replicates A, B and C on the left side of the box, and as replicates D, E and F on the right side of the box .... 58 12 Phase 1. Flow diagram for the determination of D10 values of PSP toxins from shellstocks of green mussels (Sample A) and from sem(-purified toxin extract (Sample B) 59 13 Arrangement of samples of semi-purified toxin extract contained in plastic bottles inside a styropore box (13a, top view; 13b, front view). The samples are arranged as replicates A, B and C starting from the left side of the box 60 14 Phase 2. Flow diagram for determination of effects of different Ionizing radiation on the quality characteristics of shellstocks of green mussels 66 15 Arrangement of a styropore box containing samples of frozen shellstocks of green mussels on the turntable which will be irradiated with the calculated D10 value ... 68 16 Phase 3. Flow diagram for determination of fatty acids In irradiated shellstocks of green mussels 70 17 D10 value of PSP toxins obtained from shedstocks of green mussels (Sample A) with high initial toxin level ... 73 18 D-io value of PSP toxins obtained from semi-purified toxin extract (Sample B) in mussels with high initial toxin level 75 19 D10 value of PSP toxins obtained from shellstocks of green mussels (Sample A) with low Initial toxin level (Trial 1) 78 XIV Figure Page 20 D10 value of PSP toxins obtained from semi-purified toxin extract (Sample B) in mussels with low initial toxin level (Trial 1) 80 21 D10 value of PSP toxins obtained from shellstocks of green mussels (Sample A) with low initial toxin level (Trial 2) 83 22 Diq value of PSP toxins obtained from semi-purified toxin extract (Sample B) in mussels with iow initial toxin level (Trial 2) 85 23 Chromatogram of fatty acid standards 94 24 Chromatogram of fatly acid components in frozen shellstocks of green mussels subjected to different doses of ionizing radiation with initially high toxin level. . . 96 25 Chromatogram of fatty acid components in frozen shellstocks of green mussels subjected to different doses of ionizing radiation with initially low toxin level (Trial 1) 100 26 Chromatogram of fatty acid components In frozen shellstocks of green mussels subjected to different doses of ionizing radiation with initially low toxin level (Trial 2) 103 27 Chromatogram of fatty acid components in shellstocks of green mussels: (a) unirradiated or control; (b) irradiated at the D10 value of 28.5 KGy 107 28 Schematic diagram for determination of Aerobic Plate Count in shellstocks of green mussels 131 29 Schematic diagram for determination of Coltforms In she list ocks of green mussels 134 xv INTRODUCTION The recurrent outbreaks of toxic red tide Pyrodinium blooms in the Philippines have not only caused a number of mortalities but immense economic difficulties to the coastal fishing industries of affected areas particularly those involved in the cultivation of green mussels. The first recorded outbreak of paralytic shellfish poisoning (PSP) in the Philippines occurred in June to September 1983 in the Samar Sea, where the causative organism was Pyrodinium bahamense var. compression, and about 20 deaths and nearly 700 illnesses resulted mostly from eating bivalves and a few from pianktivorous fishes. Since then the red tide outbreaks were reported yearty in the Philippines causing deaths and difficulties to the fishermen In terms of livelihood sources. PSP results from concentration of the poisons followed by human consumption. There is no antidote and the poisoning can be fatal (Yentsch and Incza, 1980 as cited by Estudlllo and Gonzales, 1984). The Field Epidemiology and Training Program of the Department of Health (DOH-FETP, 1997) reported 107 cases of illnesses and 8 cases of deaths in 1995, 121 cases of illnesses and 7 deaths in 1996, and 12 cases of illnesses and 2 deaths in 1997 due to red tide incidents. During the last quarter of 1999, a Pyrodinium bloom occurred in Cancabato Bay in Tacloban City, Leyte and the ban on the harvest of mussel was lifted only in February 2000 (BFAR, 2000). Post-harvest treatments applicable to eliminate or reduce toxicity of contaminated mussels were studied. Some of these were the use of purification systems and improved processing methods. Since the Pyrodinium is made up of strongly developed wall or theca of cellulose plates which renders it heat resistant, improved processing methods such as thermal processing was not able to reduce the toxicity level to the acceptable limit of 40 jig /100 g mussel meat (Acevedo et a!., 1993). A study conducted by Genesera (1992) on depuration of PSP affected green mussels showed that the method was able to reduce the toxin concentration to the acceptable level only after 43.5 hours of depuration. Another processing method which may find application in detoxifying the red tide affected mussels is irradiation. It is presumed that the biological effects of radiation are due to chemical changes within the organism. Radiation damage is mainly associated with the impairment of metabolic reactions. The wholesomeness of irradiated foods refers to the nutritional value and lack of mutagenicity, teratogenicity and toxicity of these foods (Thayer, 1990 as cited by Thayer, 1994). In 1997, the Joint FAO / IAEA /WHO Study Group on High Dose Irradiation, agreed that the wholesomeness of foods irradiated to high doses should have "absence of adverse effects" which will support the safety of the procedure, such as preservation of palatability, the protection of nutritional components in foodstuffs and control of radiolysis which is achievable when the high dose irradiation of foodstuffs Is carried out under specified conditions. It is therefore imperative that other postharvest treatments be evaluated in terms of detoxification potential for PSP in contaminated mussels. The application of irradiation as a processing tool to detoxify PSP in red tide contaminated mussels need to be studied. The study principally aims to determine the effects of irradiation, using Cobalt-60, on the toxicity of paralytic shellfish poison (PSP) on shelistocks of green mussels and in semi-purified PSP toxins. Specificialry, the study has the following objectives: 1. to determine and compare the radiation resistance of PSP toxins In terms of D10 value obtained from shellstocks of green mussels and from semi- purified toxin extract; 2. to determine the effect of the Initial toxicity level on the radiation resistance of PSP toxins in shellstocks of green mussels and in the semi-purified toxin extract; 3. to determine the effects of varying doses of ionizing radiation on the physico-chemical, microbiological and sensory characteristics of the green mussels; and 4. to determine the changes in the fatty acid components of irradiated shellstocks of green mussels using different doses of ionizing radiation. Limitations of the study: • Samples of green mussels were obtained only in the vicinity of Barrio Luz and Put ing Buhangln in Limay, Bataan. • Only one (1) trial was conducted on the shellstocks of green mussels with high PSP toxin level of about 847.01 ug/100 g meat. • The PSP concentration of the irradiated mussels was determined only by the Standard Mouse Bioassay (SMB) method. • The fatty acid components were determined instead of the volatile flavors due to unavailability of standards and of extracting reagent as required in a method in the analysis of volatile flavor components In clams. REVIEW OF LITERATURE Mussels Description of musseis. The green bay mussel, Perna viridts, is locally termed as "tahong". The green mussel is a sessile form of bivalve moilusk. The soft unsegmented body Is protected by a hard shell that is never shed but accumulates layer upon layer as the animal grows. The shell is In two separate halves which is kept together by a powerful muscle. Contraction of the large muscle situated at the center of the body causes the opening and closing of the mussel. The color of the outer margin of the subtriangular shell is bright green which becomes darker towards the center (BFAR, 1986). The mussei is roughly oval in shape and rather pointed towards the hinge. The color of the meat of a male mussel is light to pale orange while the female mussel acquires a dark orange color. Mussels breathe by means of two pairs of gills. These gills sometimes served as an excretory organ of the bivalve. Mussel movement, though slow and for short distances, is by means of a foot and byssus thread. When byssal threads are damaged or cut, mussel secretes new threads. Mussels feed by the dilatory action of the labial palps in vigorous motion. Mussel Distribution. The Philippine Council for Agricultural Research and Development (PCARD.1977) gives the different natural grounds, farming sites and prospective sites of mussels in the Philippines (Figure 1). The green bay mussel "tahong" grows extensively and intensively in certain areas of the Philippines. They are most often found in Manila Bay, along the shore areas of ®-m!/f2: Figure 1. A map of the Philippines showing the fanning areas of mussels and oysters (Philippine Council for Agricultural Research and Development, 1977). Navotas, Malabon, Paranaque, Manila, Pasay and Las Plflas. They are also found in Sorsogon, Tacloban, Leyte and the towns lining Bacoor Bay in Cavite. Mussel also grows in the bays and inlets in the Northern Coast of Panay Is., in Western Negros Is., in Maqueda Bay and Jiabong, Samar (BFAR, 1986). Habitat. Mussels grow In combination with oysters which thrive in brackish to marine water coves, bays and estuaries. They grow in areas where there is an adequate tidal current for transport of food and oxygen and free from disturbances like big waves and strong winds. They can tolerate a salinity range of 27 to 35 ppt and a temperature of 26°C. Water should be clear, quiet and greenish with average light penetration and should contain enough food for the mussels. Muddy to sandy bottoms have been found to give higher yields. Depths of 10 to 20 feet are favourable for constructing plots for spat collection (Bureau of Fisheries and Aquatic Resources, 1987). Feeding habit. Mussels feed by Uttering phytoplankton such as dinoflagellates, diatoms and zooptankton which are naturally present in estuaries and nearshore waters. When feeding the mussels open their valves and draws in water through the siphon. The incoming water passes over the gills where food particle is caught and strained by the mucus sheet, then directed by a hairlike cilia towards the mouth or directed along the rejection paths to the exterior where it is eliminated as their pseudofaeces. Mussels are susceptible to contamination by microorganisms due to their filter feeding habit, hence accumulate and concentrate within their digestive system microbiological flora of the surrounding environment (Bureau of Fisheries and Aquatic Resources, 1987). Reproduction. Mussels spawn throughout the year with peak months from April to May and from October to November. During spawning, the 6 spawners release millions of minute eggs and sperms in the water where fertilization takes place. The fertilized eggs develop into a free swimming larvae for 15 to 29 days before they mature. The free swimming larvae known as spats or seeds attach themselves to any hard object when ready lo settle. The spats develop shells and remain stationary In the cultch or collector for the rest of their lives (Bureau of Fisheries and Aquatic Resources, 1986). Culture of mussels. In the culture of mussels, the following conditions must be met (Bureau of Fisheries and Aquatic Resources, 1987): 1. Location. Ideal sites are brackish to saltwater coves, bays and estuaries. They should be free from water disturbances like big waves and strong winds. However, an adequate tidal current should aid the transport of food and supply of oxygen to the animals. Sufficient breeding stock and spatfall in the area are needed to produce enough young mussels for the farm. 2. Water condition. Water should be clear, quiet and greenish with average light penetration and should contain enough food for the mussels. Temperature should fluctuate from 26°C to 30°C and salinity should range from 27 to 35 ppt. 3. Bottom character. Muddy to sandy bottoms have been found to give higher yields. Depths of 10 to 12 feet are favourable for constructing plots for spat collection. Methods of mussel farming. In the Philippines, the most common method of mussel farming are as follows (Bureau of Fisheries and Aquatic Resources, 1987; Philippine Council for Agricultural Research and Development, 1977): 1. Stake method (lulos"). This is the traditional and most common method of mussel culture. This method makes use of bamboo poles as collectors. Holes are punched at the upper end of each segment to fill the hollow portion of the bamboo with water to facilitate staking and prevent it from floating. The basal end of the bamboo pole is sharpened with a diagonal cut and staked 0.5 to 1 meter apart in a soft muddy bottom. 2. Rope-web method. The method was found effective and durable because of its reusability. Synthetic ropes with a length of 12 mm diameter are made into webs tied vertically to bamboo poles. A web consists of two parallel ropes with a length of 5 meters each and positioned 2 meters apart. They are connected to each other by a 40 meter long rope tied or fastened in a zigzag fashion at an interval of 40 cms between knots along each of the parallel ropes. Mussel production. Table 1 presents a summary of total mussel production and value of production from 1986 to 1998 as obtained from the Philippine Fisheries Profile and Fishery Statistics (BFAR, 1999). Red Tide and Paralytic Shellfish Poisoning Red tide. Red tide refers to the reddish-brown discoloration of seawater caused by the proliferation of microscopic organisms called dinoflagellates (Hallegraeff and Maclean, 1989). Certain blooms of algae are termed red tides when the tiny pigmented plants grow in such an abundance that they change the color of the seawater to red, brown or even green. The name is misleading, however, because many toxic events are called red tides even when the waters show no discoloration. Likewise, an accumulation of nontoxic, harmless algae can change the color of ocean water. The picture is even more Table 1. Mussel production and value of production (Bureau of Fisheries and Aquatic Resources, 1998; Bureau of Agricultural Statistics, 1998). Year Production Value (metric tons) ('000 P) 1998 15,533 (a) 1997 11,658 44,113 1996 21,027 51,682 1995 14,688 46, 360 1994 11,355 119,357 1993 25,070 244,513 1992 17,217 278, 247 1991 17,345 207,077 1990 17,515 194,113 1989 16,405 152,279 (a) Value in pesos, not yet available from the Bureau of Agricultural Statistics. The volume of production was taken from the BFAR-Phllippine Fisheries Profile, 1999. complicated: some phytoplankton neither discolor the water nor produce toxins but kill marine animals in other ways. Many diverse phenomena thus fall under the "red tide" rubic (Anderson, 1984). A bloom develops when these single-celled algae photosynthesize and multiply, converting dissolved nutrients and sunlight into plant biomass. The dominant mode of reproduction Is simple asexual fission - one cell grows larger then divides into two cells, the two split into four, and so on. Barring a shortage of nutrients or light, or heavy grazing by tiny zooplankton that consume the algae, the population size can increase rapidly (Anderson, 1984). In some cases, a milliliter of seawater can contain tens or hundreds of thousands of algal cells. Spread over large areas, the phenomenon can be both spectacular and catastrophic. Some species switch to sexual reproduction when nutrients are scarce. They form thick-walied, dormant cells, called cysts, that settle on the seafloor and can survive there for years. When favourable growth conditions return, cysts germinate and reinoculate the water with swimming cells that can then bloom. Although not all red tide species form cysts, many do, and this transformation explains Important aspects of their ecology and biogeography. The timing and location of the bloom can depend on when the cysts germinate and were they are deposited, respectively. Cyst production facilitates species dispersal as well; blooms carried into new waters by currents or other means can deposit "seed" populations to colonize previously unaffected areas. The cyst stage has provided a very effective strategy for the survival and dispersal of many other red tide species as well (Anderson, 1984). Causative organism. The species causing red tide in the Philippines is named Pyrodinium bahamense var. compressum. It thrives in coastal waters and lagoons under conditions of high salinity and does not survive in freshwater fishponds. Surface sediments collected from Maqueda Bay in the Samar Sea, Philippines contained many cysts which are identical to Pyrodinium bahamense var. compressum based on the characteristic epicystal archeophyle, and 10 intratabular, hollow and slender processes. Many thecate cells of this species also occurred in the same sample. This is the first reliable record of the cysts of Pyrodinium bahamense var. compression in Southeast Asia. Figure 2 shows two Pyrodinium cell types. In surface sediments, there are two types of cysts, namely living cysts which are filled with protoplasm and may germinate after a considerable resting period, and empty cysts which are already hatched and bear an encystment aperture, the archeopyle. At present the two archeopyle type is one of the most important characters for the identification of dinoflagellate cysts. However, to produce a strain of a certain dinoflagellate species from a cyst, the living cyst is needed before germination, in that case the cyst must be observed and identified for experiments without any information concerning the archeopyle type (Matsuoka, 1989). Pyrodinium was present in all plankton samples collected in Maqueda, Viilareal and Carigara Bays, and Samar Sea during the recurrence of red tide in June 1987. The denser population of the organism was observed in some parts of Carigara Bay and waters along the coasts of Western Samar. In Maqueda and Viilareal Bays, the densities of Pyrodinium were less than 10,000 cells/ L, except at the northern part where the values ranged from 14,857 to 43,502 cells/L. A maximum count of 8.57 x 106 cells/L was recorded between Biliran and Canahauan islands, while counts of more than 2 x 106 cells/L were observed In Carigara Bay. Red tide sightings coincided with the areas of high Pyrodinium counts (Gonzales ef at, 1989). Mechanism of red tide. Red tides often occur when heating or freshwater runoff creates a stratified surface layer above colder, nutrient-rich 11 — npical spine apical horn ventral pore girdle lists • lef! sulcal list anfapica! spines Typical Atlantic Typical single Pacific specimen specimen ivar. l>a/iamensti ) (va r. compressum ) Figure 2. Pyrodinium celltypes (Taylor and Fukuyo, 1989) 12 waters. Fast-growing algae quickly strip away nutrients in the upper layer, leaving nitrogen and phosphorus only below the interface of the layers, called the pycnocline. Nonmotile phytoplankton cannot easily get to this layer, whereas motile algae, including dinoflageHates, can thrive. Many swim at speeds in excess of 10 meters per day, and some undergo daily vertical migration: they reside in surface waters by day to harvest sunlight like sunbathers, then swim down to the pycnocline to take up nutrients at night. As a result, blooms can suddenly appear in surface waters that are devoid of nutrients and would seem incapable of supporting such prolific growth (Anderson, 1984). The presence or absence of the cyst probably plays a major role as the biological basis for the start or initiation of a red tide. Favourable combinations of some environmental factors also contribute. They can be topographical, like a shallow bay or lagoon; meteorological, like periods after strong rain with temperature increases; or oceanographical, like stagnant water after upweiling (Hermes, 1983). For the support or maintenance phase of the bloom, some physical and biological factors can be cited as possibly involved or in some better Investigated cases responsible. These are the concentration by wind and currents into dense patches or streaks; presence of convergences, density discontinuity layers between water masses or frontal zones can lead to aggregations of the algae (Hermes, 1983). The Incidence of red tide seems to be associated with one monsoon or the other in each country. Wind-driven upwelling is proposed to be a dominant factor in bloom initiation. The sequential appearance of toxic Pyrodinium blooms in Papua New Guinea, Western Borneo and the Philippines suggests a northerly spread of blooms if not of the organism itself. Coincidence of major blooms with 13 ENSO (El Nino-Southern Oscillation) event years suggests an association of the two phenomena and perhaps a northerly movement of ENSO anomalies since the 1970s (Maclean, 1989). Based on the incidence of red tide and ENSO events between 1972 and 1987, Maclean (1989) interpreted that the ENSO events probably affected different parts of the western Pacific to varying degrees on each occasion, creating a short- or long-term environment suitable for toxic blooms in one locality or another. The prevailing northerly winds cause cold-nutrient-rich water to rise up from the deeper regions of the ocean to the surface, a process known as upwelling. Swept along with the upwelled water are dinoflagellate cysts, the resting stage of the organism, which lie dormant in the sediments on the sea floor. The high nutrient concentrations in the upwelled water, together with ideal conditions of temperature, salinity and light, trigger the germination of cysts, so that the dlnoflageHates begin to grow and divide. The rapid increase in dinoflagellate numbers, sometimes to millions of ceils per liter of water, is what is known as a "bioom" of phytoplankton. Concentration of the bloom by wind and currents, as well as the dinoflageHates' ability to swim to the surface, together lead to the formation of a red tide (van der Vyver and Pitcher, 1997). If conditions in the surface waters become unfavourable for the dinoflagellates, for example if the nutrients are depleted or the bloom is dispersed by wind and currents, the dinoflage Hates will again form cysts and sink to the sea floor. During red tide blooms the observed water temperature ranged from 29.6 to 32.5°C. The bottom temperature ranged from 26.4 to 29.8°C. The observed surface salinities ranged from 31.15 to 33.88 ppt whereas the bottom salinities ranged from 33.78 to 35.28 ppt. Water temperature and salinity values 14 measured are within the range of the observed values for Pyrodinium blooms elsewhere, i.e., 24.4 to 31.9 °C and 24.7 to 36.8 ppt in Papua New Guinea (Maclean, 1977); and 24.5 Jo 29.4 °C and 24.3 to 32.08 ppt in Brunei. Earlier studies of red tide showed that Pyrodinium can tolerate salinities as low as 14 ppt for a few days . Laboratory experiments conducted In Papua New Guinea during the 1973 or 1974 blooms showed that the Pyrodinium band formed within the salinity range of 28.6 and 31.5 ppt, can tolerate salinities as high as 40.7 ppt (Maclean, 1977). Pyrodinium is atypical, having an optimum salinity of around 35.7 ppt. This suggests that Pyrodinium favors high salinity, as noted above. The lower salinities and reverse temperature gradient associated with river runoff are merely indicators of the source of nutrients and not catalysts of the widespread blooms (Maclean, 1977). The observed surface dissolved oxygen ranged from 3.19 to 5.40 mL/L, whereas the bottom dissolved oxygen values ranged from 0.45 to 3.86 mL/L. A review of culture methods for Pyrodinium bahamense conducted by Blackburn and Oshima (1989) showed that this tropical dinoflagellate requires temperatures of 24-30°C, dilute nutrient media and soil extract, while a pH optimum of 8 has also been observed. In 1996, Bajaras and Relox reported some hydrologicai and climatological parameters that accompanied the annual blooms of Pyrodinium bahamense var. compressum. Figure 3 shows the hydrographic parameters and 15 Figure 3. Hyrographic parameters and Pyrodinium bahamense population in Manila Bay from January 1992 to December 1994 (Bajaras and Relox, 1996) 16 phytoplankton population from January 1992 to December 1994, wherein Pyrodinium cell density, shellfish toxicity, nutrient (P-PO4), water temperature, salinity, rainfall and wind force were plotted as a function of time. The results revealed a trend/pattern in the blooms of Pyrodinium in the Bay. Prior to the bloom formation, an Increase in surface water temperature of >3°C was noted during the 1992 blooms, whereas in 1993 and 1994 >1°C was recorded. Meanwhile, salinity measured during the 1992 blooms was >34 ppt, and in 1993 and 1994, It was >31 ppt. In 1992, the peak of the bloom was observed in June while in 1993 and 1994, the peak was in June and Jury, respectively. Discoloration of the surface water was observed in June, 1992 (21,027 cells/L) and in June, 1993 (26, 711 cells/L) while patches or streaks was observed in Jury 1994 (1,991 cells/L). The bloom in 1994 was smaller Jn magnitude compared to the 1992 and 1993 blooms. Blooms of Pyrodinium in the Bay persisted for 2 months. Pyrodinium cells in the Bay were first detected at the onset of the rainy season, and as the rain persists, the development of the bloom progresses. Observation over a three-year period showed that the major peaks of the bloom occurred in months with maximum amount of rainfall (Figure 3). Surface patches of red tide dinoflagellates become visible in bright sunlight when cell concentrations approach 106/L. If these dinofiagellates are bioluminiscent their presence at night will produce visible luminous wakes behind ships at concentrations of 103/L and the bioluminiscent color can be identified as blue at concentrations of 104/L (Seliger, 1989). Outbreaks of paralytic shellfish poisoning (PSP). The first occurrence of PSP in the Philippines was in June to September of 1983 in the Samar Sea (Hermes, 1983) which was caused by Pyrodinium bahamense var. 17 compressum (Hermes and Vllloso, 1983). Bajaras and Relox (1996) conducted a monitoring program on red tide in Manila Bay from January 1992 to December 1994. Results of the monitoring revealed a trend/pattern in the bloom formation. Bloom formation occurred in the same geographical region of the Bay, but varies in time of occurrence. Bloom initiation occurred at the western section of the Bay. As the bloom progressed, the distribution of Pyrodinium cells followed the direction of the water. Pyrodinium cells were adverted northward, eastward and southward. Concurrent with the blooms, high level of toxicity in green mussels was also noted, resulting in massive paralytic shellfish poisoning (PSP) incidents. Figure 4 shows the distribution of Pyrodinium babamense var. compressum in the Bay during the 1992 bloom (Bajaras and Relox, 1996). Low concentrations (30-60 cells/L) of Pyrodinium were first detected In the northwestern section of the Bay, and were confined only in this section In mid- May. By June the bloom developed and spread clockwise to the entire Bay. High concentrations of the organism were observed particularly in western, northern, and southern sections. The bloom persisted for 2 months. By July the bloom in the eastern, western, and southern sections dwindled and only in the northwestern section did concentrations of the organisms remain high. The presence of Pyrodinium cells in the entire Bay waned by August. Minimal cell density was observed in northwestern and eastern part of the Bay, and a month after, the entire Bay was already free of the Pyrodinium. The distribution of Pyrodinium cells during the 1993 blooms Is shown in Figure 5 (Bajaras and Relox, 1996). The first appearance of the bloom in the Bay was in late-March, wherein a cell density of 5 cells/L was detected at the 18 Figure 4. Horizontal distribution of Pyrodinium cells in Manila Bay during the 1992 blooms (• - sampling stations, Bajaras and Reiox, 1996). i7 A *4 r\\ •M -•y " •/fu .•—^i 4^> JUL Figure 5. Horizontal distribution of Pyrodinium cells in Manila Bay during the 1993 blooms (• - sampling stations, Bajaras and Relox, 1996). Figure 6. Horizontal distribution of Pyrodinium cells in Manila Bay during the 1993 blooms (• - sampling stations, Bajaras and Relox, 1996). western section. Bloom development also occurred in this region by late-May, and the organisms dissipated to the eastern and southern sections. The cell density in the western sections (90-200 cells/L) was higher than in the eastern part (40 cells/L). By June the organism dispersed to the northern section, while a bloom developed in the eastern and southern sections. In the western section, cell density of Pyrodinium declined. Low level cell density (40 cell/L) were observed in the entire Bay by July, and in the succeeding month the organism disappeared totally. Bloom formation during 1994 was similar to the 1993 blooms (Figure 6, Bajaras and Relax, 1996). Pyrodinium was first detected in the western section in late-March, and bloom development also occurred in the same area in late-May. The bloom dispersed northward and southward by June . The peak of the bloom was in July, with the entire Bay invaded by the organism. High concentration of Pyrodinium ceils were observed along and/or near the coasts of the northwestern, eastern, and southern regions of the Bay, white in the central part, ceil density was low. The bloom prevailed for a month, and by August the entire Bay was free of Pyrodinium. Paralytic shellfish poison (PSP). Humans can become ill from eating seafood products contaminated with red tide organisms. The most notorious causing Illnesses are bivalve shellfish such as the green mussel, oysters, scallops, cockles and limpets but gills and guts of small fish such as sardines and anchovy can also become contaminated with red tide. Fish, shrimps, crabs, prawns and other products grown in ponds or freshwater are safe because red tide organisms do not survive under conditions of low salinity. The predominant toxins in dinoflageHates are nerve poisons which fall 20 into two categories. The first type blocks the normal entry of sodium ions into nerves, thus preventing normal nerve signals. These poisons commonly concentrate in the tissues of fitter-feeding shellfish (mussels, clams, etc.) which have fed on the red tide. Shellfish are relatrvefy insensitive to the toxins. Concentration of the poisons, followed by human consumption of the contaminated product can lead to Paralytic Shellfish Poisoning (PSP). There is no antidote, and the poisoning can be fatal (Dale et a!., 1987). The accumulation of dinofiagellate derived toxins in moliuscan shellfish represents a serious health threat if proper control measures are not implemented. The periodic development of toxicity in normally nontoxic seafoods presents serious problems to harvesters, seafood processors, consumers and regulatory agencies, in a majority of cases, the toxicity stems from the presence of toxins derived from the marine food web (Sullivan, 1988). It is now known that the primary producers of the toxins involved in PSP are a number of species of dinoflage Hates (unicellular photosynthetic algae), which as a group represent an important link in the marine food chain. Filter feeding shellfish are particularly prone to accumulation of dinofiagellate derived toxins since these algae serve as a primary food source. Paralytic she Irish poisoning is caused specifically by toxins from certain dinoflagellates which are normally present In very low concentration in the sea. Occasionally, in response to a favourable change in their environment, they undergo a rapid population increase, and their numbers may attain several millions per liter, forming visible patches on the surface, or "red tides". Humans are poisoned by eating bivalve shellfish which have been exposed to a red tide, and thus have ingested large numbers of dinoflagellates and stored their toxin. 21 The symptoms are quite characteristic (Maclean, 1973). Of the economically important seafood toxin problems, more is known about PSP than any of the others. PSP involves the accumulation of a number of alkaloid toxins (Figure 7) in shellfish. The primary producers of ihe PSP are several species of dinoflagellates such as Protogonyaufax spp., Gymnodinium catenation and Pyrodinium bahamense (Taylor, 1985). Chemical structure of saxltoxln. The toxins produced by these dinoflagellates are termed collectively as saxitoxins (Hall and Reichardt, 1984). The structures of the parent molecule, saxltoxin and its derivatives are shown in Ffgure 7 (Taylor, 1988; Yasumofo et aL, 1984) and the relative abundance of the toxic components in shellfish and dinoflagellates are shown in Table 2 (Yasumoto et a/., 1984; Oshima et a/., 1984). Saxitoxin is a dibasic HCI salt which is highly soluble in water. It Is represented by the formula C10H17O4N7 . 2HCI and has a molecular weight of 372 (Villao, 1988). The dinoflagellate Pyrodinium bahamense var. compressum and bivalves collected at Palau contained saxitoxin, neosaxitoxin, gonyautoxins V and VI and an unidentified toxin code-named PBT. Chemical structures of gonyautoxins V and VI and PBT were confirmed to be carbamoyl-N- sulfosaxitoxin, carbarn oy!-N-sutfoneosaxitoxin and decarbamoylsaxitoxin, respectively (Oshima et al, 1984). A considerable base of knowledge has been established on the chemical and biochemical nature of PSP and is the subject of several recent reviews (Hall and Reichardt, 1984; Shimizu, 1987; Harada et a!., 1982). It is apparent that the 12 suifocarbamoyi and carbamate toxins (Figure 7) comprise the major "suite" of toxins produced by dinoflagellates. In addition, the 22 R1 R2 R3 Carbamaie N-SuifocarbamoyI Decarbamoyl toxins toxins toxins H H H STX B1 dc-STX OH H H NEO B2 dc-NEO OH H OSO" GTX I C3 dc-GTX ! H H OSO" GTX I! C 1 dc-GTX II H OSO' H GTX I!) C2 dc-GTX III OH OSO H GTX IV C4 dc-GTX IV R4: R4: H R4: O- HO- 0 o Figure 7. Toxins associated with PSP. Several (de-NEO, de-GTX I/IV) have not been reported in the literature, but are postulated to occur based on presence of the other (Taylor, 1988) 23 Table 2. Composition of paralytic shellfish toxins in organisms of tropical waters (Yasumoto ef a/., 1984) Organism Locality | GTX1 | GTX2 j GTX3 | GTX4 | GTX5 \ GTX6 [ neoSTXt STX" dcSTX | TST Dinoflagellate Pyrodinium bahamense var. compressa Palau Protogonyaulax tamarensts Ofunato ++++ Pelecypods Spondylus butferi Palau Tridacna crocea Palau Septifer biiocularis Palau Decapods Zosimus aeneus Ishigaki + Atergatis floridus Ishigaki Pfatipodia granulosa Ishigaki Eriphia scabricufa Ishigaki + Thalamita sp. Ishigaki + Gastropods Turbo mamorata Ishigaki +•+++ T&ctus pyramis Ishigaki Rhodophyta Jania sp. -1 Ishigaki ++++ 24 decarbamoyl toxin derivatives can be present in shellfish due to enzymatic action on the corresponding suifocarbamoyi toxin derivatives or carbamate toxins. Consequently, any of the 18 toxins may be present in shellfish, depending on which toxins are produced by a particular strain of dinoflagellate in the local area, the presence of selective uptake and storage of the various toxins in the shellfish and any subsequent metabolism of the toxins in the shellfish tissue (Sullivan, 1988). Mode of action of saxttoxln. The toxins produced by the Pyrodmium are the saxitoxin and neosaxitoxin. Saxitoxin is the most potent neurotoxin found in dinoflagellate blooms. It is a white, very hygroscopic solid, soluble in water, slightly soluble in methanol and ethanol, but practically insoluble in most non- polar organic solvents. It is a very basic substance, being a dibasic salt with maximum toxic it y at pH 3. Absorption occurs through the gastro-intestinai tract and rapid excretion of the active toxin occurs through the kidneys (Licudan, 1994). The primary mode of action of the toxins in mammals is their binding to sodium channels in nerve cell membranes followed by interruption of normal depolarization. It has been found that the degree of binding is directly proportional to the degree of toxicity of the various PSP toxins. Therefore, by measuring the degree of sodium channel binding exhibited by the toxins in a shellfish extract, a very accurate estimate of the total toxicity of the shellfish could be obtained (Sullivan, 1988). The systemic action of saxitoxin Is secondary to the widespread blockage of impulse generation in the peripheral nerves and skeletal muscles. 25 Saxitoxin affects the excitable membrane of single nerves and muscle fibers by means of reversible selective blocking of the voltage activated sodium channels through which the downhill movement of sodium ions accounts for the initiation of electrical impulse. There are no effects on the flow of potassium or chlorine ions. The differences in the structures of the various saxitoxins alter the rate at which they bind to and depart from the binding sites on the sodium channels (Licudan, 1994). In isolation, the toxin losses its deadly sting when a molecule of hydrogen is left to react with the hydroxyl (OH) group at position R (Villao, 1988). In higher organisms such as mammals, saxitoxin blocks the sodium (Na4) channel which controls the movement of sodium ions in and out of the cell. This channel is part of a complex cell membrane machinery which also includes the sodium-potassium (Na^K4) pump and the potassium (K4) channel. All of them function synergistically to carry out depolarization in nerve cells necessary to execute the proper transmission of nerve impulse. With the sodium channel blocked, passage of a nerve impulse along a nerve or muscle fiber would be completely inhibited. This, in effect explains the resultant muscular paralysis experienced by PSP victims. Furthermore, the toxin has been found to irritate the mucus membranes of the eyes, nose, throat and nostrils causing spasmodic coughing and respiratory difficulties (Villao, 1988). On the other hand, bivalves like green bay mussels are not affected physiologically by this toxin. Nature has provided these shelled mollusks with a defense mechanism in the form of saxitoxin binding sites located in the so-called digestive or dark gland, also called hepatopancreas. The gland is able to accumulate the toxin in this part of the shellfish anatomy thus preventing the 26 substance from diffusing to the shellfish's nervous system and carrying out Its Insidious action there. The shellfish's natural moat renders the toxin- contaminated shellfish indistinguishable from the non-contaminated shellfish. Unfortunately, it is the higher order consumers, lacking any protective mechanism against the toxin, who bear the brunt of the toxin's fatal blows (Vlllao, 1988). The algal toxin saxftoxin is a neurotoxin that acts at sodium channel receptor sites 1 and 5, respectively. This toxin can be used as molecular probe to identify functional regions within the sodium channel since it alters the three properties essential for normal channel gating: voltage-dependent activation, inactivation and ion conductance. Saxitoxin inhibits sodium ion conductance by binding to specific amino acids located at the extracellular opening of the ion- conducting pore of the sodium channel in each of the four (alpha) subunit domains, as shown by the results of the study conducted by Trainer et al. (1996). Toxicology / paihophyslology. In a number of animal species, the LDso values by oral administration range from 100 to 800 jig saxitoxin/kg bodyweight or 80 jig/100g. In mice the LD50 is about 263 pg per kg by the oral route. Man is four times more susceptible than mice. The American Organization of Analytical Chemists has set the maximum tolerable level of toxin at 80 jjg/kg of shellfish meat. Any sample greater than 80 jig/kg (about 400 mouse units/1 OOg of sample) was considered not fit for human consumption. A mouse unit is the amount of poison titrated to kill a 30 g mouse in 15 minutes. Clinical picture. The clinical signs and symptoms of PSP may be as mild as tingling sensation of the lips, or as fatal as complete paralysis leading to 27 respiratory failure. Typically, the symptoms begin 5 - 30 minutes after Ingestion of the shellfish as tingling sensation around the lips, gums and tongue, in moderate and severe cases, this is regularly followed by a feeling of numbness of the fingertips and toes. Within 4-6 hours, the same sensation may progress to the arms, legs and neck so that voluntary movements can be made only with great difficulty. In fatal cases, death is usually caused by respiratory paralysis within 2 - 12 hours of consumption of the PSP containing food (Licudan, 1994). In 1988-89 epidemic in the Philippines, Pastor et ai. (1989) noted that the illness generally affected three systems: the gastrointestinal, neural and respiratory systems. The illness started as vomitting followed by numbness descended from the upper to the lower extremities that led to a light-healed or floating sensation. Later, inability to walk properly was experienced, followed by dyspnea, dysphagia and dysophnia. Death was also due to respiratory paralysis. The symptoms can also be classified according to their severity as: 1) Mild - tingling sensation or numbness of the neck and face, prickly sensation in the fingertips and toes; headache, dizziness and nausea; 2) Moderate - incoherent speech, progression of prickly sensation to the arms and legs; stiffness and incoordination of the limbs; general weakness and feeling of lightness, slight respiratory difficulty; rapid pulse; 3) Severe - muscular paralysis, pronounced respiratory difficulty; choking sensation, high probability of death in the absence of ventllatory support (Pastor et a/., 1989). Table 3 shows the symptoms of PSP. 28 Table 3. Symptoms of paralytic shellfish poison (PSP) (Pastor ef a/., 1989). Gastrointestinal Motor Disturbances Sensory Disturbances Vomlttlng Inability to ambulance Numbness Abdominal pain Paresis of extremities General body malaise Watery diarrhea Dyspnea Dizziness Nausea Dysphagia Light headed sensation Hypersalivation Dipiopia Headache Paralysis Paresthesia Dysphonia Felt hot Dythesia Short tongue sensation Pruritis Mussels may remain toxic for some time after the occurrence of this type of red tide. If the red tide disappears completely the mussels may take only a few weeks to flush the toxins from their systems. However, if the red tide organisms remain in the water at low concentrations the mussels may remain toxic for several months (van der Vyver and Pitcher, 1997). Economic Implications. During red tide blooms the following consequences are observed: 1) Shellfish Bans - Marketing of shellfish is definitely affected whenever shellfish bans are imposed. Bans also result in loss 29 of jobs/unemployment for fishermen directly Involved with production of shellfish. Other people/industries involved in related shellfish activities, such as marketing, processing, ice making, middlemen, and suppliers (bamboo and other materials for culture) are also affected. Unemployment becomes a liability to the government. Bans also pose problems for international trade and discourage expansion of the local fishing industry and development in aquaculture (White ef a/., 1984). 2) Finfish - Kills of adults / larvae may have effects on the population over the years, in certain areas, and on certain species. Fins landings may decline. An example was the decrease in herring catch from the Bay of Fundy 3-4 months after a fish kill in 1976. The settling of dead herring on the bottom of the bay caused other fish to avoid the area although it is not known by what mechanism (White ef a/., 1984). 3) Consumer Wariness - When the red tide conquered the waters of Maqueda Bay, Samar in June 1983, aside from claiming the lives of the first PSP victims, it wrought heavy damage upon the fish and musse! industry which incurred an estimated loss of R30 million. In 1987, it recurred in the same province off Sierra Island outside of Maqueda Bay, bringing with it once more the scourge of the red tide curse. And in mid-1988, it dislocated some 37,000 shell farmers and fishermen whose only source of livelihood depended upon the marine riches of the 90,000 hectare-Manila Bay. Despite a 25% drop in the prices of aquatic food sources, vendors continued to experience as much as 50% loss in sales that many of them had taken to eating raw fish and mussels in public to demonstrate edibility and non-toxicity of their food items. Losses had been paged at P25 million, and because of this vendors grow more apprehensive each day over the possible devastation and phase out of the R60 million industry (Villao, 1988). 30 The occurrences of red tide resulted to financial losses to fishermen and mussel growers, in 1986, BFAR had a compilation of financial losses due to red tide. Prices of all seafood dropped to 40% of normal prices and landings fell by up to 80%, while mussels were virtually unsaleable. Japan and Singapore were said to have banned shrimp imports from the Philippines. If mussel fishermen and growers in Manila Bay lost 3 months of their crop, their economic loss would have been some P- 20 million (US$ 1.0 = P-21). At the nation's largest fish landing, Navotas in Manila Bay, losses during the height of the 'scare' would have been over P- 6.0 million per day. Perhaps as much again would have been lost by fish brokers and market vendors. If banned shrimp were not finally exported, losses of P-10 million per day were accumulating in that sector. Major losses also occurred in many related industries, such as in the vinegar industry (because the toxin was found to be enhanced in acid conditions), while fuel or revenue fell by P-,3000 for each trip cancelled by a large trawler (Maciean, 1989). Pyrodinium bahamense var. compression ravaged Manila Bay at five sites that were eventually declared calamity areas: Bulacan, Lamao in Limay, Bataan, Pararlaque, Pasay and Navotas. Had it not been for the special role it plays in the ecological food chain, it would not have posed such great a problem. Having been naturally endowed with photo synthetic pigments, Pyrodinium bahamense as well as the other one thousand species of dinoflagellates, is highly capable of converting energy from the sun into usable chemical energy lodged into the molecules of carbohydrates. Thus, it is part of the important base of the food chain occupied by primary producers which are responsible for supplying nutrients to other consumer organisms represented by vertebrates and invertebrates (Villao, 1988). 31 In Philippine waters, among the vertebrates heavily dependent on dinofiageHates and other plankton species are certain representatives of the pelagic fish group, which includes the Indo-Pacific mackerel (RastreiSger brachysomus), sardines (Sardinefla), indian mackerel (Rastrelliger kanagurta) and anchovies (Stoiephorus spp.). Among the invertebrates, on the other hand, are the filter-feeder bivalves like green bay mussels (Perna viridis), oysters (Crassostrea) and scallops (Amusium). Thus, during population blooms of Pyrodinium bahamense, it is these diverse aquatic species that accumulate the most number of Pyrodinium cells, dependent as they are on these cells, along with other plankton, for their survival. Each has been found to accumulate at least 200 Pyrodtnium cells during red tide outbreaks (VJIJao,1988). The Philippine government responded by offering funds to various non- governmental organizations for production loans to subsistence fishermen. Regular Red Tide Bulletins were disseminated to the media and interested parties such as restauranteurs. An interdepartmental group was formed to coordinate future government activities related to red tides and funds have been allocated for a number of research projects (Maclean, 1989). Standard Mouse Bloassay for paralytic shellfish toxins. Toxicological evaluation of foods deals with the nature of the adverse effects of chemicals on living organisms and assesses the probability of their occurrence. Two assumptions underlie the use of animal models in evaluating natural and added toxicants in foods. The first assumption is that the effects of the compound on laboratory animals, when properly qualified, also apply to humans. The second assumption is that when evaluating potential hazards to humans the use of high doses of toxic agents in animal models is necessary and appropriate for 32 the effect to occur frequently enough to be detected because practical considerations limit the number of animals that can be used relative to the size of the human population at risk (Clifford, 1985). The mouse bioassay is the most commonly used method for monitoring toxins in shellfish. The detection limits of the method depend on the the mouse strain used and commonly range from 38 to 58 ^g saxitoxin-equivalents per 100 grams shellfish tissue (STX-equ . 100"1). The accuracy is regarded as ±20% of the calculated value at 80 ug STX-equ . 100 g'1. A minimum of three mice is required for each assay. The mice must be within specifed weight limits and require care in the laboratory before use. The mouse bioassay has also been used to measure toxin levels in dinoflageHates. The data are expressed as saxitoxin-equivalents per cell; these numbers are derived from populations, with the total analytical value being divided by the cell or cyst count. The inherent sensitivity of the mouse bioassay limits Its usefulness for the dinoflagellate investigator because large numbers of cells and/or cysts are needed (Hurst ef al., 1985). Selection of animals. The rat is the most widely used of all animals for food analysis. The coefficient of variation of the body weights at different ages is between 10 and 15% from weaning to maturity. Because of this, experimental groups containing approximately 10 animals per group are adequate to detect successfully (p < 0.05) treatment differences in body weights as small as 15%. The popularity of the rat as a model system is also due to its prolific reproduction and comparative ease of management in domestication (Clifford, 1985). 33 Storage of PSP toxins. Based on AOAC (1995) the PSP (saxitoxin) standard solution is indefinitely stable when stored in cool place or, the working standard solution is stable for several weeks at 3 - 4°C. Experiments conducted by Arafiles etaf. (1984) also made use of chilling temperatures (3 - 4°C) to maintain toxin stability. Food Irradiation The process of food Irradiation. Food irradiation is the process wherein food is exposed to the energy source in such a way that the precise and specific dose is absorbed. It is necessary to know the energy output of the source per unit of time, to have a defined spatial relationship between the source and the target, and to expose the target material for a specific time (World Health Organization, 1988). Food irradiation can have a number of beneficial effects, including delay of ripening and prevention of sprouting; control of insects, parasites, helminths, pathogenic and spoilage bacteria, moulds and yeasts; and sterilization, which enables commodities to be stored unrefrigerated for long periods. Before this new food processing technology could be introduced, clear evidence and assurance had to be obtained that not only would it produce the desired results but also that it would not have any unacceptable toxicological, nutritional, and microbiological effects. Irradiation of food can be conducted using low dose or high dose irradiation. Low dose irradiation utilizes an overall dose of 10 kGy, whereas, high dose irradiation is defined as those exposures providing target absorption of radiation greater than or equal to 10 kGy (Takeguchi, 1997). 34 In 1981, the World Health Organization (WHO), together with the Food and Agriculture Organization of the United Nations (FAO) and the International Atomic Energy Agency (IAEA) concluded that the "irradiation of any food commodity up to an overall average dose of 10 kGy presents no toxicological hazard; hence, toxicological testing of foods so treated is no longer required." It is found that irradiation up to 10 kGy "introduces no special nutritional or microbiological problems." The conclusions of the Expert Committee, then clearly established the wholesomeness of irradiated food up to this maximum absorbed dose of 10 kGy (WHO, 1994). Ionizing radiation. Irradiation is the use of ionizing radiations (also called ionizing energy), either from radioactive isotopes of cobalt or cesium, or from devices that produce controlled amounts of beta rays or X rays, on food. This process does not make the food radioactive. Different doses (levels) of radiation are used for different purposes. In addition to extending shelf life, some applications of irradiation eliminates hazards in food or change the physical properties of foods in desirable ways (American Council on Science and Health, 1988; International Consultant Group on Food Irradiation, 1991). The radiation used is called "ionizing radiation," because it produces electrically charged particles, or the energy is high enough to dislodge electrons from atoms and molecules and to convert them to electrically charged particles called ions. Ionizing radiations, including X rays, gamma rays, and beams of high velocity electrons (also called beta rays) produced by electron accelerators, have a higher energy than other, non-ionizing radiations such as light, microwaves, and radio waves. Irradiation causes a variety of changes in living cells. High doses kill the 35 cells, thus killing microorganisms or insects. Lower doses alter biochemical reactions, such as those involved in fruit ripening, and interfere with cell division, which is necessary for the reproduction of parasites and the sprouting of vegetables. ionizing radiation is a form of energy that is lethal to microorganisms, through a series of drastic physical attacks at the atomic nucleus of their constituents, which results in a variety of serious alterations to their life processes (celi permeability and reproduction) (Nickerson etal., 1983). Naturally occurring and man-made radionuclides, also called radioactive isotopes or radioisotopes, are unstable, and emit radiation as they spontaneously disintegrate, or decay to a stable state. The radionuclide used almost exclusively for the irradiation of food by gamma rays is cobalt-60. It is produced by neutron bombardment in a nuclear reactor of the metal cobalt-59, then doubly encapsulated in stainless steei "pencils" to prevent any leakage during its use in a radiation plant. Radiation dose is the quantity of radiation energy absorbed by the food as it passes through the radiation field during processing. It is now generally measured by a unit called the Gray (Gy). In early work the unit was the rad (1 Gy = 100 rads). International health and safety authorities have endorsed the safety of irradiation for all foods up to a dose level of 10,000 Gy (10 kGy). In terms of energy relationships, one gray equals one joule of energy absorbed per kilogram of food being irradiated (International Consultant Group on Food Irradiation, 1991). Mechanism of food irradiation. Radiation destroys microbial contaminants, including spoilage organisms, by the partial or total inactivation of 36 the genetic material of the living cells in food, either by its direct effects on DMA or through the production of radicals and ions that attack DMA. It is important to remember that food irradiation under the recommended conditions does not involve the atomic nucleus itself, but rather the electron cloud surrounding the nucleus, which initiates a purely chemical reaction. The consequent effects on biological materials are the sum of the direct or primary, and the indirect or secondary effects (World Health Organization, 1994). The primary effect is produced by energetic electrons (emanating from the source or produced through Compton scattering) and may result in one or more of three outcomes: (1) ionization (removal of an electron); (2) dissociation (loss of a hydrogen atom); or (3) excitation (raising of the molecule to a higher energy level). As a result of the highly reactive free radicals produced, a number of secondary reactions may occur, e.g. recombination, dimerization or electron capture, involving other molecules present in the complex mixture constituting the food. Disproportionation may also occur, producing a substance which may not have been present originally. The breaking of chemical bonds by radiation is called radio lysis. It produces unstable reactive products that are subsequently converted to stable end-products. The tendency is for there to be a linear relationship between the radiation dose and the amount of radiolytic products generated, i.e. a doubling of the radiation dose will double the amount of such products formed. Virtually all radiolytic products have been found to be the same as thermolytic and photolytic products produced by heating and exposure to light, respectively (World Health Organization, 1994). 37 Factors affecting radlotytlc products. Factors oiher than dose strongly influence the nature and the amount of radioiytic products formed. The presence of water or oxygen and the relative amounts thereof can have a profound influence on the radioiytic process. The temperature and the physical state of the food can also affect the outcome of the process. Freezing, for example, has a protective effect during irradiation by preventing the products of water radlolysis from reacting with the substrate. On warming, these products (hydroxyl radicals) tend to react preferentially with each other rather than with the substrate, so that damage to the latter is often less when it is irradiated in the frozen state. Anaerobic conditions also influence the nature of the radioiytic products, since the presence of oxygen during irradiation can generate highly reactive superoxide radicals, peroxy radicals and hydrogen peroxide. Another application of radiation sterilization to high protein foods (beef, chicken, ham, pork) was in combination with iow temperature of -40°C (Taub et a/., 1978). Results showed that the irradiated foods retained the same features of taste, aroma, and color as the unirradiated samples stored in the frozen state. Chemically, the amount of change occurring is relatively small, despite the high doses of electron or gamma irradiation. The reason for diminution in chemistry is the almost complete elimination of indirect effects and the reduction in net effects of direct actions when the aqueous phase is frozen, which prevents molecular diffusion. Effects of Irradiation on food. Irradiation may affect any of the following aspects of food depending on its condition during the process: 38 1) chemical components of food, 2) sensory characteristics, and 3) nutritional quality. An interesting observation is that while individual food components, such as amino acids, vitamins and sugars, can be destroyed by irradiation, they are invariably less susceptible to damage when irradiated in the complex, and evidently protective, matrix of an intact food product (World Health Organization, 1988). Another effect of irradiation is on the sensory characteristics of food. The chemical changes that radiation produces in food may lead to noticeable effects on flavour. The extent of these depends principally on the type of food being irradiated, on the radiation dose, and on various other factors, such as temperature, during radiation processing (World Health Organization, 1988). The high radiation dose required for steritzafion has been associated with unwanted flavor changes in meat with more unwanted changes occurring in the lean rather than the fat portion of the meat, presumably because of its higher fat content. Colour is another property of meat that can be changed by irradiation. Doses higher than 1.5 kGy may cause a brown discoloration of meat exposed to air (World Health Organization, 1988). The nutritional changes in food as a result of irradiation are primarily related to dose. The composition of the food and other factors, such as temperature and the presence or absence of air also influence nutrient loss. The use of low temperature and exclusion of air during irradiation as protective measures, especially at high doses, can mitigate nutrient losses (World Health Organization, 1988). 39 Effects of Irradiation on microorganisms. Microorganisms (especially Gram-negative bacteria, such as saimoneilae) can be destroyed by irradiation. Bacterial spores, however, are killed only by high doses, which means that the highly lethal foodborne disease, botulism, is not necessarily prevented by Irradiation (World Health Organization, 1988). A given radiation dose will kill a certain proportion of the microbial population exposed to it, regardless of the number of microorganisms present. This property, or result, of radiation treatment implies that the higher the pretreatment population of spoilage bacteria, for example, the higher the population wil! be after the food has been irradiated. And, of course, if spoilage has already begun, radiation can do nothing to reverse it. Consequently, as with any other method of food preservation, irradiation is not a substitute for food hygienic practice in food production and processing (World Health Organization, 1988). During irradiation, the portion of microbial population destroyed by the process depends on several factors, including the temperature at which the radiation treatment is carried out. Higher temperatures make organisms more sensitive to radiation; some microorganisms are more affected by radiation when the moisture content of food is high. At a given dose, microorganisms are less sensitive to radiation when incorporated in food than when suspended in water (World Health Organization, 1988). One goal of food irradiation is to reduce the contaminant microbial population. The mean lethal dose (MLD) Is defined as the dose required to kill 63% of a population (037). However, D10, the dose required to kill 90% of the population, is more commonly used. D10 depends on a variety of factors 40 including the food to be irradiated, the temperature, the presence of oxygen, and the water content. All these factors, including post-irradiation storage conditions (for nonsterilizing doses), determine the ultimate fitness of the food for human consumption. Though radiation may inactivate organisms or even sterilize food, many of fheir products, whether mycotoxins or bacterial toxins, are radiation-resistant and cannot be inactivated at practical dose levels, so that toxin-contaminated food cannot be detoxified through irradiation, in this regard, irradiation does not differ from conventional methods of pasteurization or sterilization by heat, which also do not destroy most microbial toxins (World Health Organization, 1994; Hui, 1992). Activity In biological systems. When ionizing radiation acts upon a biological system, it does so at the cellular level. The chemical changes resulting from the absorption of radiation that are of interest are those that interfere with the complex set of activities that occur in cells. Ceils have been considered to be the unit of life. They are highly organized bodies having a continuous enclosing membrane which defines the celfs limits. Materials pass through this membrane in a controlled manner in accordance with the cell functions. The celTs interior is filled with cytoplasm, an unspeciaiized protoplasm, a viscous translucent material. Inside the cell there usually is a continuous membrane network. Within this network are a number of inclusions, which may be different in different kinds of cells. Each inclusion performs a particular function in the activity of the cell. Cells also contain enzymes which participate in the ceil activities (Urbain, 1986). For a living biological system, chromosomal DNA is the most critical 41 target of irradiation, although other cellular components may also be affected. The direct effect of irradiation on nucleic acid molecules is either ionlzation or excitation, indirect effects on DNA include excitation of water molecules which then diffuse in the medium and may make contact with the chromosomal material. An exposure of 0.1 kGy results in 2.8% of the DNA being damaged whereas 0.14% of enzymes and 0.005% of amino acids are altered with the same dose (World Health Organization, 1994). Although some effects occur with sublethal doses, the use of sufficient radiation always leads to death, and often, as a consequence, to death of the organism of which it is a component. As shown in Table 3, the iethal dose varies with the organism. It is to be noted that the lethal dose decreases with increasing complexity and size of the organism (Urbain, 1986). Table 4. Approximate lethal doses for various organisms (Urbain, 1986). Organism Dose (kGy) Mammals 5-10 Insects 10- 1000 Vegetative bacteria 500 -10,000 Sporulating bacteria 10,000 - 50,000 Viruses 10,000-200,000 Since our interest in the irradiation of foods is generally directed to the inactivation of organisms that contaminate the food, it is fortunate that only a small amount of energy in the form of ionizing radiation is required for this 42 inactivation. While the food mass receives this lethal energy, It is generally insufficient to cause significant alteration of the food. In this sense food irradiation is a selective process, since it does have an effective impact on the targeted contaminants without having a comparable impact on the food, in terms of what happens to the food, irradiation can be described as a "gentle" process (Urbafn, 1986). Radiation resistance (Dto value) of organisms. The sensitivity of the organism to radiation is conveniently expressed in terms of the number of grays (Gy) required to accomplish the kill of a fraction of a population. This is done rather than attempting to determine the amount of radiation required to kill 100% of the population. Not only would A be difficult to do this experimentally, but the amount of radiation required is dependent on the number of organisms in the population. Therefore, a procedure is used that kills 90% of the population present. The result is expressed as the D10 value, or the treatment required to reduce the population by a factor of 10. In the case of treatment with ionizing radiation D10 is commonly called the decimal reduction dose, or the D10 value (IAEA, 1982). In a given population, as dose is increased, the fraction of survivors become smaller. In the concept of the "target theory," a radiation-induced event such as an ionization in or close to some particular entity of the organism causes its inactivation. The effectiveness of the radiation in producing lethality is dependent upon it striking the "target." With low doses only some of the organisms present will receive "hits." With increasing doses more will be inactivated in direct proportion to the amount of radiation. With increases in dose, some targets previously hit will be hit again and the efficiency of the radiation in producing organism inactivations will be reduced. While the actual number of 43 organisms inactivated per dose increment diminishes, the same fraction of organisms present is inactivated with each successive increment. The relationship between dose and surviving fraction is exponential, and a plot of dose versus the logarithm of the survivng fraction is a straight line, as shown in Figure 8 (Urbain, 1986). While the dose to effect a 100% mortality of organisms present in a food could be an objective of Irradiation, it will be recognized that this dose is dependent upon the initial number of organisms present. Further, determination of the exact dose to effect 100% mortality of a bacterial population, for example, is very difficult experimentally. Plate-counting procedures for bacteria, for example, are not effective when the counts are below 10 per milliliter. To provide an index of the radiation sensitivity of an organism and to estimate the dose needed for a particular quantitative effect in treating a food, the Di0 value has been devised. If No Is the initial number of the organisms present and N the number after application of a dose D, and D10 is the dose needed to reduce the population to 10% of No, then N D log 10 = No D10 The D10 value will be recognized as the dose needed to secure one log cycle eduction of the population (The dose for 37% survival (D37) also has been used. D10 equals 2.303 D37). The slope of the regression line of Figure 8 is -I/D10. The D10 value is an index of the radiation sensitivity of the organism. It is a characteristic of the organism and enables comparison of the sensitivities of 44 DOSE (RAD) Figure 8. Schematic diagram of survival curves, N= survivors, No= Initial number of organisms, n= extrapolation number 45 different organisms. A dose equal to one D10 value will produce a 90% inactivation of a population. If fewer survivors are desired, then additional increments of dose can be applied: A dose equal to two D10 values will yield a 1% survival; three, 0.1%; and so on (Urbain, 1986; International Atomic Energy Agency, 1982). The D10 value as just described makes use of the target theory. Essentially it assumes that only direct action of the radiation is involved. It assumes that each organism is acted upon independently. While the D10 value concept is very useful, its premises do not always hold, mainly in that indirect action of radiation does play an important role in the inactivation of some organisms under some circumstances. For this reason the relevant circumstances applicable to the D-io value must be considered in its use. Like any other microorganism, irradiation is applicable to bacteria. Bacteria are single-celled organisms. They are prokaryotic; that is, they possess no nucleus. Reproduction is by mitosis (cell division). Each bacterium is characterized by a particular D10 value, reflecting its inherent sensitivity to radiation. Since indirect action of radiation enters into the inactivation of a bacterium, different D10 values may be obtained with different relevant environmental circumstances. The Dm values for a number of spore-forming bacteria are shown in Table 4. The target theory concept explains the inactivation of bacteria on the basis of a single "hit" within the cell boundaries. In this situation, D10 values are independent of indirect action and associated environmental factors, and the relationship shown in Figure 8 holds. Indirect action, however, does occur and it is necessary, therefore, to specify the particular circumstances which apply to a given Dio value. 46 Table 5. D10 values of spore-forming bacteria (Urbain, 1986) Organism Dio (Gy) Medium Temperature Bacilus coagulans 1290 Buffer 2-12 (thermoacidurans) Bacilus pumilus E 601 1550-2110 Dried from buffer RT Bacilus subtilis 2600 Saline — B. subtilis 350 Pea puree — B. subtilis 1700 Evaporated milk — Clostridium botuiinum Type A 33 3450 - 3600 Cooked beef 25 33 3730 - 3850 Cooked beef 0 33 4300 - 4340 Cooked beef -50 33 5770 - 5950 Cooked beef -196 36 3360 Buffer 10 62 2240 Buffer 10 12885 2410 Buffer 10 Type B 9 2270 Buffer 10 40 3170 Buffer 10 41 3180 Buffer 10 51 1290 Buffer 10 53 3290 Buffer 10 Type E Alaska 1370 Beef stew RT VH 1280 Beef stew RT Beluga 1360 Beef stew RT 8E 1380 Beef stew RT 1304E 1310 Beef stew RT Iwanai 1250 1250 Beef stew RT Clostridium perfringens Type C 3181 2100 Aqueous RT Type D 8503 1800 Aqueous RT Type E 8081 1200 Aqueous RT 47 Factors affecting D10 values. Among the more important environmental factors affecting inactivation of bacteria are the following (Urbain, 1986): 1. Medium Composition. a. Water Content. In foods water may or may not be present, if present, indirect action of radiation can occur through the radiolytic products of water, commonly through the action of the OH- radical. Freezing of the water in a food produces an effect similar to water removal. The consequence of either freezing or water removal is to increase the resistance of the bacterium io radiation. b. Food Components. The food components other than water also affect indirect action of radiation on bacteria. The food components may be regarded as competing with the bacteria for interaction with the active radiolytlc products of water. Because of the compositional complexity of most foods, this competitive action is highly significant in the D10 value, and the application of D10 values obtained with simpler systems to foods can lead to error. It is commonly necessary to measure the Di0 value of a bacterium in the particular food of interest in order to define a process for irradiation. c. pH. The pH of the medium can affect free-radical formation and, in this way, the indirect action, it can also change certain aspects of the bacterium. This is illustrated in Figure 9 on Clostrkiium botuSnum 33A spores in borate buffer solution. The explanation for the change in radiation sensitivity with pH is based on (1) the effect of pH on the radiolysis of water and (2) the effect of pH in the bipolymer nature of the spores of C. botulinum. As may be anticipated, the role 48 10 Figure 9. Relationship of the survival spores of Clostrldium botulinum 33A with pH. Spores suspended in buffer solution and irradiated at -50°C with 9 kGy (Urbain, 1986). 0.5 0.4 - 0.3 \ \ \\ 0.2 - \i \ 0.1 I 1 . t 1 1 -200 -150 -100 -50 0 SO 100 IRRAOIAriON TMPtlAUK |°C| Figure 10. Change with irradiation temperature of Dio values of spores of Clostridium botuMnum 33A in ground beef. (— approximately 107 spores /dose; approximately 109 spores/dose) (Urbain, 1986). 49 of water in these effects is temperature dependent and Is related to the mobHity of radicals as determined by the physical state of the water at different temperatures. d. Oxygen. Due to the nature of the oxygen molecule in that it has unpaired electrons, oxygen adds on to many radicals, thereby preventing radical recombination and dfmerizaiion. In this way, under given conditions, the presence of oxygen enhances the indirect action of radiation, increasing its effectiveness and causing a reduction in the D10 value. This effect is illustrated in Table 5. Table 6. Effect of oxygen on D10 values of four strains of Salmonella (Urbain, 1986). Dio (Gy) Strain Aerated Anoxic Salmonella galSnarium 132 363 S. senftenberg 130 389 S. typhimurium 208 619 S. paratyphi B 192 659 2. Conditions During Irradiation a. Temperature. In foods having an appreciable water content, it can be anticipated that temperature will affect the radiation sensitivity of bacteria that are present. The change of D10 value of Streptococcus faecium A21 in phosphate buffer with temperature is shown in Table 6. The change of D-io values with 50 temperature for the spores of Ctostridium botuinum 33A in Figure 10. The change of D10 values with temperature demonstrates the importance of the indirect action in high-moisture foods. It afso emphasizes the necessity for specifying the temperature of irradiation in measuring the radiation sensitivity of a bacterium. Table 7. Change of D10 value of Streptococcus faecium A21 in phosphate buffer with temperature (Urbain, 1986). Irradiation temperature Dio (°C) (kGy) __ __ -140 3.7 - 80 3.3 - 30 2.4 5 0.09 A similar study was conducted by Grecz ef s/. (1966) on combination treatment of spores of Cl botullnum with heat plus radiation. Radiation resistance of spores of Cl botulinum is strongly affected by the temperature during irradiation. Very low radiation resistance was consistently observed when samples were in the liquid state. Below 0°C, the resistance of spores increased because the solidly frozen medium presumably decreased the diffusion of free radicals. When spores were subjected to low levels of radiation (0.6 - 0.8 Mrad) the heat resistance of the surviving spores remarkedly decreased. Spores of Cl. botullnum 33A in phosphate buffer were irradiated to 0.6, 0.8 and 1.0 Mrad at an 51 irradiation temperature range of +25 to -196°C and subsequently heated at 99°C. Survival curves revealed that all spores irradiated at +25 and 0°C were highly sensitive to heat with D-m = 5.5 min (0.6 Mrad), D-io = 3.0 min (0.8 Mrad) and D10 = 2.3 min (1.0 Mrad). For non-irradiated controls D10 was 23 min. Cbstridium botuSnum toxin Type E was not inactivated at 32°F using 2 Mrad, with higher doses inactivation proceeded at a linear rate with Dio of 2.1 Mrad. The effect of irradiating at temperature up to 117.5°F was only to shorten or eliminate the initial lag. Inactivation of the toxin was rapid with a Dio value of 0.33 Mrad (Licciardello et a/., 1969). b. Dose Rate. In systems as complex as foods, dose rate is not likely to have significant effect on the radiation sensitivity of bacteria. This is so largely because radical interaction with food components will predominate over a wide range of dose rates. Bacterial toxins. Some foodborne bacteria (e.g., Staphylococcus and Cfostrkiium botuSnum) produce toxins. These toxins are proteins and as they occur in the complex systems of food, they require doses for inactivation that are very great. The Dio values in buffer solutions of the toxins of C. botuSnum types A and B have been observed to range from 6.2 to 31 kGy. Values 100 times higher were obtained in broth and even greater values in cheese. These values Indicate that Inactivation of bacterial toxins by radiation is not feasible. For this reason irradiation of foods must be based on treating only foods that do not contain preformed bacterial toxins, unless other measures that are effective in inactivating the toxins are used additionally (Urbain, 1986). 52 Toxin production by CbstrkSium botulinum Type E in fish was studied by Hobbs (1966). Radiation pasteurization at 0.3 Mrad resulted in an extension of the storage life of the fish but had little effect on the rate of toxin production. In fish irradiated at this level it is possible for toxin to develop in about the same time as it becomes organoleptically unacceptable. Whether toxin is produced before the fish becomes unacceptable or not depends on several factors, the most important being level of contamination and storage temperature. The results indicate that as the level of contamination and the storage temperature are decreased, the "safety margin" between fish becoming unacceptable before development of toxin increases. Irradiation as a food process. It should be noted that irradiation, like any other food processes, is not a substitute for good manufacturing practices (GMP) required in the manufacture of food products. While these processes can destroy bacteria, they may not totally destroy preformed toxins and viruses already in the food. Treatments such as heat pasteurization, chemical fumigation, and irradiation, however, are effective in destroying or suppressing microbial contamination of food. Irradiation is especially effective as a control measure for parasitic diseases transmitted through solid food, especially those of animal origin (International Consultant Group on Food Irradiation, 1991; Rose etaL, 1988). Irradiation is shown capable of serving as an effective sanitizing treatment improving the sanitary quality of shellfish and providing an increased margin of safety for shellfish consumers. ^Co irradiation of the hard-shelled clam, Mercenaria mercenaria, and the oyster, Crassostrea vfrgfn!ca, significantly reduced virus carriage numbers without unduly affecting shellfish survival rates 53 or desirable organoSeptic qualities. Study results showed Ionizing radiation capable of providing an extra, highly effective safeguard of shellfish sanitary quality when combined with traditional depuration treatment. D10 values were as low as 1.0 kGy and can effectively eliminate Vibrio c ho ferae and V. parahaemolytjcusfrom shelffish (Mallett etal., 1991). Ionizing radiation was found to effectively reduce the number of microorganisms and preserve the quality of frozen prawns ( Hau et a!., 1992) when doses of 2.5, 5.0 and 7.5 kGy were used. Likewise, Nerker and Bandekar (1990) found that Salmonella in frozen shrimp can be completely eliminated at 3 kGy except for APC. Dose and doslmetry. The purpose of dosimetry in irradiation treatment is to ensure that the required absorbed dose of a particular commodity has been delivered, it is accomplished by dose mapping and process load configurations to determine the minimum absorbed dose, their locations in the process load, and the appropriate treatment time needed to satisfy established requirements (North American Plant Protection Organization, 1996). Dosimetry must consider variations due to density and composition of the material treated, variations in shape and size, variations in orientation of the product, stacking, volume and packaging. Food Irradiation costs. Any food processes will add cost. In most cases, however, food prices would not necessarily rise just because a product has been treated. Many variables affect food costs, and one of them is the cost of processing. Canning, freezing, pasteurization, refrigeration, fumigation, and irradiation will add cost to the product. These treatments will also bring benefits 54 to consumers in terms of availability and quantity, storage life, convenience, and improved hygiene of the food (International Consultant Group on Food irradiation, 1991). 55 METHODOLOGY Raw Materials Newly harvested mussels (Perna viridis) were obtained from Barrio Luz and Barrio Pitting Buhangin in Urn ay, Bataan during the bloom of red tide. Sampies used for all phases in the experiment were obtained within the vicinity, such that, all the samples were taken from nearby musse! farms at three different sampiing dates in 1998. Samples were obtained in July 1998 when the toxicity level was high, and in October (Trial 1) and November (Trial 2) 1998 when the toxicity level was low. The musseis utilized in the study were harvested in shellstocks with sizes ranging from 50 to 80 mm. Approximately 33 kgs. of green musseis were used for each sampling. Before transporting to the laboratory, the adhering dirt (i.e., sand, seaweeds, and other surface dirts) in the mussels were removed by splashing with clean tap water. The mussels were packed in polyethylene bags (25 cm x 35 cm) with a thickness of 0.70 mm and sealed with cellulose scotch tape. Each bag had a net weight of 750 g of shellstocks of green musseis. The packages were placed in styropore boxes with crushed ice io maintain a temperature of 0 - 4°C during transport to the College of Home Economics Quality Control Laboratory where the samples were frozen upon arrival. The samples were kept frozen until subjected to irradiation and/or analysis. Preparation of Samples for Irradiation Samples of shellstocks of green mussels were divided into three lots identified as follows: One lot, or Sample A, was irradiated at varying doses as shellstocks. Eighteen (18) kgs were packed in polyethylene (PE) bags consisting of 750 g mussels per bag. Six bags were placed in a styropore box as shown in Figure 11. Three (3) bags were layered one on top of the other on the left side of the box as replicates A, B, and C, and another three bags were arranged similarly on the right side of the box as replicates D, E? and F. Four boxes were prepared. Each box contained six (6) bags of the sample and exposed to a dose of ionizing radiation, such as 5 kGy. After irradiation, replicates A, B and C were used for the extraction of PSP toxins, while replicates D, E, and F were used for the sensory, microbiological, and chemical evaluation as well as the determination of the fatty acid component of the irradiated samples. The Standard Mouse Bioassay (SMB) method was performed on the extracts following the procedure described In Appendix A (AOAC, 1995), and the D-m values were determined (Figure 12). Another lot, or Sample B, was prepared for toxin extraction prior to irradiation. Seven and a half (7.5) kgs of frozen shellstocks of mussels were used in the extraction of semi-purified PSP toxins. The toxins were extracted following the procedure described in Appendix A. After extraction, three 200 mL extracts, used as replicates A, B and C, were filled into 250 mL plastic bottles and placed in a styropore box with ice and water mixture to maintain the temperature of the extract at 0 - 4°C during irradiation as shown in Figure 13. Four boxes were prepared. Each box contained three bottles of the sample and subjected to a 57 11a 11b Figure 11. Arangement of packed shellstocks of green mussels in a styropore box (11a, top view; 11b, front view). The packs are arranged from top to bottom as repScates A B and C on the left side of the box, and as repBcates D, E and F on the right side of the box 58 Shellstocks of green mussels A. Remove dirt by tap water Pack in PE bags (0.75 kg/pack) Freeze to -18°C Control (Unirradiated Sample A Sample B Pack in styropore boxes Extract toxin from 7.5 kgs sheilstocks fAooendix A) Maintain shelistocks' temDerature at -18°C Fifl in 250 mL plastic bottles and place in a stvrooore box Maintain extract temperature ai4°C irradiate in styropore boxes (kGy) 10 10 20 Mouse test and determine toxicity Determine DID value Figure 12. Phase 1. Flow diagram for the determination of D^ values of PSP toxins from sheBstocks of green mussels (Sample A) and from semi-purified toxin extract (Sample B) 59 13a 13b Figure 13. Arrangement of samples of semi-purified toxin extract contained in plastic bottles inside a styropore box (13 a, top view; 13b, front view). The samples are arranged as replicates A, B and C starting from the left side of the box. 60 dose of ionizing radiation, such as 5 kGy. After irradiation, the SMB method was conducted following (he procedure described in Appendix A (AOAC, 1995), and the D10 value was determined (Figure 12). The remaining third lot was irradiated at a dose level determined to be the D10 value of the PSP toxins. Four and a half (4.5) kgs of the shellstocks were packed in PE bags containing 750 g mussels per bag and frozen. The six (6) bags were arranged and coded similarly in a styropore box as in Sample A prior to irradiation. After Irradiation, the samples were analyzed as in Sample A. Control samples of frozen green mussels were prepared similarly as in Sample A except that the samples were not subjected to different radiation doses. Three replicates were prepared and the concentration of PSP toxins were determined by the SMB method. Preparation of Samples for Toxin Extraction The samples were prepared as follows: The shellfish was cleaned thoroughly with fresh water. The shell was opened by cutting \he adductor muscles. The meat was removed from the shell by separating first the adductor muscles and tissue connected at the hinge. Care was observed so as not to cut or damage the body of the moilusk at this state. About 100 g meat was collected per replicate. The meat was allowed to drain for 5 minutes in a strainer. Pieces of shells were discarded. The meat was blended until homogeneous. In mussels, meat recovery was approximately 25% or 187.5 g meat in 750 g shelfstocks. 61 Determination of PSP toxin concentration by Standard Mouse Bloassay (SMB) Method. The PSP toxin concentration of the samples was determined by the Standard Mouse Bioassay Method for Paralytic Shellfish Toxins as described in AOAC, 1995, Section 959.08 (Appendix A). Irradiation of Samples Samples of shellstocks of green mussels (Sample A) and of PSP toxins from semi-purified toxin extract (Sample B) were irradiated in the batch-type Gammabeam 651PT Co60 gamma irradiator. The activity of the gamma irradiator was about 120,000 curies in January 1998. Shellstocks of green mussels (Sample A). Before irradiation, the frozen mussels were arranged in four (4) styropore boxes. Each box was subjected to a dose of gamma radiation, i.e. 5,10, 15 or 20 kGy. Each box had six (6) bags of frozen mussels (Figure 11). To maintain the mussels In the frozen condition, dry ice was placed under and on top of the samples. This is also done to allow less interference with the radiation coming from the gamma source. Two boxes were positioned side by side on one side of the gamma source and the other two boxes were positioned similarly at the opposite side of the source. During irradiation, two ethanol chlorobenzene (ECB) dosimeters were placed in the minimum position and two in the maximum position. The ECB dosimeters were used in both dose mapping and process control. The dosimeter solution contains 24 volume% monochiorobenzene, 4 volume% distilled water and 71.92 volume% absolute ethanol, 0.04 volume% acetone and 0.04 volume% benzene. The absorbed dose was determined using an oscillotitrator 62 developed by INISO Hungary (RADELKIS OK- 302/1 Oscillotftrator). A batch of these dosimeters was calibrated against Fricke dosimeters before use. In dose mapping, shellstocks of green mussels, which were toxin-free, were used as dummy. ECB dosimeters were distributed in each plane (front, middle and back) to determine the minimum and maximum dose positions. Dose mapping was done three (3) times before irradiation of the actual samples. During the routine Irradiation process, two ECB dosimeters were placed In the minimum position and two (2) in the maximum position. The dose uniformity within a box was made sure not to exceed ±10% of the required absorbed dose. The dose rate in July 1998 was determined to be 3.06 kGy per hour which was used as basis in calculating for irradiation time of the dose to be applied. The process made use of two-sided irradiation wherein all the boxes were rotated 180° at half the irradiation time. The boxes were removed when the total Irradiation time was completed. After irradiation, the frozen green mussels were brought back to the College of Home Economics with dry ice to maintain a temperature of -18°C or less during transport. The mussels were stored in a freezer until analyzed. Semi-purffled toxin extract (Sample B). Four styropore boxes were used in the irradiation of samples, each box representing a dose of gamma radiation of 5,10,15 and 20 kGy. Two hundred (200) mL of the toxin extract was filled into 250 mL capacity plastic bottles with screw-type caps. Each box contains three bottles, as replicates A, B and C, which were positioned horizontally on the lengthwise side of the box (Figure 13). The procedure for dose mapping of the semi-purified toxin extract was similar to that followed for the dose mapping of shelistocks of green mussels. 63 Phase 1. Radiation Resistance of Paralytic Shellfish Poison (PSP) Toxins Comparison of the radiation resistance of PSP toxins obtained from shellstocks of green mussels and from semi-purified toxin extract was conducted by irradiation of the samples at different doses: 5, 10, 15, and 20 kGy. The distance of the styropore boxes from the source of radiation or shroud is 35 centimeters from the center of the box. The Dto value or the decimal reduction dose was determined by plotting the PSP toxin concentration versus applied radiation dose on a semilog paper. Radiation resistance of PSP toxins at high toxlclty level. Mussel samples were collected only once at this toxicrty level. They were collected during the height of the red tide bloom in July 1998 In Barrio Luz, Limay, Bataan. The samples were exposed to ionizing radiation of 5, 10, 15 and 20 kGy. The initial PSP concentration was about 847.01 pg/100 g meat. Radiation resistance of PSP toxins at low toxlclty level. Mussel samples were collected in the months of October (Trial 1) in Puting Buhangin, and in November (Trial 2) 1998 in Barrio Luz both in Limay, Bataan when the concentration of PSP toxins was low. The samples were exposed to the same doses of ionizing radiation as above. The PSP concentration was 60.09 p.g/100g meat In Trial 1 and 66.86 u.g/100g meat in Trial 2. Determination of decimal reduction dose (D™ Value). The method to determine the sensitivity of the PSP toxins to radiation is expressed in terms of the decimal reduction dose or commonly called the Devalue, it is the radiation dose required to reduce the concentration level of the toxin by 90%, or to reduce the toxin concentration to 10%. The D10 value is obtained by plotting the average 64 PSP toxin concentration in the logarithmic scale versus radiation dose in the linear scale on a semilog paper. Dio is the reciprocal of ihe slope of the straight line portion of the curve. It is also the dose required for the curve to traverse one logarithmic cycle, or it is the difference in dose (X2 - xi) equivalent to 90% reduction in PSP concentration or 10% retention (Figure 8). Before a straight line was obtained, the regression line by the least square method was calculated. Phase 2: Effects of Ionizing Radiation on the Quality Characteristics of Shellstocks of Green Mussels The sheilstocks of mussels were evaluated in terms of its physico- chemical, sensory, and microbial qualities as well as the fatty acid component after exposure to varying doses of radiation of 5, 10,15 and 20 kGy, and at the value of the toxin as determined in Phase 1. Effects on shellstocks of green mussels subjected to different doses of Ionizing radiation. From Ihe six (6) bags of shellstocks contained in a styropore box (Figure 11) in Phase 1, the remaining three bags identified as replicates D, E and F were taken and the effect of different doses of ionizing radiation on the physico-chemical, sensory and microbiological qualities of the shellstocks was determined following the procedures described below on effects on shellstocks irradiated at the Dio value, and shown in Figure 14. 65 Shellstocks of green mussels Remove dirt by clean tap water Drain Pack in PE bags ( 0.75 kg / bag) and freeze to -18°C internal temp. Irradiate using different doses of ionizing radiation to compare Irradiate using the Devalue effect of dose vs. toxicitvand to determine Dm value of toxin obtained in Phase 1 Transport to laboratory in styropore box, maintain temperature at -18°C Evaluate qua Sty Physico-chemical test Micro test Sensory test Remove meat from Steam shelHish to an internal temperature of 70°C Aerobic Colifcrms Plate Homogenize Evaluate quaSty ( use scoresheei in Appendix E Analyze pH Moisture content Figure 14. Phase 2. Flow diagram for determination of effects of different ionizing radiation on the quality of shellstocks of green mussels 66 Effects on shellstocks of green mussels Irradiated at the D10 value. This study required about 6.0 kg or eight (8) bags of frozen green mussels containing 750 g mussels per bag for each sampling obtained from the whole lot of 33 kg per sampling. Two bags were taken before irradiation and analyzed for Aerobic Plate Count and coliforms. The remaining six bags were placed in a styropore box arranged similarly as in Phase 1 and exposed to an irradiation dose equivalent to the D10 value. The position of the styropore box on the turntable is shown in Figure 15. Replicates A, B, and C were used to verify the remaining toxicity of the sample after applying the computed D10 value obtained in Phase 1 by following the procedure described in Appendix A. Replicates D, E, and F were used for quality assessment of the irradiated samples (i.e., physico- chemical, sensory, and microbiological tests) as shown in Figure 14. After irradiation the following tests were conducted on the shellstocks of green mussels: Physico-chemical tests. The pH and moisture content were analyzed following the standard procedures described in AOAC, 1995, Sections 973.41 and 950.46 (Appendices B and C). Microbial tests. Analysis for Aerobic Plate Count ( Appendix D ), and coliforms (Appendix E ) were conducted as described in USFDA Bacteriological Analytical Manual, 1995. Sensory tests. Two hundred fifty (250) g of irradiated mussels were taken from each pack in replicates D, E, and F. The samples were prepared for evaluation as described in Appendix F. Sensory tests were carried out at the Food Development Center using a trained panel of 6 members who were familiar with the characteristics of irradiated mussels. A descriptive scoresheet, as shown in Appendix G, was used which was developed from a training session conducted on familiarization of the mussel samples. 67 Figure 15. Arrangement of a styropore box containing samples of frozen shelistocks of green mussels on the turntable which will be irradiated with the calculated D10 value. 68 Phase 3. Changes in the Fatty Acid Profile of Irradiated Shelistocks of Green Mussels The changes in the fatty acid composition of the unirradiated and irradiated shellsiocks of mussels were determined using a gas chromatograph with flame ionization detector. Effect on shellstocks of green mussels exposed to different doses of Ionizing radiation. The fatty acid content of the samples subjected to different doses of ionizing radiation was determined by using the procedures described in AOAC, 1995, Section 948.15 on Determination of Crude Fat in Seafoods (Appendix H) and Section 969.33 on Fatty Acids in Fats and Oils (Appendix I) as shown in Figure 16. Replicates 0, E, and F from each dose were used in the analysis. Effect on shellstocks of green mussels Irradiated at the D10 value. From the remaining samples in replicates D, E, and F from Phase 2, the fatty acid components of mussels irradiated at its D10 value was determined (Figure 16). identification of fatty acids. Determination of the fatty acid content of the samples was based on the procedure described m AOAC, 1995 Section 969.33 for Fatty Acids in Oils and Fats (Appendix I) using gas chromatography. A programmed temperature gas chromatography was used In the analysis. The gas chromatograph is a multipurpose high-efficiency gas chromatograph controlled by a micro-computer. Automatic cooling and recovery to the initial column temperature are included in the program. The standard operating conditions used were as follows: 69 Instrument : Shimadzu Gas Chromatograph GC- 9A with Shimadzu C- R6A Chromatopac (Printe r) Detector : Flame lonization Detector Column : 10% Polyethylene gtycol (PEG) 20 M Chart Speed : 3 Volume Injected; 2 Mi- Attenuation : 4 Temperature Injection Port: 230°C Shelfetocks of green mussels Remove dirt by tap water Drain Pack in PE bags (0.75 kg / bag ) Irradiate Irradiate at 5,10,15 and 20 kGy Irradiate at the D10 vakie Keep in styrobox (-18°C) durirta transoort to laboratory Extract oil (Appendix H) Esterify oil extract (Appendix I) Analyze for fatty acids using gas chromatoaraohv Figure 16. Phase 3. Flow diagram for determination of fatty acids in rradiated sheBstocks of green mussels 70 RESULTS AMD DISCUSSION Phase 1. Radiation Resistance of Paralytic Shellfish Poison (PSP) Toxins The radiation resistance in terms of D^ value of PSP toxins in shellstocks of mussels and in semi-purified toxin extract was determined and compared by exposing the samples to varying doses of ionizing radiation of 5, 10,15 and 20 kGy. The SMB method was conducted to determine the PSP toxin concentration after irradiation. Radiation resistance of PSP toxins at high toxlctty level. Samples with high PSP toxicity level were obtained from Bo. Luz, Limay, Bataan in July 1998 during the peak of the red tide bloom, with an initial PSP content of 847.01 jig/100 g meat (Table 8). The samples were divided into two lots. One lot was prepared as frozen shellstocks of green mussels and the other lot was prepared Into semi-purified toxin extract. The shellstocks of green mussels (Sample A) were frozen and subjected to different doses of gamma radiation of 5, 10, 15 and 20 kGy. After irradiation, the PSP content was determined to be 700.20, 370.58, 316.08 and 237.25 jjg/100g meat, respectively at the above doses (Table 8). The D10 value obtained for Sample A was 28.5 kGy (Figure 17). On the other hand, after irradiation at above doses the average PSP concentration of samples of semi- purified toxin extract (Sample B) were 188.77,182.58, 241.14 and 126.27 jig/100 g meat, respectively (Table 9). The D10 value which was obtained after plotting the PSP concentration versus applied dose was 17.5 kGy (Figure 18). Results showed that the shellstocks of green mussels had a higher Di0 value as compared with the semi-purified toxin extract. Since the semi-purified toxin extract is in aqueous solution, the presence of water increased the indirect action of radiation through presence of radiofytic products of water (Urbain, 1986) which contributed to the destruction of the toxins. Table 8. Concentration of PSP toxins from sheiistocks of green mussels (or Sample A) with high initial toxin ievel Average Average % PSP Ave. % Dose Replicate ug PSP/100g meat PSP PSP Retention PSP (kGy) (ug/100 g (ug/100 g ( N / No ) Retention 1 2 meat) per meat) per per Per dose 2 replicate dose 1 replicate Control 729.27 964.76 847.01 - 100.00 100.00 5 A 574.00 542.16 560.08 700.20 66.12 82.67 B 784.04 659.30 721.67 85.20 C 890.44 747.26 818.85 96.68 10 A 361.50 355.96 358.73 370.58 42.35 43.75 B 375.19 316.09 345.64 40.81 C 403.00 411.73 407.37 48.10 15 A 349.14 353.74 351.44 316.08 41.49 37.32 B 306.95 306.62 306.79 36.22 C 312.62 267.43 290.02 34.24 20 A 191.62 201.58 196.60 237.25 23.21 28.02 B 239.41 241.73 240.57 28.40 C 276.60 273.14 274.87 32.45 -) means not applicable Average PSP (jig/100g meat) per replicate per dose Average %PSP Retention of three replicates per dose 72 P3P Concertration bigfmL) (a Table 9. Concentration of PSP toxins from semi-purified toxin extract (or Sample B) obtained in mussels with high initial toxin level Average Average % PSP Ave. % Dose Replicate VQ PSP/100g meat PSP PSP Retention PSP (kGy) (pg/100 g (ug/100 g (N/No) Retention meat) per meat) per per per dose 2 1 2 1 replicate dose replicate Control 729.27 964.76 847.01 - 100.00 100.00 5 A 150.89 147.87 149.38 188.77 17.64 22.29 B 221.80 238.76 230.28 27.19 C 245.37 127.97 186.67 22.04 10 A 226.40 199.23 212.82 182.58 25.13 21.56 B 235.27 210.68 222.98 26.32 C 111.78 112.11 111.94 13.22 15 A 216.93 300.86 258.89 241.14 30.57 28.47 B 203.01 250.89 226.95 26.79 C 265.08 210.08 237.58 28.05 20 A 109.07 134.68 121.87 126.27 14.39 14.91 B 167.20 148.51 157.86 18.64 C 93.66 104.51 99.09 11.70 (-) means not applicable 1 Average PSP (jig/100g meat) per replicate per dose 2 Average %PSP Retention of three replicates per dose 74 Sml-Loivlihml« 0 ) Cvdn i 10 lo itif Inch Doan (kGy) Figure 18. D10 value of PSP toxins obtained from semi-purified toxin extract (Sample B) with initially high toxin level 75 In shellstocks of green mussels, (he toxins are present in real food so there is substantial quenching of radiation-derived free radicals that occurred in the medium which resulted in the protection of individual protein molecules (Wilkinson and Gould, 1996). Also, the frozen condition of the samples during Irradiation affected the radiation resistance of the PSP toxins because the solidly frozen medium presumably prevented the diffusion of free radicals produced during irradiation which is a similar reaction in bacterial spores as in Cfostridium botufinum (Grecz, 1966). The diffusion of free radicals was necessary for reactions to proceed, and in this case the destruction of toxins. The consequence of freezing, which is also similar to water removal, was to increase the resistance of the bacterium to irradiation (Urbain, 1986), which Is a similar behavior in PSP toxins. Radiation resistance of PSP toxins at low toxlclty level. Samples with low toxicity level were gathered in October 1998 in Puting Buhangin (Trial 1) and in Barrio Luz (Trial 2) both in Limay, Bataan. The initial PSP content of the samples in Trial 1 was averaged at 60.09 jig/100 g meat (Table 10). After irradiation at doses of 5,10,15 and 20 kGy, the average PSP concentration was determined to be 51.24, 44.74. 36.39 and 26.93 jxg/100g meat> respectively (Table 10). After plotting the PSP concentration versus applied dose on the semilog paper, the obtained Devalue was 57.5 kGy (Figure 19). In the semi- purified toxin extract, the average PSP concentration of the toxins after being subjected to the above doses were 34.81, 30.60, 29.04 and 29.12 ng/100 g meat, respectively (Table 11), and a D-io value of 43.0 kGy (Figure 20) was obtained. The Dio values were observed to be much higher than the D^ values of samples with inttial high PSP concentration. Similarly, the Dio value was higher in the shelistocks than in the semi-purified toxin extract. 76 The D10 value of samples of green mussels with a high initial level of PSP concentration prior to irradiation was lower when compared with the D10 value of green mussels having a low initial level of PSP concentration. Since the PSP toxins is made up of several toxins, the bio-conversion of these toxins by irradiation is possible. Some of the toxins may be sensitive to irradiation while the others may be less sensitive. In this case, most of the toxins in mussels with high initial toxicrty may be more sensitive to irradiation than the toxin composition of mussels with low initial toxin level. Table 10. Concentration of PSP toxins from shellstocks of green mussels (or Sample A) with low initial toxin level (Trial 1) pgPSP/100gmeat Average Average %PSP Ave. % Dose Replicate PSP PSP Retention PSP (kGy) (ug/100 g (jjg/100 g (N/No) Retention meat) per meat) per per per dose 2 1 2 replicate dose1 replicate Control 65.83 54.35 60.09 - 100.00 100.00 5 A 44.96 42.69 43.82 51.24 72.93 85.27 B 52.40 54.02 53.21 88.55 c 58.40 54.98 56.69 94.34 10 A 48.23 49.52 48.88 44.74 81.34 74.45 B 42.22 43.68 42.95 71.48 C 42.44 42.34 42.39 70.54 15 A 35.85 35.66 35.74 36.39 59.48 80.55 B 37.12 36.18 36.65 60.99 C 36.22 37.32 36.77 61.19 20 A 24.67 25.96 25.32 26.93 42.14 44.82 B 29.05 29.70 29.38 48.89 C 26.51 25.67 26.09 43.42 (-) means not applicable 1 Average PSP (jig/100g meat) per replicate per dose 2 Average %PSP Retention of three replicates per dose 77 Co Dose (kGy) Figure 19. D10 value of PSP toxins obtained from shellstocks of green mussels (Sample A) with initially low toxin level (Trial 1) Table 11. Concentration of PSP toxins from semi-purified toxin extract (or Sample B) obtained in mussels with low initial toxin level (Trial 1) Average Average % PSP Ave. % Dose Replicate MS PSP/100g PSP PSP Retention PSP (kGy) meat 0ig/100 (pg/100 ( N / No ) Retention g meat) g meat) per per dose2 per per replicate 1 1 2 replicate dose Control 65.83 54.35 60.09 - 100.00 100.00 5 A 33.71 35.48 34.60 34.81 57.58 57.92 B 35.69 34.44 35.07 58.36 C 35.07 34.44 34.75 57.83 10 A 30.78 31.95 31.37 30.60 52.20 50.92 B 29.46 30.88 30.17 50.21 C 32.95 27.56 30.26 50.36 15 A 30.07 28.91 29.49 29.04 49.08 48.32 B 28.59 27.56 28.08 46.73 C 28.56 30.52 29.54 49.16 20 A 30.14 29.96 30.05 29.12 50.00 48.46 B 29.00 29.58 29.29 48.74 C 26.87 27.18 28.02 46.63 (-) means not applicable 1 Average PSP (jig/100g meat) per replicate per dose 2 Average %PSP Retention of three replicates per dose 79 5 1 CD CO CO 2 0) ID cd I CD J3 o CN i il 80 The Initial PSP toxin concentration of the samples for Trial 2 was 66.86 jig/100 g meat (Table 12). Doses of gamma radiation applied were 5, 10,15 and 20 kGy. The PSP concentration of toxins in the shellstocks of green mussels after application of the above doses were 33.18, 31.34, 31.54 and 28.42 }ig/10Qg meat, respectively (Table 12). The Dio value was determined to be 43.5 kGy (Figure 21). In the semi-purified toxin extract, the PSP concentration were reduced to 43.02, 31.12,30.65 and 29.86 jxg/100g meat, respectively (Table 13), after the above doses were applied. The Dio value of this sample was 43.0 kGy (Figure 22). Results of the radiation sensitivity of PSP toxins obtained from the three different sampling periods showed that the D-m values of PSP toxins in the sheilstocks of green mussels were found to be higher than in the semi-purified toxin extract. The frozen condition of the substrate or the shellstocks had a protective effect on the toxins due to unavailability of water in the frozen state. When water is unavailable, the radiolytic product is less as compared to a substrate where water is present or available as in the semi-purified toxin extract. When water is present the indirect action of radiation through presence of radiolytic products of water is increased resulting to destruction of more sensitive toxins. The dinoffagellate Pyrodinium bahamense var. compressum is made up of several toxins such as saxitoxin, neosaxitoxin, gonyautoxins V and VI and an unidentified toxin code-named PBT which is collectively called PSP toxins. The biochemistry as well as their behavior or sensitivity to irradiation may differ so that the more sensitive toxins may have been destroyed by irradiation and the more resistant toxins were retained which were detected by the SMB method. 81 Table 12. Concentration of paralytic shellfish poison toxins from shellstocks of green mussels (or Sample A) with low initial toxin level (Trial 2) Average Average % PSP Ave. % Dose Replicat pgPSP/100g PSP (pg/100 PSP Retention PSP (KGy) e meat g meat) per Qjg/100 g ( N / No ) Retention replicate meat) per per per dose2 dose 1 replicate 1 2 Control 65.64 68.08 66.86 - 100.00 100.00 5 A 38.98 34.16 36.57 33.18 54.70 49.63 B 29.52 29.29 29.40 43.97 C 30.81 36.34 33.58 50.22 10 A 31.74 29.25 30.50 31.34 45.62 46.88 B 33.74 29.44 31.59 47.25 C 31.87 31.97 31.93 47.76 15 A 34.08 32.82 33.45 31.54 50.03 47.18 B 29.14 31.10 30.12 45.05 C 30.19 31.92 31.06 46.46 20 A 29.76 29.08 29.42 28.42 44.00 42.51 B 28.16 27.24 27.70 41.43 C 28.26 28.03 28.14 42.09 (-) means not applicable 1 Average PSP (^tg/100g meat) per replicate per dose 2 Average %PSP Retention of three replicates per dose 82 PSP Concentration (jig/ml) -n 3 so c CD O —ti TJ en Io 0) Q. 3 a> o o c/i O o CD C3 so 3 > §. 3 2. i Table 13. Concentration of paralytic shellfish poison (PSP) toxins from semi- purified toxin extract (or Sample B) obtained in mussels with low initial toxin level (Trial 2) pgPSP/100g Average Average % PSP Ave.% Dose Replicate meat PSP PSP Retention PSP (kGy) (fjg/100 g (tig/100 g ( N / No ) Retention meat) per meat) per per per dose 2 1 2 1 replicate dose replicate 100.00 Control 65.64 68.08 66.86 - 100.00 5 A 49.18 45.57 47.38 43.02 70.86 64.35 B 42.25 40.19 41.22 61.65 C 40.48 40.45 40.47 60.53 10 A 34.67 37.19 35.93 31.12 53.74 46.55 B 27.44 26.11 26.78 40.05 C 30.32 31.00 30.66 45.86 15 A 27.35 30.21 28.78 30.65 43.04 45.84 B 35.13 29.61 32.37 48.41 C 31.09 30.52 30.80 46.07 20 A 30.13 30.28 30.20 29.86 45.17 44.67 B 29.58 29.37 29.48 44.09 C 29.33 30.49 29.91 44.74 (-) means not applicable ^ Average PSP (p.g/100g meat) per replicate per dose 2 Average %PSP Retention of three replicates per dose 84 I 1 1 CO OD _CD Q. CD I •o o CD Q | CD 1/! o 4= <1> •i Is d CM 85 Phase 2. Effects of Ionizing Radiation on the Quality Characteristics of Shelistocks of Green Mussels The quality of the shellstocks of green mussels was evaluated after subjecting to different doses of ionizing radiation and at the D10 value of the PSP toxins. Samples for evaluation were taken from replicates D, E and F from each box receiving a dose of radiation. Effects on shellstocks of green mussels subjected to different doses of Ionizing radiation. Results of the chemical tests for the unirradiated (control) and irradiated samples of shellstocks of green mussels for Phase 1 are shown in Table 13. In Phase 1, the average pH value for frozen unirradiated samples was 6.24 (Table 13). The pH values of the irradiated samples ranged from 6.75 to 6.78. The moisture content of the frozen unirradiated samples was 78.5%. The moisture content values of the irradiated samples ranged from 77.2 to 77.5%. in Trial 1 of samples with low toxicity level, the average pH value for frozen unirradiated samples was 6.25 (Table 13). The pH values of the irradiated samples ranged from 6.76 to 6.80. The moisture content of the frozen unirradiated samples was 77.5%. The moisture content values of the irradiated samples ranged from 76.5 to 76.9%. In Trial 2 of samples with low toxicity level, the average pH value of the frozen unirradiated samples was 6.22 (Table 13). The pH values of the irradiated samples ranged from 6.77 to 6.79. The moisture content of the frozen unirradiated samples was 78.8%. The moisture content values of the irradiated samples ranged from 77.3 to 77.5%. 86 Results showed that the difference in the pH values and moisture content of the irradiated samples to the unirradiated was nil which implies that these chemical characteristics were not affected by irradiation. Table 14. Chemical characteristics of frozen irradiated and unirradiated shellstocks of green mussels Type of Sample Treatment Dose Applied Chemical Characteristics 1 (kGy) PH Moisture content (%) Mussels with Unirradiated None 6.24 78.5 high initial toxin (ControD level Irradiated 5 6.76 77.5 10 6.75 77.4 15 6.77 77.4 20 6.78 77.2 Trial 1 Unirradiated None 6.25 77.5 (Mussels with (Control) low initial toxin level) Irradiated 5 6.77 76.9 10 6.76 76.7 15 6.76 76.5 20 6.80 76.8 Trial 2 Unirradiated None 6.22 78.8 (Mussels with (Control) low initial toxin level) Irradiated 5 6.78 77.5 10 6.77 77.3 15 6.79 77.6 20 6.78 77.5 Values are averages of 6 determinations per treatment 87 The color of the unirradiated (control) samples for Phase 1 was described as bright red orange while the color of samples irradiated at 5, 10, 15 and 20 kGy was described as brownish red orange (Table 14). The brown discoloration which was observed in the mussel meat exposed to air may be due to application of gamma radiation higher than 1.5 kGy (World Health Organization, 1988). The odor characteristics of the samples were described as follows: The intensity of the characteristic mussel odor was moderate. No stored/fishy odor was detected in the unirradiated samples while a perceptible intensity of this odor was observed in the irradiated samples. The perceived fishy odor may be a result of the oxidation of lipids in mussels during irradiation since mussels has a fat content of 7.5%. The fishy odor may have been contributed by various oxidized compounds such as alkanals (C5-C10); alk-2-enals (C5-C10); hepta-2-t,4-t- dienal; 2-alkanones (C3-C11); oct-1-en-3«one; deca-2-t,4-c,7-t-triena!; pent-1- en-3-one (Saxby, 1996). No burnt odor, which is a characteristic of irradiated products, was detected in the samples. Likewise, presence of other off-odors was not noted in all samples. The frozen condition of the samples during the irradiation process had a protective effect on its characteristics. Since water is bound when frozen, the indirect action of radiation occurring through the radiolytic products of water was very minima! (Urbain, 1986). The Aerobic Plate Count (APC) and coliform count of the unirradiated mussels were 43,000 cfu/g and 9.81 MPN/g for samples with high initial toxicity level; 44,000 cfu/g and 4.62 MPN/g, respectively, for Trial 1 of samples with low initial toxicity level; and 18,000 cfu/g and 4.56 MPN/g, respectively, for Trial 2 of samples with low initial toxicity level (Table 15). The microbial load was reduced Fable 15. Color and odor characteristics of frozen irradiated and unirradiated shellstocks of green mussels Type of Treatment Dose Color Odor Characteristics n Sample AppBed Charac- Intensity of Presence of Presence of Presence (kGv) teristic charac- stored/ fishy burnt-fike of other off- teristic odor odor odor mussel odor Mussels Unirradiated None Bright red Moderate None Not None with high (Control) orange appicable IHltlQinitialt toxin Irradiated 5 Brownish Moderate Perceptible None None level red orange 10 Brownish Moderate Perceptible None None red orange 15 Brownish Moderate Perceptible None None red orange 20 Brownish Moderate Perceptible None None red orange Trial 1 Unirradiated None Bright red Moderate None Not None (Mussels (Control) orange appicable with low initial Irradiated 5 Brownish Moderate Perceptible None None toxin red orange level) 10 Brownish Moderate Perceptible None None red orange 15 Brownish Moderate Perceptible None None red orange 20 Brownish Moderate Perceptible None None red orange Trial 2 Unirradiated None Bright red Moderate None Not None (Mussels (Control) orange applicable buHh IrttM initial irradiated 5 Brownish Moderate Perceptible None None toxin red orange level) 10 Brownish Moderate Perceptible None None red orange 15 Brownish Moderate Perceptible None None red orange 20 Brownish Moderate Perceptible None None red orange Corresponding descriptors are based on the average of 3 replicates (D, E and F) per treatment as evaluated by 6 trained paneists 89 Table 16. Microbiological characteristics of frozen irradiated and unirradiated shellstocks of green mussels Type of Treatment Dose Applied APC Conforms Sample (kGv) (col/g)1 (MPN/g)1 Mussels Unirradiated None 43,000 0.918 with high (Control) initial toxin level Irradiated 5 2,000 0 10 990 0 15 620 0 20 360 0 Trial 1 Unirradiated None 44,000 0.462 (Mussels (Control) with low initial toxin Irradiated 5 4,200 0 level) 10 3,300 0 15 470 0 20 270 0 Trial 2 Unirradiated None 18,000 0.456 (Mussels (Control) with low initial toxin Irradiated 5 4,000 0 level) 10 2,600 0 15 360 0 20 260 0 1 Values are averages of 2 determinations (Appendices Q and R) when the samples were irradiated at 5, 10,15 and 20 kGy. Remarkable results were obtained when the samples were analyzed for coliforms. Coliforms were no longer detected in all irradiated samples. Results showed that coliforms are sensitive when subjected to above doses of gamma radiation, which also confirms the findings of IAEA (1982) that Escherichia cofi has a low D10 value of 100 to 200 Gy. 90 Effects on sheiistocks of green mussels Irradiated at the Dm value. The effect of the obtained D10 value on the quality characteristics of sheiistocks of green mussels was conducted only in samples with high initial toxin level. The effect of the D10 value was not determined in the samples with low initial toxin level since the obtained D10 values were very high and the samples may not pass the requirement for wholesomeness of irradiated foods. Sheiistocks of green mussels in Phase 1 with high initial toxin level were irradiated at the D10 value after the radiation resistance of the PSP toxins was determined. The D10 value was obtained from the graph to be 28.5 kGy. After irradiation the samples were evaluated in terms of the PSP toxin concentration, physico-chemical characteristics (pH and moisture content), sensory and microblal qualities as shown in Table 16. Results of the SMB method showed that the concentration of PSP toxins of the irradiated samples was reduced from the Initial concentration of 847.01 to 136.92 j±g/100g meat. Irradiation at its Devalue was unable to reduce the PSP concentration to the acceptable limit of 40 pg/100g meat. The initial leve! of PSP toxins in the samples is another factor for dose determination, or the amount of radiation required is dependent on the number of organisms in the population as in the case of bacteria (IAEA, 1982). Sheiistocks of green mussels with high initial level of PSP toxins will still have a higher level of PSP concentration even after the application of the Dio value since only 90% of the toxin is destroyed or 10% is retained. Conversely, if the PSP concentration is low initially, the concentration may reach the acceptable limit when the Dio value is applied. 91 Table 17. Effects of irradiation at the D10 value of 28.5 kGy on the quality characteristics of frozen shellstocks of green mussels with high initial toxin level Quality Characteristic Evaluation 1. Sensory Characteristics 1 1.1 Color Brownish red orange 1.2 Odor • Intensity of characteristic mussel odor Moderate • Presence of stored/fishy odor Perceptible • Presence of burnt-like odor N°ne • Presence of other odor None 2. Microbial Characteristics 2 2.1 Aerobic Plate Count (cfu/g) 1355 3.2 Coliforms (MPN/g) 0 3. Chemical 2 4.1 pH 6.70 4.2 Moisture content (%) 73.40 1 Corresponding descriptors are based on the average of 3 replicates (D, E and F) per treatment as evaluated by 6 trainedpanelists 2 Values are averages of 2 replicates Results of sensory evaluation showed that the sensory characteristics of the samples irradiated at its Dio value were similar to the samples irradiated at different doses of ionizing radiation. This implies that a high dose of 28.5 kGy did not produce any unacceptable sensory qualities in the product provided that the temperature is maintained low, which in this case is not greater than -18°C during irradiation. 92 The samples had a much lower microbial load after subjecting to an irradiation dose of 28.5 kG, as compared with samples irradiated at different doses of 5, 10, 15 and 20 kGy. At its D10 vaiue the aerobic plate count was further reduced to 1355 cfu/g and was negative for collforms. The average pH and moisture content of the samples were 6.70 and 73.4%, respectively. The values were observed to be lower than samples irradiated at different doses of ionizing radiation. Phase 3. Changes In the Fatty Acid Profile of Irradiated Shellstocks of Green Mussels Eleven fatty acid standards were eluted by the gas chromatographic conditions used in the study (Figure 23). The fatty acid standards used for the identification of fatty acids present in the musse! samples were obtained from coconut oil, as this oil contains most of the fatty acids present in mussels. The retention times of the unidentified fatty acids in the mussel samples were compared with the retention time of the standard fatty acids. To quantify the different tentatively identified fatty acids in the sample, exact amounts of the pure standards were injected, and the retention time values were used as reference for that particular fatty acid. The concentration of the fatty acids present in the samples were computed based on the values of the area given by the computer print-out. The areas of all the peaks present were added and the concentration of each fatty acid was computed by dividing the area for each fatty acid by the total area and multiplying by 100. 93 FDD Respbr.se (,?, fa LSutic Myristic 13,7U Palmitic Stearic Oleic Linoleic Arachidic Behenic Figure 23. Chromatogram of standard fatty acids 94 Effect on sheHstocks of green mussels subjected to different doses of ionizing radiation. Frozen samples of unirradiated green mussels consisted of twelve fatty acids. These were identified as lauric acid, myristic acid, palmitic acid, stearic acid, oieic acid, iinoleic acid and six other unidentified fatty acids (Figure 24). Table 18 shows the concentration of the fatty acids present in these samples. Results showed that palmitic acid was the major fatty acid in the frozen samples of mussels with a concentration of 35.2032%, followed by component E, myristic acid, stearic acid, oleic acid and component B. These acids represent the five major fatty acid components in this sample. In samples with high initial toxin level, mussels irradiated at 5 kGy retained most of the fatty acids present in the unirradiated samples of green mussels (Table 18). Arachidic acid was detected along with two unidentified fatty acids. Palmitic acid, however, was still the major fatty acid component of mussels irradiated at 5 kGy with a concentration of 33.0325%, which is slightly lower than the unirradiated mussels. On the other hand, Hnoleic acid was the major component of mussels subjected to 10 kGy of ionizing radiation with a concentration 47.2352 %. Palmitic acid which was previously greater in concentration was reduced to 16.3982% at this dose. At 15 kGy, palmitic acid was no longer detected in the sample. At 20 kGy further reduction in the concentrations of the identified fatty acids was observed. Generally, the concentration of the fatty acids decreased with application of higher doses of ionizing radiation. Figure 24 shows the different fatty acid components of fresh or unirradiated green mussels irradiated at different doses of ionizing radiation. 95 *.«/ l*uric A «•« P*l»itic B ti.tn C • UiMI 01«iC u-vc Olelc Linolelc ""• D •Mtl-I Artchlrflc -» y 13 » I- F (b) (c) (e) F^jure 24. Chromato^am of fatty acid components In frozen sheBstocks of green mussels subjected to different doses of Ionizing radiation with Initially high toxin level: (a) Unlrradiated or control, (b) 5kGy, (c) 10kGy, (d) 15 kGy, and (e) 20kGy 96 Table 18. Tentatively identified fatty acids and their concentrations in the frozen irradiated and unirradiated shelistocks of green mussels in samples with high initial toxin level Name of Type of Sample Fatty Acid Unirradiated Irradiated Mussels (Control) 5 kGy 10 kGy 15 kGy 20 kGY Concen- Concen- Percent Concen- Percent Concen- Percent Concen- Percent 1 1 1 1 tration (%) tration difference tration (%) difference tration (%) difference tration (%) difference (%)1 from from from from Control Control Control Control Laurie 0.8756 2.1804 149.02 — — — — — — Myristic 14.3838 6.2474 (- 56.57) 3.0308 (-78.43) 0.8917 (-93.80) 0.8851 (-93.85) Component 0.8364 0.9675 15.67 0.5239 (-37.36) 11.9154 1324.61 2.1658 158.94 A A — Palmitic 35.2032 33.0325 (-6.17) 16.3982 (-53.42) — — — Component 4.3811 6.0922 39.06 1.3657 (-68.83) 10.7024 144.29 9.3573 113.58 r> ts 9.7589 12.1388 24.39 5.9869 (-38.65) 2.4151 (-75.25) 2.4220 (-75.18) Stearic Olelc 5.8522 4.5075 (-22.98) — — — — — — Linoleic 2.3571 2.2850 (-3.06) 47.2352 1903.95 2.0836 (11.60) 1.9842 (-15.82) Component 2.5462 4.0180 57.80 2.2977 (-9.76) 33.4693 1214.48 34.5107 1255.38 Component 3.4392 1.7920 (-47.89) 11.2309 226.56 7.0763 105.75 7.3055 112.42 D 1 Values iare averages of 2 determinations (-) Means not detected 97 Type of Sample Unirradiated Irradiated Mussels (Control) Name of 5 kGy 10 kGy 15 kGy 20 kGY Fatty Acid Concen- Concen- Percent Concen- Percent Concen- Percent Concen- Percent tration (%) tration difference tration (%) difference tration (%) difference tration (%) difference (%) from from from from Control Control Control Control Arachidic — 3.3580 ND 1.4469 ND — — 0.84424 ND Component 17.2966 8.4975 (-50.87) 3.8682 (-77.64) 10.1721 (-41.20) 10.4411 (-39.63) c 3.2697 3.8572 17.97 1.5714 (-51.94) 7.0763 137.42 — Component — rc — Component 7.0550 ND 1.7737 ND 3.5681 ND 11.7525 ND Component — 3.9702 ND 3.2704 ND 6.9998 ND 3.2472 ND LJ n Component — — — — 3.0601 ND 6.7842 ND i i — — — Component — — — — 3.3937 ND J 1 Values are averages of 2 determinations (-) means not detected ND means not determined as this fatty acid was not detected in the unirradiated mussels 98 In Trial 1 of mussel samples with low initial toxin level, most of the identified fatty acids in the fresh unirradiated samples of musseis were still detected in the samples irradiated at 5 kGy except for oleic acid (Table 19 and Figure 25). Palmitic acid was the major fatty acid found in mussels irradiated at 5 kGy. At this dose, oleic acid was no longer detected, but arachidic acid and two other unidentified fatty acids were instead detected. At 10 kGy, most of the fatty acids detected at 5 kGy were still present at this dose level except for lauric acid. At 15 kGy, palmitic acid was no longer detected. The concentration of most of the previously identified fatty acids were reduced at this dose. At 20 kGy, a slight reduction in the concentration of the fatty acids were observed. Results showed that the concentration of the fatty acids in the irradiated mussels decreased with an increase in the applied dose. In Trial 2 of samples with low initial toxin level, palmitic acid was the major fatty acid component of samples irradiated at 5 kGy with a concentration of 30.1060% (Table 20). Lauric acid which was present \n the frozen unirradiated sample was not detected in mussels subjected to this dose. Arachidic acid, however, was also detected (Figure 26). At 10 kGy the concentration of most fatty acids decreased except for myristic and palmitic acid. At higher doses of 15 and 20 kGy slight changes in the concentration of most fatty acids were observed except for stearic acid where a continuous increase in concentration was noted. 99 ni« c tl-nt g M.fj« Ql«ie I Unolelc ) «1-?U (a) (b) (c) (e) Figire 25. Chromato^am of fatty acid components in frozen sheSstocks of green mussels subjected to different doses of Ionizing radiation wfth Initially low toxin level (Trial 1): (a) Unfrradfeted or control, (b) 5kGy, (c) 10kGy, (d) 15 kGy, and (e) 20kGy 100 Table 19. Tentatively Identified fatty acids and their concentrations in the frozen irradiated and unirradiated shellstocks of green mussels in samples with low initial toxin level (Trial 1) Name of Type of Sample Fatty Acid Unirradiated Irradiated Mussels (Control) 5 kGy 10 kGy 15 kGy 20 kGY Concen- Concen- Percent Concen- Percent Concen- Percent Concen- Percent tration (%)1 tration difference tration (%)1 difference tration (%)1 difference tration (%)1 difference (%)1 from from from from Control Control Control Control Laurie 0.8756 1.2363 41.19 — — — — — — Myristic 14.3838 7.6568 (-46.77) 6.0104 (-58.21) 0.9208 (-93.60) 0.9282 (-93.55) Component 0.8364 1.7162 105.19 1.0390 24.22 11.9934 1333.93 2.1239 153.93 A Palmitic 35.2032 42.3484 20.30 33.3205 (-5.35) — — — — Component 4.3811 4.6647 6.47 2.8766 (-34.34) 10.4461 138.44 9.2731 111.66 n D Stearic 9.7589 10.7735 10.40 12.3533 26.58 2.3870 (-75.54) 2.3369 (-76.05) Oleic 5.8522 — — — — — — — — Llnoleic 2.3571 0.6708 (-71.59) 9.3998 298.77 1.9746 (-16.23) 1.8657 (-20.85) Component 2.5462 5.3093 108.52 4.5565 78.95 33.7087 1223.88 39.1490 1437.55 c 0.6614 (-80.77) 94.70 7.8729 128.92 7.7177 124.40 Component 3.4392 6.6961 D 1 Values are averages of 2 determinations (-) Means none detected 101 Name of Type of Sample Fatty Acid Unirradiated Irradiated Mussels (Control) 5 kGy 10 kGy 15 kGy 20 kGY Concen- Concen- Percent Concen- Percent Concen- Percent Concen- Percent tration (%)1 tration difference tration (%)1 difference tration (%)1 difference tration (%)1 difference (%)1 from from from from Control Control Control Control Arachidic — — — 2.8694 ND — — 0.8248 ND Component 17.2966 2.3351 (-86.41) 7.6710 (-55.65) 10.1128 (-41.53) 10.7692 (-37.74) tr t 3.2697 57.98 3.1234 (-4.47) 6.8111 108.31 4.7759 46.07 Component 5.1654 Component — 10.8314 ND 3.5560 ND 3.4989 ND 11.5376 ND Component — 5.1406 ND 6.5279 ND 7.3145 ND 3.2161 ND H Component — — — — — 2.9591 ND 7.1264 ND i i Component — — — — — — — 3.3554 ND J 2 Values are averages of 2 determinations (-) Means none detected ND Means not determined as this fatty acid was not detected in the unirradiated mussels 102 - (b) (c) (<*) (e) Rgure 26. Chromatop^m of fatty acid components in frozen sfteBstocks of green mussete subjected to dtfferent doses of ionLdng radlatton with biitiaity low toxin level (Trial 2): (a) Untrradfated or control, (b) 5kGy, (c) 10kGy, (d) 15 kGy, and (e) 20kGy 103 Table 20. Tentatively identified fatty acids and their concentrations in the frozen irradiated and unirradiated shellstocks of green musseis in samples with low initial toxin level (Trial 2) Name of Type of Sample Fatty Acid Unirradiated Irradiated Mussels (Control) 5 kGy 10 kGy 15 kGy 20 kGY Concen- Concen- Percent Concen- Percent Concen- Percent Concen- Percent tration (%)1 tration difference tration (%)1 difference tration (%)1 difference tration (%)1 difference (%)1 from from from from Control Control Control Control Laurie 0.8756 — 2.8733 228.16 0.6784 (-22.52) Myristic 14.3838 1.9942 (-86.14) 7.6080 (-47.11) 8.6647 (-39.76) 10.6143 (-26.21) Component 0.8364 0.7930 (-5.19) 0.9174 9.68 0.7970 (-4.71) 1.0314 23.31 A A 33.6823 32.2624 Palmitic 35.2032 30.1060 (-14.48) (-4.32) (-8.35) 38.6786 9.87 Component 4.3811 0.8710 (-80.12) 6.2981 43.76 2.9662 (-32.30) 3.2936 (-24.82) a D Stearic 9.7589 9.6800 (-0.81) 9.9694 2.16 10.5766 8.38 11.5373 18.22 Oteic 5.8522 4.4533 (-23.90) 3.9172 (-33.06) 5.3571 (-8.46) 0.2223 (-96.20) Llnoleic 2.3571 3.0648 30.02 3.2103 36.20 2.7339 15.99 3.2784 39.09 Component 2.5462 3.6836 44.67 9.8184 285.61 2.6089 2.46 3.4750 36.48 /^ Component 3.4392 22.1294 543.45 3.6792 6.98 2.9091 (-15.41) 2.785 (-19.02) D 1 Values are averages of 2 determinations (-) Means none detected 104 Name of Type of Sample Fatty Acid Unirradiated irradiated Mussels (Control) 5 kGy 10 kGy 15 kGy 20 kGY Concen- Concen- Percent Concen- Percent Concen- Percent Concen- Percent tration (%)1 tration difference tration (%)1 difference tration (%)1 difference tration (%)1 difference (%)1 from from from from Control Control Control Control Arachidic — 11.7244 ND 8.5600 ND 3.8472 ND 3.8832 ND Component 17.2966 3.4567 (-80.01) 3.9465 (-77.18) 16.1946 (-6.37) 3.7669 (-78.57) t 146.00 156.69 151.06 Component 3.2697 8.0436 8.3931 8.2090 10.6419 225.47 F 2 Values are averages of 2 determinations (-) Means none detected ND Means not determined as this fatty acid was not detected in the unirradiated mussels 105 Effect on shelistocks of green mussels irradiated at the DID value. The identified fatty acids in the frozen unirradiated green mussels were not detected in the samples irradiated at the computed D10 value of 28.5 kGy such as lauric acid, myristic acid, oleic acid and linoleic acid. The present fatty acid components were mostly unidentified. Palmitic acid, however, was still the major component of this type of sample. Table 20 and Figure 27 shows the fatty acid profile of mussels irradiated at 28.5 kGy. Table 21. Tentatively identified fatty acids and their concentrations in frozen shelistocks of green musseis irradiated at the Dio value of 28.5 kGy in samples with initial high toxin level Name of Fatty Unirradiated Irradiated Mussels Acid (Control) Concentration Concentration Percent (%) (%) difference from Control Lauric 0.8756 — Myristic 14.3838 Component A 0.8364 0.5212 (-37.69) Palmitic 35.2032 50.2049 42.61 Component B 4.3811 1.3110 (-70.08) Stearic 9.7589 13.4524 37.85 Oleic 5.8522 Linoleic 2.3571 Component C 2.5462 4.4006 72.83 Component D 3.4392 3.5710 3.82 Arachidic — 5.0670 ND Component E 17.2966 12.4736 27.88 Component F 3.2697 3.1035 (-5 08) Component G 5.8946 ND ^ Values are averages of 2 replicates (-) Means not detected ND Means not determined as this fatty acid was not detected in the unirradiated mussels 106 FH> Response FID Response- Myristic ti.tu B t3.it Palmitic =— u.n Palmitic 13-tii Stearic Arachidic bo. MS D Figure 27. Chromatogram of fatty acid components in shelistocks of green mussels: (a) unfefadiated or control, (b) irradiated at the D10 value of 28.5 kGy 107 Radiation products are derived from irradiated food constituents an^ the Products increase with applied doses. Results showed the effect of varying levels of gamma irradiation on the concentration of fatty acids in mussels. There Was a decrease in the concentration of most of the fatty acids in mussels as dose level increased. Apparently, these fatty acids were degraded or destroyed to $ome extent by irradiation. Also, there was a concomitant increase in the concentration of the other fatty acids as dose level increased. 108 SUMMARY AND RECOMMENDATIONS The radiation resistance of paralytic shellfish poison (PSP) toxins from Pyrodinium bahamense var. compressum in shellstocks of green mussels was determined after exposure of the shellstocks and of the semi-purified toxin extract to varying doses of ionizing radiation of 5, 10, 15 and 20 kGy. The radiation resistance was measured by determining the Dio value or decimal reduction dose of the PSP toxins. The radiation resistance of PSP toxins from the shellstocks of green mussels was higher as compared to radiation resistance of PSP toxins in the semi-purified toxin extract as shown in the Dio values obtained. The D10 value of the PSP toxins in the shellstocks was 28.5 kGy while the Dio value in the semi- purified toxin extract was 17.5 kGy in samples with high initial level of PSP toxins. Similar observations were obtained when the radiation resistance of the toxins were determined in mussels with low initial PSP toxin level. A much higher Dio value in shellstocks of green mussels was obtained as compared with the D10 value in semi-purified toxin extract. The D-m values in the shellstocks were found to be 57.5 kGy in Trial 1 and 43.5 kGy in Trial 2, while the PSP toxins in the semi-purified toxin extract was 43.0 kGy for Trials 1 and 2. Based on the results, shellstocks with high initial PSP concentration would have a lower Dio value by about 50%, as compared with shellstocks with low initial PSP toxin concentration. Irradiation of frozen shellstocks of green mussels using different doses of ionizing radiation of 5, 10, 15 and 20 kGy did not render the product unacceptable. A brownish discoloration in the irradiated mussels was also observed. However, the only characteristic which was observed in the irradiated samples was the presence of fishy odor which was not detected in the control samples. No burnt odor was detected in the samples even at a high dose of 20 kGy which is usually detected in shrimps even at a low dose of less than 5 kGy. Similar observations were noted in shellstocks irradiated at its D10 value of 28.5 kGy. The microbial load of the samples were remarkably reduced at higher doses of gamma radiation. Changes in the pH and moisture content of the irradiated samples were iess than one unit. Changes in the volatile fatty acid content were observed in the irradiated samples. Generally, a reduction in concentration of the previously identified fatty acids was observed as higher doses of gamma radiation were applied. One limitation of this study is that it was only able to tentatively identify fatty acids present in coconut oil. Behenic and arachidic acid were added as standards as these acids showed the same retention times as the unknown. There were several other fatty acids present in the samples but were not identified due to lack of standards. Further study could be done on this part. Irradiation was not able to totally detoxify mussels of PSP toxins. However, with a much lower toxin content as a result of irradiation, further studies can be conducted In slightly detoxified green mussels by determining 110 which processing method can be used in conjunction with irradiation to be able to utilize PSP affected mussels. Another trial can be performed on PSP contaminated mussels during the bloom of red tide when the toxicity level is high since only one trial was conducted during this season. Determination of PSP toxins in mussels can also be done using receptor binding assay for saxitoxins or by high performance liquid chromatography (HPLC) which are detection methods comparable to the Standard Mouse Bioassay . In the SMB method the detection limits depend on the mouse strain used and commonly ranges from 38 to 58 p.g saxitoxin- equivalents per 100 grams shellfish tissue with accuracy regarded as ± 20% of the calculated value. In the receptor binding assay for PSP toxins, there is competition between the labelled toxin and uniabelled toxin in the sample or standard for specific binding sites for PSP. A standard curve is plotted and the concentration is extrapolated from the standard curve. 111 REFERENCES Acevedo, T.P., Tolentino, E.G. and Bacay, M.C.H. 1993. Effects of Food Processing on toxins from Red Tide in Molluscan Bivalves and Crustaceans. Project No. 09007 NS. UP Office of Research and Coordination. 74 pp. American Council on Science and Health. 1988. 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Effect of irradiation temperature on inactivation of Ctostridium botu&num toxin Type E by gamma rays. Journal of Applied Bacteriology. 32(4):476-480. Licudan, F.L. 1994. All about red tide and paralytic shellfish poisoning. JPMA. 9(3 & 4): 259-265. Maclean, J.L. 1989. An overview of Pyrodinium red tides in the Western Pacific. In Red Tides Biology, Environmental Science, and Toxicology (Okaichi, T., Anderson, D.M. and Nemoto, T., eds.). ICLARM Conference Proceedings 21. Fisheries Department, Ministry of Development, Brunei Darussalam and International Center for Living Aquatic Resources Management, Manila, Philippines. Eisevier Science Publishing Co., Inc. New York, pp. 1-7. Maclean, J.L. 1989. Indo - Pacific red tides, 1985 - 1988. Marine Pollution Bulletin. 20 (7): 304-310. Maclean, J.L. 1984. indo - Pacific red tides. In Toxic Dinoflageliate Blooms (Taylor, 1. and Selinger, H.H., eds.). Eisevier, North Holland, pp. 173-178. Maclean, J.L. 1973. Red tide and paralytic shellfish poisoning in Papua New Guinea. Papua New Guinea Agricultural Journal. 24(4):131-138. 116 Mallett, J.C., Beghian, .E., Metcalf, T.G. and Kaylor, J.D. 1991. Potential of irradiation technology for improved shellfish sanitation. Journal of Food Safety. 11:231-245. Matsuoka, K. 1989. Morphological features of a cyst Pyrodinium bahamense var. compression. in_ Red Tides Biology, Environmental Science, and Toxicology (Okaichi, T., Anderson, D. M. and Nemoto, T., eds.). ICLARM Conference Proceedings 21 Fisheries Department, Ministry of Development, Brunei Darussalam and International Center for Living Aquatic Resources Management, Manila, Philippines. Elsevier Science Publishing Co., Inc. New York. pp. 219-229. Nerker, D.P. and Bandekar, J.R. 1990. Elimination of Salmonella from frozen shrimps by gamma radiation. Journal of Food Safety. 10(3):175-180. Nickerson, J.T.R., Licciardello, J.J. and Ronsivalli, L.J. 1983. Radurization and radicidation: fish and shellfish, in Preservation of Food by Ionizing Radiation._Vol. IIL (Josephson, E.S. and Peterson, M.S., eds.). CRC Press, Inc. Boca Raton, Florida, pp. 15-74. North American Plant Protection Organization. 1996. NAPPQ Standards for Phytosanitary Measures. Napean, Ontario, Canada, pp. 1-15. Oshima, Y. 1989. Toxins in Pyrodinium bahamense var. compressum and infested marine organisms. In Red Tides Biology, Environmental Science, and Toxicology (Okaichi, T., Anderson, D. M. and Nemoto, eds.). ICLARM Conference Proceedings 21. Fisheries Department, Ministry of Development, Brunei Darussalam and International Center for Living Aquatic Resources Management, Manila, Philippines. Elsevier Science Publishing Co., Inc. New York. pp. 73-79. Oshima, Y., Kotaki, Y., Harada, T. and Yasumoto, T. 1984. Paralytic shellfish toxins in tropical waters. ]n Seafood Toxins (Ragelis, E.P., ed.). American Chemical Society, Washington, D.C. pp. 161-170. Pastor, N. I.S., Gopez, I.L., Quizon, M.C., Bautista, N.B., White, F.M., Lofrenco, V.S., Benabaye, R.M., White, M.E. and Dayrit, M.M. 117 1989. Epidemics of paralytic shellfish poisoning in the Philippines, [n Biology, Epidemiology and Management of Pyrodinium Red Tides. Proceedings of the Management and Training Workshop, Bandar Seri Begawan, Brunei Darussalam. 23-30 May 1989. Fisheries Department, Ministry of Development, Brunei Darussalam and International Center for Living Aquatic Resources Management, Manila, Philippines, pp.165-172. PCARRD. 1977. Characteristics of Mussels and Oysters. The Philippine Recommends: Mussels and Oysters. Rose, S.A., Modi, N.K., Trauter, H.S., Bailey, N.E., Stringer, M.F., Hableton, P. 1988. Studies on the irradiation of toxins of Cfostricfium botuHnum and Staphyfococcus aureus. Journal of Applied Bacteriology. 65(3):223-229. Kochhar, S.P. 1996. Oxidative pathways to the formation of off-flavours. In Food Taints and Off-Flavours (Saxby, M. J., ed.). Blackie Academic & Professional. UK. pp. 168-225. Seliger, H.H. 1989. Mechanisms of red tides of Pyrodinium bahamense var. compressum in Papua New Guinea, Sabah and Brunei Darussalam. In Red Tides Biology, Environmental Science, and Toxicology (Okaichi, T., Anderson, D.M. and Nemoto, T., eds.). ICLARM Conference Proceedings 21. Fisheries Department, Ministry of Development, Brunei Darussalam and Internationa! Center for Living Aquatic Resources Management, Manila, Philippines. Elsevier Science Publishing Co., Inc. New York. pp. 53-72. Shimizu, Y. 1987. Chemistry of paralytic shellfish toxins. In Handbook of Natural Toxins: Marine Toxins and Venoms (Tu, A.T., ed.). Marcel Dekker, Inc. New York. Sullivan, JJ. 1988. Methods of analysis for DSP and PSP toxins in shellfish: a review. Journal of Shellfish Research. 7(4):587-595. Taub, I.A., Kaprielian, R.A. and Haltiday, J.W. 1978. Radiation chemistry of high protein foods irradiated at low temperature, [n Food Preservation by Irradiation. Vol. I. IAEA, Vienna, pp. 371-383. 118 Taylor, F.J.R. 1988. Marine toxins of microbiological origin. Food Technology. 42(3):94-98. Taylor, F.J.R. 1985. The taxonomy and relationships of red tide flagellates, [n Toxic Dinflageilates (Anderson, D. M., White, A.W. and Baden, D. G., eds.). Elsevier Science Publishing. New York, pp. 11-26. Taylor, F.J.R. and Fukuyo, Y. 1989. Morphological features of the motile cell of Pyrodinium bahamense. In Biology, Epidemiology and Management of Pyrodinium Red Tides. Proceedings of the Management and Training Workshop, Bandar Seri Begawan, Brunei Darussalam. 23-30 May 1989. Fisheries Department, Ministry of Development, Brunei Darussalam and Internationa! Center for Living Aquatic Resources Management, Manila, Philippines, pp. 207-217. Trainer, V.L., Baden, D.G. and Caterall, W.A. 1996. Brevetoxin and saxitoxin binding to sodium channel transiently expressed in human kidney cells. |n Harmful and Toxic Algal Blooms (Yasumoto, T., Oshima, Y. and Fukuyo, Y., eds.). Proceedings of the Seventh International Conference on Toxic Phytoplankton. Sudai, Japan, pp. 467-470. Urbain, W. M. 1986. Food Irradiation. Academic Press, Inc. London, pp. 83-115. van der Vyver, I. and Pitcher, G. 1997. Red tide and shellfish poisoning. Internet Enviro Facts: Red Tides. 5 pp. Vlllao, R.S. 1988. The red tide menace. Dlllman Review. 36 (6): 15- 20. White, A.T., Anraku, M. and Hooi, K. 1984. Toxic Red Tides and Shellfish Toxicity in Southeast Asia. Proceedings of a consultation meeting held in Singapore. 11-14 Sept. 1984. Southeast Asian Fisheries Development Center and International Development Research Center. Singapore, pp. 10-11. World Health Organization. 1994. Safety nd Nutritional Adequacy of irradiated Food. World Health Organization (WHO). Geneva, pp. 1-161 119 World Health Organization. 1988. Food Irradiation. A Technique for Preserving and Improving the Safety of Food. World Health Organization. Geneva, pp. 18-76. Yasumoto, T., Raj, U. and Bagnis. 1984. Seafood Poisonings in Tropical Regions. Laboratory of Food Hygiene. Faculty of Agriculture. Tohoku University. 24 p. Yentsch, CM. and Incze, L.S. 1980. Accumulation of algal biotoxins in mussels. In Mussel Culture and Harvest. A North American Perspective ( Lutz, R. A., ed.). Developments in Aquacuiture and Fisheries Science 7. Elsevier. pp. 223-246. 120 APPENDICES APPENDIX A Standard Mouse Bioassay for Paralytic Shellfish Toxins (AOAC, 1995, Section 959.08 on Paralytic Shellfish Poison, Biological Method) Materials: a. Paralytic shellfish poison (saxitoxin) standard solution - 100 jug/mL. Available from Division of Contaminants Chemistry, Natural Products and Instrumentation Branch (HFF-423), Food and Drug Administration, 200 C St., SW, Washington, DC 20204, as acidified 20% alcohol solution. Standard is stable indefinitely in cool place. b. Paralytic shellfish poison working standard solution - 1 jig/mL. Dilute 1 mL standard solution to 100 mL with distilled water. Solution is stable several weeks at 3-4°C. c. Mice - Healthy mice, 19-21 g, from stock colony used for routine assay. If < 19 g or > 21 g, apply correction factor to obtain true death time (See Somer's Table, Appendix A). Do not use mice weighing >23 g and do not re- use mice. Standardization of Bioassay: Dilute 10 mL aliquots of 1 ug/mL standard solution with 10, 15, 20, 25 and 30 mL water, respectively, until intraperltoneal injection of 1 mL doses into few test mice causes median death time of 5-7 min. pH of dilutions should be 122 2-4 and must not be > 4.5. Test additional dilutions in 1 mL increments of water, e.g., if 10 mL diluted with 25 ml water kills mice in 5-7 min, test solutions diluted 10+ 24 and 10+ 26. Inject group of 10 mice with each of 2 or preferably 3 dilutions that fall within median death time of 5-7 min. Give 1 mL dose to each mouse by intraperitoneal injection and determine death time as time elapsed from completion of injection to last gasping breath of mouse. Repeat assay 1 or 2 days later, using dilutions prepared above which differed by 1 mL increments of water. Then repeat entire test, starting with testing of dilutions prepared from newly prepared working standard solution. Calculate median death time for each group of 10 mice used on each dilution. If all groups of 10 mice injected with any 1 dilution gave median death time <5 or >7 min, disregard results from this dilution In subsequent calculations. On the other hand, if any groups of 10 mice injected with 1 dilution gave median death time falling between 5 and 7 min, include all groups of 10 mice used on that dilution, even though some of median death times may be <5 or >7 min. From median death time for each group of 10 mice in each of selected dilutions, determine number of mouse units/ml from Sommer's Table. Divide calculated jig poison/1 ml by mouse units/1 mL to obtain conversion factor (CF value) expressing pig poison equivalent to 1 mouse unit. Calculate average of individual CF values, and use this average value as reference point to check routine assays. Individual CF values may vary significantly within laboratory if techniques and mice are not rigidly controlled. This situation will require continued use of working standard or secondary standard, depending on volume of assay work performed. 123 Use of Standard with Routine Assays of Shellfish: Check CF value periodically as follows; If shellfish products are assayed less than once a week, determine CF value on each day assays are performed by injecting 5 mice with appropriate dilution of working standard. If assays are made on several days during a week, only 1 check need to be made each week on dilution of standard such that median death time fails within 5-7 min. CF value thus determined should check with average CF value within ±20%. If it does not check within this range, complete group of 10 mice by adding 5 mice to the 5 mice already injected, and inject second group of 10 mice with same dilution of standard. Average CF value determined for second group with that of first group. Take resulting value as new CF value. Variation of >20% represents significant change in response of mice to poison, or in technique of assay. Changes of this type require change in CF value. Repeated checks of CF value ordinarily produce consistent results within ±20%. If wider variations are found frequently, the possibility of uncontrolled or unrecognized variables in method should be investigated before proceeding with routine assays. Extraction: Weigh 100 g well mixed material into tared beaker. Add 100 ml 0.1N HCI, stir thoroughly and check pH. (pH should be <4.0, preferably about 3.0. If necessary adjust pH as indicated below.) Heat mixture, boil gently 5 min, and let cool to room temperature. Adjust cooled mixture to pH 2.0-4.0 (never >4.5) as determined by BHD Universal Indicator, phenol blue, Congo red paper, or pH meter. To lower pH, add 5N HCI dropwise with stirring; to raise pH, add 0.1N 124 NaOH dropwise with constant stirring to prevent local alkalinization and consequent destruction of poison. Transfer mixture to graduated cylinder and dilute to 200 ml. Return mixture to beaker, stir to homogeneity, and let settle until portion of supernate is translucent and can be decanted free of solid particles large enough to block 26-gauge hypodermic needle. If necessary, centrifuge mixture or supernate 5 min at 3,000 rpm or filter through paper. Only enough liquid to perform bioassay is necessary. Mouse Test: intraperitonealry inoculate each test mouse with 1 mL acid extract. Note time of inoculation and observe mice carefully for time of death as indicated by last gasping breath. Record death time from stopwatch or clock with sweep second hand. One mouse may be used for initial determination, but 2 or 3 are preferred. If death time or median death time of several mice is <5 min, make dilution to obtain death times of 5-7 min. If death time of 1 or 2 mice injected with undiluted sample is >7 min, a total of ^3 mice must be inoculated to establish toxic it y of sample. If large dilutions are necessary, adjust pH of dilution by dropwise addition of dilute HCI (0.1 or 0.01 N) to pH 2.0-4.0 (never >4.5). Inoculate 3 mice with dilution that gives death times of 5-7 min. Calculation of Toxlclty: Determine median death times of mice, including survivors, and from Somer's Table (Appendix A) determine corresponding number of mouse units. If test animals weigh <19 g or >21 g, make correction for each mouse units corresponding to death time for that mouse by weight correction factor for that 125 mouse from Somer's Table; then determine median mouse unit for group. (Consider death time of survivors as >60 min or equivalent to <0.875 mouse unit in caicuiating median). Convert mouse units to jig poison/mL by muitipiying by CF value. ug poison/100 g meat = (ug/ml) x dilution factor x 200 Consider any value >40 ug / 100 g as hazardous and unsafe for human consumption. 126 Table 22. Somer's Table on death time and mouse unit relations for paralytic shellfish poison Death Tme a Mouse Units Death Tme a Mouse Units Death Tme a Mouse Units 1:00 100.00 4:00 2.50 9:00 1.16 10 66.20 05 2.44 30 1.13 15 38.30 10 2.38 20 26.40 15 2.32 10:00 1.11 25 20.70 20 2.26 30 1.09 30 16.50 25 2.21 35 13.90 30 2.16 11:00 1.075 40 11.90 35 2.12 30 1.06 45 10.40 40 2.08 50 9.33 45 2.04 55 8.42 50 2.00 55 1.96 2:00 7.67 5:00 1.92 12:00 1.050 05 7.04 05 1.89 13:00 1.030 10 6.52 10 1.86 14:00 1.015 15 6.06 15 1.83 15:00 1.000 20 5.66 20 1.80 16:00 0.990 25 5.32 30 1.74 17:00 0.980 30 5.00 40 1.69 18:00 0.972 35 4.73 45 1.67 19:00 0.965 40 4.48 50 1.64 20:00 0.960 45 4.26 21:00 0.954 50 4.06 6:00 1.60 22:00 0.948 55 3.88 15 1.54 23:00 0.942 3:00 3.70 30 1.48 24:00 0.937 05 3.57 45 1.43 25:00 0.934 10 3.43 30:00 0.917 15 3.31 7:00 1.39 40:00 0.898 20 3.19 15 1.35 60:00 0.875 25 3.08 30 1.31 30 2.98 45 . 1.28 35 2.88 40 2.79 8:00 1.25 45 2.71 15 1.22 50 2.63 30 1.20 55 2.56 45 1.18 death time expressed in minutes:seconds 127 APPENDIX B DETERMINATION OF pH (AOAC, 1995, Section 973.41) Apparatus and Reagent: pH meter (pH Scan 3) Standard buffer solutions 50 mL beakers Blender Stirring rod Procedure: 1. Standardize instrument with standard buffer with pH near that of sample and then with that of 2 others to check linearity of electrode response. 2. Blend 25 g sample with 25 mL distilled water. 3. Filter mixture through cheesecloth and squeeze out remaining liquid. 4. Stir sample. Analyze sample as soon as possible, preferably within few hours. Do not open sample bottle before analysis. 5. Record pH readings and calculate for average readings of three determinations. 128 APPENDIX C DETERMINATION OF MOISTURE CONTENT (AOAC, 1995 Section 950.46 Air Drying Method) Apparatus: Drying oven Aluminum dish Dessicator Procedure: 1. Weigh 2g sample in a previously tared aluminum dish. 2. Dry sample to constant for 2 to 4 hours in a mechanical convection oven at approximately 125°C. 3. Avoid excessive drying. 4. Cover dish, cool in dessicator and weigh. Report loss in weight as moisture. 129 APPENDIX D AEROBIC PLATE COUMT (USFDABAM, 1995) Apparatus: Petri plates Test tubes Erlenmeyer fiasks incubator set at 35°C Pipettes Media: Butterfield's Phosphate-Buffered Dilution Water Plate Count Agar Sampling Procedure: 1. Sterilize all sampling tools like spoons, scissors, forceps, and knives preferably by autoclaving at 121°C for 15 minutes. Further sterilization for subsequent use may be done by dipping in alcohol and then flaming them. 2. Wash hands with soap and water then disinfect with alcohol. 3. To get mussels from a plastic bag, open sealed PE bag with a sterile scissors. 4. Temper frozen samples for no longer than 15 minutes at room temperature so that sample can be obtained conveniently while not altering the quantity and quality of microfiora present. 5. Asepticalry transfer 50 grams sample to a sterile polyethylene plastic bag. 130 using sterile spoon. 6. Work as fast and as aseptically as possible during sampling. Weigh 50 g mussel meat into a sterile polyethylene plastic bag Add 450 mL Butterfieid's phosphate buffer dilution water Homogenize in stomacher 2 minutes (101 dilution) 4 Transfer 10 mL into a dilution bottle containing 90 mL phosphate buffer dilution blank (102 dilution) 4 Transfer 10 mL into a dilution blank containing 90 mL phosphate buffer dilution blank (1Q3 dilution) Transfer 1 mL aliquot from each dilution of 10 and 10 for lower expected counts, 10'2 and 10'3 for normal routine counts, and even higher for higher expected counts 4 Add approximately 15-20 mL Plate Count Agar tempered to 45°C Mix contents carefully 4 Let agar solidify. Incubate inverted plates at 35°C for 48 hours 4 Count colonies with 25-250 colonies per plate Report results in col/g of sample Figure 28. Schematic diagram for determination of Aerobic Plate Count in shellstocks of green mussels 131 APPENDIX E COLIFORtWS (USFDA BAM, 1995) Apparatus: Test tubes Durham tubes Erlenmeyer flasks Pipettes Incubator set at 35°C Media: Butterfleld's Phosphate Buffer Lauryl suffate Tryptose (LST) Broth Brilliant Green Lactose Bile (BGLB) Broth Sampling Procedure: 1. Sterilize all sampling tools like spoons, scissors, forceps, and knives preferably by autoclaving at 121°C for 15 minutes. Further sterilization for subsequent use may be done by dipping In alcohol and then flaming them. 2. Wash hands with soap and water then disinfect with alcohol. 3. To get mussels from a plastic bag, open sealed PE bag with a sterile scissors. 132 4. Temper frozen samples for no longer than 15 minutes at room temperature so that sample can be obtained conveniently while not altering the quantity and quality of microflora present. 5. Asepticalry transfer 50 grams sample to a sterile polyethylene plastic bag using sterile spoon. 6. Work as fast and as asepticalry as possible during sampling. 133 Sample (50 g) Add 450 mL Butterfield's phosphate buffer dilution blank Homogenize 2 minutes (10"1) Transfer 10 mL to erienmeyer flask containing 90 mL phosphate buffer dilution blank(102 dilution) Transfer 10 mL to erienmeyer flask containing 90 mL phosphate buffer dilution blank (103) Transfer 1 mL aliquots to 3 Lauryl Sulfate Tryptose (LST) tubes for each 3 consecutive dilutions 10-1 sample dilution 10-2 sample dilution 10-3 sample dilution 4 i i v 1mL 1mL 1mL 1mL 1mv 1m L 1m• L 1^r mL 1mL LST tube Incubate 24-48 ±2 hours at 35°C No gas production Gas proauction Negative Positive Transfer a loopful to BGLB broth Incubate 24-48 ±2 hours at 35° C i , No gas production Gas production Y Y Negative Positive 4 Determine count using MPN table Report as MPN/g Figure 29. Schematic diagram for determination of Coliforms in shelistocks of green mussels 134 APPENDIX F SENSORY EVALUATION OF FROZEN IRRADIATED SHELLSTOCKS OFGREEN MUSSELS (FDC Report No. S-980813) 1. Sensory Test: Attribute Rating Test 1.1 The following color descriptors are used: • Bright red orange • Dull red orange 1.2 The following odor descriptors are used: • Intensity of characteristic mussel odor • Presence of stored/fishy odor • Presence of burnt-like odor • Presence of other off-odor 2. Panel 2.1 Type of panel: trained/experienced laboratory panel 2.2 Number of panelists: 6 3. Sample Preparation 3.1 Method of preparing the sample for sensory test a. The frozen sample is removed from the package and the content is washed thoroughly with coid water until all ice crystals that can be seen of felt is removed. The sample is then allowed to drain for two (2) minutes. b. For each code, about 250 grams of irradiated mussels is placed in a casserole with about 4 cups of water. The sample is boiled in a covered casserole for about six (6) minutes. 3.2 Amount of sample per panelist: 4 pieces mussels with about 70 mL broth are placed in a covered glass container 135 3.3 Serving temperature of sample: 60°C 3.4 Coding: Three digit number randomly chosen from the "Table of Random Numbers" are used to code the containers of the samples. 4. Method of Sample Evaluation by the Panelists 4.1 Individual sensory panel booths are used by each panelist for independent judgment. 4.2 The following instructions are given to the panelists: a. Remove the cover from the glass and as soon as the cover is removed, smell immediately. • Smell in short sniffs of about 2 to 3 seconds using both nostrils • Sniff one to two times • Keep mouth closed while sniffing b. Evaluate the odor of the sample according to the descriptors in Section 1. The intensity of the odor is rated according to Table 22 as: none, perceptible, slight, moderate, strong. Record results on the scoresheet. c. Rest for at least 30 seconds in between evaluation of successive samples. d. Hands should have no lotion/perfume or any other kind of smell. e. Panelists are also instructed not to taste the sample. 5. Method of Data Analysis The intensity rating for color descriptor is analyzed by frequency while for odor descriptor a numerical score is assigned by the analyst as defined in Table 22. The numerical score for intensity is averaged. The average numerical score for intensity is reported in terms of its equivalent definition in Table 22. 136 Table 23. Scoring system for the intensity of odor descriptors For a positive characteristic For a negative descriptor Intensity Score Definition Intensity Score Definition Rating Rating Strong 5 Very readily None 5 Not present detectable Moderate 4 Readily Perceptible 4 Just detectable, detectable not readily and re- recognizable, cognizable, weak distinct Slight 3 Detectable Slight 3 Detectable and and re- recognizable, cognizable, weaK Perceptible 2 Just detect- Moderate 2 Readily able, not Detectable and readily re- recognizable, cognizable distinct None 1 Not present Strong 1 Very readily Detectable and recognizable, very distinct 137 APPENDIX G DESCRIPTIVE SCORESHEET FOR STEAMED GREEN MUSSELS 1 Name: Date: Instruction: You are given colled samples of mussels. Please check the characteristic that best describes the samples and then check if sample Is acceptable (marketabie ) or not acceptable (unmarketable). Color bright red orange dull red orange others (specify) Acceptable (marketable) Not acceptable (unmarketable) Discoloration none perceptible * slight moderate extensive Acceptable (marketable) Not acceptable (unmarketable) 138 Intensity of Characteristic Odor of Mussels strong moderate slight * perceptible none Acceptable (marketable) Not acceptable (Unmarketable) Presence of Stored/Fishv Odor none perceptible * slight moderate strong Acceptable (marketable) Not acceptable (marketable) Not acceptable (unmarketable) Presence of Burnt Odor none perceptible slight I moderate strong 139 Acceptable _; (marketable) Not acceptable (unmarketable) Presence of Ammoniacal Odor or Other Off-odor Indicative of Spoilage None Perceptible • slight 2IH ""._" IT moderate strong Acceptable (marketable) Not acceptable (unmarketable) means still at marketable level Thank you! 1 The scoresheet for mussels was developed at the Food Development Center using trained panelists. 140 APPENDIX H DETERMINATION OF CRUDE FAT IN SEAFOODS (AOAC, 1995, Section 948.15, Acid Hydrolysis Method) Apparatus: 50 mL beaker Stirring rod Watch glass Steam bath Mojonnier fat-extraction flask with glass stopper Reagents: Concentrated hydrochloric acid Absolute ethanol Petroleum ether Procedure: 1. Weigh 8 g well mixed sample into 50 mL beaker and add 2 mL HCI. 2. Using stirring rod with extra large flat end, break up coagulated lumps until mixture is homogeneous. 3. Add another 6 mL HCI, mix, cover with watch glass, and heat on steam bath for 90 minutes, stirring occasionally with rod. 4. Cool solution and transfer to Mojonnier fat-extraction flask. 5. Rinse beaker and rod with 7 mL absolute ethanol, add to extraction flask, and mix. 141 6. Rinse beaker and rod with 25 mL ether, add in 3 portions, add rinsings to extraction flask, stopper, and shake vigorously for 1 minute. 7. Add 25 mL petroleum ether to extraction fiask, and repeat vigorous shaking. 8. Distili in a fat distillation set-up. 9. To recover fat subject Soxhlet fiask to a rotatory evaporator and transfer fat to a glass vial with cover. 142 APPENDIX I FATTY ACIDS IN OILS AND FATS (AOAC, 1995, Section 969.33, Preparation of Methyl Esters - Boron Triflouride Method) Apparatus: Reaction flasks - 50 and 125 mL flasks with outer joints Condenser - Water-cooled, reflux, with 20 - 30 cm jacket and inner joint Reagents: Boron triflouride reagent - 125 g BF3/L MeOH Methanolic sodium hyrdoxide solution- 0.5N. Dissolve 2 g NaOH in 100 mL MeOH. Heptane Saturated sodium chloride solution Preparation: 1. Weigh approximately 350 mg fat sample in a reaction flask obtained in Appendix I. 2. Add 6 mL methanolic NaOH solution and boiling chip. Attach condenser, and reflux until fat globules disappear (usually 5-10 minutes). 3. Add 7 mL BF3 solution from bulb or automatic pipet through condenser and continue boiling for 2 minutes. 4. Add 2 mL heptane thru condenser and boil 1 minute longer. 143 5. Remove heat, then condenser, and add several mL saturated NaCI solution to float heptane solution into neck of flask. 6. Transfer approximately 1 mL upper heptane solution to a glass via! with cover. 7. Store sample in a freezer until analyzed for fatty acids using a gas chromatograph. 144 APPENDIX J PSP Toxin Concentration In irradiated Shellstocks of Green Mussels (Sample A) with High Initial Toxin Level Date of Sampling: July 4, 1998 Dose Mice Death time PSP applied Replicate Weight Mouse Time Mouse Dilution concentration 1 (kGy) (g) unit (minutes: unit factor (jig poison/ seconds) 100 g meat) Control 1 16.5 0.860 4:39 2.11997 1:9 729.27 2 17.2 0.890 3:45 2.71000 1:9 964.76 5 A- 1 19.5 0.985 6:34 1.46700 1:9 578.00 A-2 19.1 0.973 6:59 1.39300 1:9 542.16 B- 1 18.4 0.946 4:42 2.07200 1:9 784.04 B-2 18.5 0.950 5:31 1.73500 1:9 659.30 C- 1 19.5 0.985 4:20 2.26000 1:9 890.44 C-2 15.9 0.834 4:22 2.24000 1:9 747.26 10 A- 1 18.3 0.942 20:1 0.95940 1:9 361.50 A-2 18.3 0.942 22:35 0.94470 1:9 355.96 B- 1 19.1 0.973 19:20 0.96400 1:9 375.19 B-2 15.2 0.795 15:36 0.99400 1:9 316.09 C- 1 20.2 1.006 14:54 1.00150 1:9 403.00 C-2 20.1 1.003 13:15 1.02625 1:9 411.73 15 A- 1 20.8 0.994 17:54 0.97568 1:8 349.14 A- 2 20.4 1.012 18:15 0.97095 1:8 353.74 B- 1 17.0 0.880 18:44 0.96892 1:8 306.95 B-2 17.1 0.885 19:52 0.96240 1:8 306.62 C-1 16.9 0.876 15:52 0.99130 1:8 312.62 C-2 14.5 0.760 17:32 0.97744 1:8 267.43 20 A- 1 13.3 0.690 15:49 0.99180 1:6 191.62 A-2 14.0 0.730 16:38 0.98620 1:6 201.58 B- 1 14.1 0.736 13:54 1.01650 1:7 239.41 B-2 15.0 0.785 19:54 0.96230 1:7 241.73 C- 1 16.0 0.840 13:4 1.02900 1:7 276.60 C-2 16.5 0.860 15:45 0.99250 1:7 273.14 1 Dilution factor is volume of toxin : volume of distiiled water 145 APPENDIX K PSP Toxin Concentration In Irradiated Semi-Purified Toxin Extract (Sample B) In Samples with High initial Toxin Leve! Date of Sampling: July 4,1998 Dose Mice Death time PSP applied Replicate Weight Mouse Time Mouse Dilution concentration 1 (kGy) (g) unit (minutes: unit factor (jxg poison/ seconds) 100 g meat) Control 1 16.5 0.860 4:39 2.11997 1:9 729.27 2 17.2 0.890 3:45 2.71000 1:9 964.76 5 A- 1 18.6 0.954 2:53 3.95400 150.885 A-2 16.9 0.876 2:46 4.22000 147.869 B- 1 17.7 0.915 2:15 6.06000 221.796 B-2 19.5 0.985 2:15 6.06000 238.764 C- 1 22.2 1.054 2:18 5.82000 245.371 C-2 19.6 0.988 3:18 3.23800 127.966 10 A- 1 20.0 1.000 2:20 5.66000 226.400 A-2 17.0 0.880 2:20 5.66000 199.232 B- 1 18.3 0.942 2:13 6.24400 235.274 B-2 17.5 0.905 2:18 5.82000 210.681 C- 1 16.9 0.876 3:20 3.19000 111.778 C-2 17.6 0.910 3:25 3.08000 112.112 15 A-1 19.8 0.994 2:23 5.45600 216.930 A-2 19.9 0.997 2:10 7.54400 300.855 B- 1 18.6 0.954 2:25 5.32000 203.011 B-2 18.8 0.962 2:10 6.52000 250.890 C-1 16.6 0.864 2:00 7.67000 265.075 C-2 17.7 0.915 2:19 5.74000 210.084 20 A-1 17.7 0.915 3:30 2.98000 109.068 A-2 17.6 0.910 3:00 3.70000 134.680 B- 1 16.6 0.864 2:33 4.83800 167.201 B-2 21.5 1.040 3:50 3.57000 148.512 C- 1 16.6 0.864 3:45 2.71000 93.660 C-2 17.6 0.910 3:34 2.87100 104.510 1 Dilution factor is volume of toxin volume of distilled water 2 (-) means not applicable 146 APPENDIX L PSP Toxin Concentration In Irradiated Shelistocks of Green Mussels (Sample A) with Low initial Toxin Level (Trial 1) Date of Sampling: October 31, 1998 Dose Mice Death time PSP concentration applied Repiicafe Weight Mouse Time Mouse (ng poison/ (kGy) (g) unit (minutes: unit 100 g meat) seconds) Control 1 16.7 0.868 5:04 1.8960 65.8300 2 16.5 0.860 6:05 1.5800 54.3520 5 A-1 13.6 0.706 6:02 1.5920 44.9581 A-2 14.1 0.736 6:39 1.4500 42.6880 B- 1 16.1 0.844 6:12 1.5520 52.4000 B-2 16.1 0.844 6:00 1.6000 54.0200 C- 1 15.0 0.785 5:10 1.8600 58.4000 C-2 15.1 0.790 5:30 1.7400 54.9840 10 A-1 14.6 0.765 6:06 1.5760 48.2256 A-2 16.6 0.864 6:44 1.4330 49.5244 B- 1 17.6 0.910 9:00 1.1600 42.2240 B-2 17.6 0.910 8:30 1.2000 43.6800 C- 1 12.1 0.626 5:39 1.6950 42.4428 C-2 14.0 0.730 6:39 1.4500 42.3400 15 A-1 17.9 0.925 18:50 0.9685 35.8345 A-2 18.7 0.958 26:00 0.9306 35.6606 B-1 18.1 0.934 15:39 0.9935 37.1172 B-2 18.1 0.934 18:50 0.9685 36.1832 C- 1 16.5 0.860 11:51 1.0530 36.2232 C-2 20.1 1.003 26:11 0.9302 37.3196 20 A- 1 12.0 0.620 15:32 0.9947 24.6686 A-2 12.5 0.650 15:09 0.9985 25.9610 B- 1 12.2 0.632 9:12 1.1490 29.0467 B-2 12.8 0.665 9:50 1.1167 29.7042 C-1 12.5 0.650 13:42 0.9579 26.5070 C-2 12.9 0.670 20:35 0.9694 25.6717 147 APPENDIX M PSP Toxin Concentration in Irradiated Semi-Purified Toxin Extract (Sample B) In Samples with Low Initial toxin Level (Trial 1) Date of Sampling: October 31, 1998 Dose Mice Death time PSP concentration applied Replicate Weight Mouse Time Mouse (jig poison/ (kGy) (g) unit (minutes: unit 100 g meat) seconds) Control 1 16.7 0.868 5:04 1.8960 65.8300 2 16.5 0.860 6:05 1.5800 54.3520 5 A- 1 17.4 0.900 24:20 0.9364 32.7104 A-2 17.8 0.920 19:15 0.9642 35.4844 B-1 15.9 0.834 11:11 1.0699 35.6935 B-2 16.1 0.844 13:34 1.0200 34.4352 C-1 15.7 0.822 11:17 1.0665 35.0665 C-2 16.1 0.844 13:34 1.0200 34.4352 10 A- 1 14.8 0.775 15:42 0.9930 30.7830 A-2 15.2 0.795 14:41 1.0048 31.9510 B- 1 14.5 0.760 18:41 0.9691 29.4616 B-2 14.5 0.780 13:57 1.0158 30.8788 C- 1 14.7 0.770 11:10 1.0698 32.9498 C-2 13.7 0.712 18:43 0.9677 27.5601 15 A- 1 14.7 0.770 17:45 0.9764 30.0731 A-2 14.8 0.775 25:44 0.9325 28.9075 B- 1 14.0 0.730 17:13 0.9789 28.5856 B-2 13.7 0.712 18:59 0.9677 27.5649 C- 1 14.0 0.730 17:13 0.9780 28.5576 C-2 15.0 0.785 18:00 0.9720 30.5208 20 A-1 15.0 0.785 20:00 0.96Q0 30.1440 A-2 15.0 0.785 21:00 0.9540 29.9556 B- 1 14.5 0.760 21:00 0.9540 29.0016 B-2 15.0 0.785 23:00 0.9420 29.5788 C-1 13.1 0.680 16:20 0.9880 26.8736 C-2 14.0 0.730 15:05 0.9992 29.1766 148 APPENDIX N PSP Toxin Concentration In irradiated Shellstocks of Green Mussels (Sample A) with Low initial Toxin Level (Trial 2) Date of Sampling: November 21, 1998 Dose Mice Death time PSP concentration applied Replicate Weight Mouse Time Mouse (fig poison/ (kGy) (g) unit (minutes: unit 100 g meat) seconds) Control 1 18.6 0.954 5:34 1.7200 65.6400 2 18.0 0.930 5:15 1.8300 68.0760 5 A- 1 15.5 0.810 8:28 1.2030 38.9772 A-2 15.5 0.810 11:47 1.0543 34.1593 B- 1 14.4 0.754 17:15 0.9788 29.5206 B-2 14.3 0.748 17:15 0.9788 29.2856 C- 1 14.0 0.730 11:45 1.0550 30.8060 C-2 14.4 0.754 8:26 1.2050 36.3428 10 A- 1 13.9 0.724 10:21 1.0960 31.7402 A-2 14.0 0.730 14:53 1.0018 29.2526 B- 1 14.8 0.775 10:33 1.0885 33.7435 B-2 13.2 0.685 11:01 1.0745 29.4413 C- 1 15.0 0.785 14:00 1.0150 31.8710 C-2 15.0 0.785 13:45 1.0188 31.9714 15 A- 1 16.3 0.852 15:00 1.0000 34.0800 A-2 16.5 0.860 21:00 0.9540 32.8176 B- 1 14.0 0.730 15:12 0.9980 29.1400 B-2 14.0 0.730 11:20 1.0650 31.1000 C- 1 13.1 0.680 10:00 1.1100 30.1920 C-2 13.5 0.700 9:20 1.1400 31.9200 20 A-1 14.8 0.775 20:00 0.9600 29.7600 A-2 14.3 0.748 18:00 0.9720 29.0822 B- 1 13.6 0.706 15:16 0.9973 28.1638 B-2 13.2 0.685 15:36 0.9940 27.2356 C-1 14.1 0.736 20:00 0.9600 28.2624 C-2 14.0 0.730 20:00 0.9600 28.0320 149 APPENDIX O PSP Toxin Concentration In Irradiated Semi-Purified Toxin Extract (Sample B) in Samples with Low Initial Toxin Level (Trial 2) Date of Sampling: November 21, 1998 Dose Mice Death time PSP concentration applied Replicate Weight Mouse Time Mouse Oig poison/ (kGy) (g) unit (minutes: unit 100 g meat) seconds) Control 1 18.6 0.954 5:34 1.7200 65.6400 2 18.0 0.930 5:15 1.8300 68.0760 5 A-1 17.4 0.900 7:09 1.3060 49.1760 A-2 16.4 0.856 7:22 1.3310 45.5734 B- 1 16.4 0.856 8:07 1.2340 42.2522 B-2 17.2 0.890 9:31 1.1290 40.1924 C- 1 17.0 0.880 9:10 1.1500 40.4800 C-2 17.3 0.895 9:30 1.1300 40.4540 10 A- 1 16.4 0.856 14:10 1.0125 34.6680 A-2 18.2 0.938 15:53 0.9912 37.1898 B- 1 13.5 0.700 17:02 0.9798 27.4344 B-2 13.3 0.690 22:08 0.9459 26.1068 C- 1 14.5 0.760 15:16 0.9973 30.3179 C-2 14.8 0.775 14:56 1.0001 31.0031 15 A- 1 12.7 0.660 12:42 1.0360 27.3504 A-2 14.1 0.736 13:15 1.0262 30.2113 B- 1 14.6 0.765 9:12 1.1480 35.1288 B-2 14.3 0.748 16:03 0.9897 29.6118 C- 1 15.0 0.785 16:00 0.9900 31.0860 C-2 15.0 0.785 18:00 0.9720 30.5208 20 A-1 14.6 0.765 16.53 0.9847 30.1318 A-2 14.9 0.780 18:21 0.9705 30.2796 B- 1 15.0 0.785 23:00 0.9420 29.5788 B-2 15.0 0.785 24:00 0.9355 29.3747 C-1 15.0 0.785 25:00 0.9340 29.3276 C-2 15.6 0.816 25:00 0.9340 30.4858 150 APPENDIX P CHEMICAL QUALITY OF FROZEM IRRADIATED SHELLSTOCKS OF GREEN MUSSELS type of Treatment Dose Replicate pH reading Moisture content sample applied (%) (kGy) Per analysis Average 1 Per analysis Average 1 usseis Unirradiated None 1 6.25 6.24 78.45 78.5 h high (Control) 2 6.23 78.56 iial toxin irradiated D-1 7.77 7.76 77.5 77.5 level D-2 7.78 77.6 E- 1 7.75 77.6 E-2 7.74 77.5 F- 1 7.77 77.4 F-2 7.75 77.4 10 D-1 6.78 6.75 77.3 77.4 D-2 6.75 77.5 E-1 6.73 77.4 E-2 6.75 77.8 F- 1 6.77 77.4 F-2 6.74 77.3 15 D-1 6.77 6.77 77.3 77.4 D-2 6.75 77.6 E-1 6.79 77.3 E-2 6.78 77.4 F- 1 6.77 77.7 F-2 6.78 77.4 20 D-1 6.78 6.78 77.4 77.2 D-2 6.79 77.1 E-1 6.77 77.3 E-2 6.77 77.2 F- 1 6.78 77.1 F-2 6.77 77.0 Values are averages of 6 determinations per treatment for the irradiated samples 151 pe of Treatment Dose Replicate pH reading Moisture content mple applied (%) (kGy) Per analysis Average 1 Per analysis Average 1 ial 1 Unirradiated None 1 6.25 6.25 77.4 77.5 ssels (Control) 2 6.25 77.6 h low if toxin Irradiated D- 1 6.76 6.77 76.8 76.9 jvei D-2 6.77 76.9 E- 1 6.77 77.0 E-2 6.76 76.9 F- 1 6.78 77.1 F-2 6.77 76.8 10 D- 1 6.76 6.76 76.6 76.7 D-2 6.75 76.8 E-1 6.78 76.9 E-2 6.76 76.7 F-1 6.75 76.6 F-2 6.78 76.8 15 D-1 6.77 6.76 76.6 76.5 D-2 6.76 76.4 E- 1 6.75 76.5 E-2 6.76 76.6 F- 1 6.77 76.6 F-2 6.74 76.5 20 D- 1 6.80 6.80 76.9 76.8 D-2 6.79 76.8 E- 1 6.81 76.8 E-2 6.79 76.7 F- 1 6.80 76.8 F-2 6.81 76.7 Values are averages of 6 determinations per treatment for the irradiated samples 152 ype of Treatment Dose Replicate pH reading Moisture content ;ample applied (%) (kGy) Per analysis Average 1 Per analysis Average 1 Trial 2 Unirradiated None 1 6.23 6.22 78.8 78.8 lussets (Control) 2 6.22 78.8 >ith low Sial toxin Irradiated D- 1 6.77 6.78 77.6 77.5 level D-2 6.76 77.5 E-1 6.78 77.5 E-2 6.79 77.4 F-1 6.78 77.5 F-2 6.77 77.3 10 D-1 6.77 6.77 77.5 77.3 D-2 6.76 77.3 E- 1 6.76 77.4 E-2 6.78 77.3 F- 1 6.76 77.2 F-2 6.77 77.3 15 D- 1 6.79 6.79 77.4 77.6 D-2 6.80 77.6 E- 1 6.79 77.7 E-2 6.79 77.5 F- 1 6.78 77.7 F-2 6.79 77.6 20 D- 1 6.77 6.78 77.5 77.5 D-2 6.77 77.5 E-1 6.79 77.5 E-2 6.78 77.4 F-1 6.78 77.6 F-2 6.77 77.6 Values are averages of 6 determinations per treatment for the irradiated samples 153 APPENDIX Q AEROBIC PLATE COUNT OF FROZEN IRRADIATED AND UNIRRADIATED SHELLSTOCKS OF GREEN MUSSELS Type of Treatment Dose Replicate Aerobic Plate Count (cfu/g) sample applied Per analysis Average 1 (kGy) Mussels with Unirradiated None 1 4.2 x 10* 4.3x10* high initial (Control) 2 4.4 x 104 toxin level Irradiated 5 1 2.1 x 103 2x103 2 1.9 x103 10 1 9.8 x102 9.9 x102 2 9.9 X 102 15 1 6.2 x102 6.2 x102 2 6.3 x102 20 1 3.4 X 102 3.6 X 102 2 3.6 x 102 Trial 1 Unirradiated None 1 4.5x10* 4.4 x 104 (Mussels with (Control) 2 4.4x104 low initial toxin level) Irradiated 5 1 4.1 x103 4.2 x 103 2 4.3 x109 10 1 3.3 x103 3.3 x103 2 3.3 X 103 15 1 4.6 x102 4.7 x 102 2 4.7 x102 20 1 2.5 x102 2.7 x 102 2 2.8 x102 Trial 2 Unirradiated None 1 1.8 x104 1.8 x104 (Mussels with (Control) 2 1.7x10* low Initial toxin level) Irradiated 5 1 4x103 4x103 2 4.1 X103 10 1 2.4 X103 2.6 x 103 2 2.7 x 103 15 1 3.6 x102 3.6 x102 2 3.6 x102 20 1 2.5 x 102 2.6 X102 2 2.6 x 102 1 Values are averages of 2 determinations per treatment 154 APPENDIX R COLIFORMS IN FROZEN IRRADIATED AND UNIRRADIATED SHELLSTOCKS OF GREEN MUSSELS For 3 fermentation tubes at each of 1, 0.1, and 0.01 g inoculates, the MPNs and 95 percent confeidence intervals for results that would be among the 99.985 percent most likely if their own MPNs were the actual bacterial concentrations (USFDA BAM, 1995). Type of Treatment Dose Replicate Positive tubes MPN/g Confidence limit sample appied 1 0.1 0.01 Low High (key) Missels tvfth Unirradiated None f 2 0 0 0.918 0.144 3.75 high initial (Control) 2 0 0 0 0.918 0.144 3.75 toxin level irradiated 5 1 0 0 0 0 0 0.950 2 0 0 0 0 0 0.850 10 1 0 0 0 0 0 0.950 2 0 0 0 0 0 0.950 15 1 0 0 0 0 0 0.950 2 0 0 0 0 0 0.950 20 1 0 0 0 0 0 0.950 2 0 0 0 0 0 0.850 Trial 1 Unirradiated None 1 0 1 0 0.305 0.015 1.07 (Mussels with (Control) 2 0 2 0 0.619 0.124 1.81 tow initial toxin level) Irradiated 5 1 0 0 0 0 0 0.950 2 0 0 0 0 0 0.950 10 1 0 0 0 0 0 0.850 2 0 0 0 0 0 0.950 15 1 0 0 0 0 0 0.950 2 0 0 0 0 0 0.950 20 1 0 0 0 0 0 0.950 2 0 0 0 0 0 0.950 Trial 2 Unirradiated None 1 0 0 1 0.301 0.015 0.960 (Mussels with (Control) 2 0 1 1 0.611 0.124 1.81 low initial toxin level) Irradiated 5 1 0 0 0 0 0 0.950 2 0 0 0 0 0 0.950 10 1 0 0 0 0 0 0.850 2 0 0 0 0 0 0.950 15 1 0 0 0 0 0 0.950 2 0 0 0 0 0 0.950 20 1 0 0 0 0 0 0.950 2 0 0 0 0 0 0.650 155 APPENDIX S SENSORY EVALUATION OF IRRADIATED SHELLSTOCKS OF GREEN MUSSELS WITH HIGH INITIAL TOXIN LEVEL A. Irradiation dose applied: S kGy 1. Intensity of Characterise Mu»ei Odor Code Panelist No. 1A IB 1C 1 3 3 4 2 4 4 4 3 4 4 4 4 4 4 4 5 4 3 4 6 4 4 4 Sum of Scores: 23 22 24 Avenge Score: 3.8 3.7 4.0 Corresponding Descriptor Moderate Moderate Moderate 2. Pretence of Stored/Fishy Odor Code Panelist No. 1A IB 1C 1 4 3 3 2 4 4 4 3 4 4 4 4 4 4 4 5 4 4 3 6 4 4 4 Sum of Scores: 24 23 22 Average Score: 4.0 3.8 3.7 Corresponding Descriptor Perceptible Perceptible Perceptible 156 3. Pretence of Burnt-Like Odor Code Panelist No. 1A IB 1C 1 5 5 5 2 4 4 5 3 5 5 5 4 5 5 5 5 5 3 5 6 5 5 5 Sum of Scores: 29 27 30 4.8 4.5 5.0 Average Score: Corresponding Descriptor: None None None 4. Presence of Other Off-Odor Code Panelist No. 1A IB 1C 1 5 5 5 2 5 5 5 5 L_ 5 5 4 5 5 5 5 5 5 5 6 5 5 5 Sum of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corresponding Descriptor: None None None Summary of Results: Intensity Rating Odor Descriptor Code 1A IB 1C 1. Intensity of Characteriilic Mussels Odor Moderate Moderate Moderate 1. Presence of Stored/Fishy Odor Perceptible Perceptible Perceptible 3. Pretence of Burnt-Like Odor None None None 4. Presence of Other Off- Odor None None None 157 B. Irradiation dose applied: 10kGy 1. Intensity of Characteristic Massd Odor Code Panelist No. 2A 2B 2C 1 3 4 3 2 4 4 4 3 4 4 4 4 4 4 4 5 4 4 4 6 4 4 4 Sum of Scores: 23 24 23 Average Score: 3.8 4.0 3.8 Corresponding Descriptor: Moderate Moderate Moderate 2. Presence of Stored/fishy Odor Code PaneHrt No. 2A 2B 2C 1 4 4 4 2 4 4 4 3 4 4 4 4 4 3 3 5 3 4 4 6 4 4 4 Sum of Scores: 23 23 23 Average Score: 3.8 3.8 3.8 Corresponding Descriptor: Perceptible Perceptible Perceptible 158 3. Presence of Bnmt-Like Odor Code Panelist No. 2A 2B 2C 1 5 5 5 2 5 5 4 3 5 5 5 4 5 5 5 5 5 5 5 6 5 5 5 Sum of Scores: 30 30 29 5.0 5.0 4.8 Average Score: Corresponding Descriptor: None None None 4. Presence of Other Off-Odor Code Panelist No. 2A 2B 2C 1 5 5 5 2 5 5 5 3 5 5 5 4 5 5 5 5 5 5 5 6 5 5 5 Sum of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corresponding Descriptor: None None None Summary of Results: Intensity Rating Odor Descriptor Code 2A 2B 2C 1. Intensity of Characteristic Mussels Odor Moderate Moderate Moderate 2. Presence of Stored/Fishy Odor Perceptible Perceptible Perceptible 3. Presence of Bumt-Like Odor None None None 4. Presence of Other Off- Odor None None None 159 C. Irradiation dose applied: 15 kGy 1. Intensity of CharartcrigdcMDMri Odor PaneHrtNo. 3A 3B 3C • - • ——- . 1 • — . - . —1 —.... — , . wm^m_ 4 ——*" '-'' ——•—-— 4 4 4 4 4 4 L 4 4 — — •-» —,—— u 4 - —— _ 3 '— • —— 4 Stan of Scores: 4 4 23 •Score: — 11 3.8 3.227 3.8 23 -—• ——, ——, ponding Descriptor: Moderate Moderate Moderate " """" ' —-'• - i •<— Z. Pmence of Stoi Code Panelist No. Smn of Scores: Average Score: Corrwponding Best >tor: Perceptible Perceptible Perceptible 160 3. Presence of Bumt-Lfite Odor Code Panelist No. 3A 3B 3C 1 5 5 5 2 5 5 5 3 5 5 4 4 5 5 5 5 5 5 4 6 5 5 5 Sum of Scores: 30 30 28 5.0 5.0 4.7 Average Score: Corresponding Descriptor: None None None 4. Presence of Other Off-Odor Code Panelist No. 3A 3B 3C 1 5 5 5 2 5 5 5 3 5 5 5 4 5 5 • «; 5 5 5 5 6 5 5 5 Sum of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corresponding Descriptor: None None None Summary of Results: RESULT SENSORY ATTRIBUTE 3A 3B 3C Odor Intensity 1. Intensity of Characteristic Mussels Odor Moderate Moderate Moderate 1. Presence of Stored/Fishy Odor Perceptible Perceptible Perceptible 3. Presence of Burnt-Iike Odor None None None 4. Presence of Other Off- Odor None None None 161 D. Irradiation dose applied: 20 kGy 1. Intensity off Characteristic Mmi«t Odor Code Panelist No. 4A 4B 4C 1 3 3 3 2 4 3 3 3 4 4 3 4 4 4 4 5 3 4 4 6 4 3 3 Sum of Scores: 22 21 20 Average Score: 3.7 3.5 3.3 Corresponding Descriptor: Moderate Moderate Slight 2. Presence of Stored/fflshy Odor Code Panelist No. 4A 4B 4C 1 4 3 3 2 4 4 4 3 4 4 4 4 4 4 4 5 4 4 4 6 4 4 4 Sum. of Scores: 24 23 23 Average Score: 4.0 3.8 3.8 Corresponding Descriptor: Perceptible Perceptible Perceptible 162 3. Presence of Burnt-Like Odor Code Panelist No. 4A 4B 4C 1 5 5 5 2 5 5 5 3 5 5 4 4 5 4 5 5 4 5 5 6 5 4 4 Sum of Scores: 29 28 28 Average Score: 4.8 4.7 4.7 Corresponding Descriptor: None None None 4. Presence of Oiher Off-Odor Code Panelist No. 4A 4B 4C 1 5 5 5 2 5 5 5 3 5 5 5 4 5 5 5 5 5 5 5 6 5 5 5 Sum of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corresponding Descriptor: None None None Summary of Remits: RESULT SENSORY ATTRIBirrE 4A 4B 4C Odor Intensity 1. Intensity of Characteristic Mussels Odor Moderate Moderate sliejit 2. Presence of Stored/Fishy Odor Perceptible Perceptible Perceptible 3. Presence of BnmtUke Odor None None None 4. Presence of Other Off- Odor None None None 163 APPENDIX T SENSORY EVALUATION OF IRRADIATED SHELLSTOCKS OF GREEN MUSSELS WITH LOW INITIAL TOXIN LEVEL (TRIAL 1) A. Irradiation dose applied: 5 kGy 1. Intensity of Characteristic Mwie> Odor Code Panelist No. 1A IB 1C 1 4 4 4 2 4 4 4 3 4 4 4 4 4 4 4 5 5 5 5 6 4 4 4 Sum of Scores: 25 25 25 Average Score: 4.2 4.2 4.2 Corresponding Descriptor: Moderate Moderate Moderate 2. Presets of Stored/EJthy Odor Code Panelist No. 1A IB 1C 1 5 5 5 2 4 4 4 3 4 4 4 4 3 3 3 5 4 4 4 6 5 5 5 Sam of Scores: 25 25 25 Average Score: 4.2 4.2 4.2 Corresponding Descriptor: Perceptible Perceptible Perceptible 164 Presence of Bamt-LIke Odor Code Panelist No. 1A IB 1C 1 5 5 5 2 5 3 5 3 4 4 4 4 5 5 5 5 5 5 5 6 5 5 5 Sum of Scores: 29 27 29 Average Score: 4.8 4.5 4.8 Corresponding Descriptor: None None None 4. Presence of Other Off-Odor Code Panelist No. 1A IB 1C 1 5 5 5 2 5 5 5 3 5 5 5 4 5 5 5 5 5 5 5 6 5 5 5 Sum of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corresponding Descriptor: None None None Summary of Results: INTENSITY RATING ODOR DESCRIPTOR CODE 1A IB 1C 1. Intensity of Characteristic Mussel Odor Moderate Moderate 2. Presence of Stored/Fishy Odor Perceptible Perceptible Perceptible 3. Presence of BurnMJke Odor None None None 4. Presence of Other Off- Odor None None None 165 B. irradiation dose applied: 10 kGy 1. Intensity of Characteristic Mwstd Odor Code Panelist No. 4A 4B ' 4C 1 3 3 3 2 4 4 4 3 4 4 4 4 4 4 5 5 5 5 5 6 4 4 4 Sma of Scores: 24 24 25 Average Score: 4.0 4.0 4.2 Corresponding; Descriptor: Moderate Moderate Moderate 2. Presence of Stored/Ifafay Odor Code Panelist No. 4A 4B 4C 1 4 5 5 • 2 . 4 5 5 • •• 3 3 3 3 4 4 4 4 5 5 5 5 6 3 3 3 Sum of Scores: 23 25 25 Average Score: 3.8 4.2 4.2 ,, Corresponding; Descriptor: Perceptible Perceptible Perceptible 166 3. Presence of Burnt-Like Odor Code Panelist No. 4A 4B 4C 1 5 5 5 1 4 5 5 3 4 5 5 4 5 5 5 5 5 5 5 6 5 5 5 Sum of Scores: 28 30 30 Average Score: 4.7 5.0 5.0 Corresponding Descriptor: None None None 4. Presence of Other Off-Odor Code Panelist No. 4A 4B 4C 1 5 5 5 2 5 5 5 3 5 5 5 4 5 5 5 S 5 5 5 6 5 5 5 Sum of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corresponding Descriptor: None None None Summary of Results: INTENSITY RATING ODOR DESCRIPTOR CODE 4A 4B 4C 1. Intensity of Characteristic Mussel Odor Moderate Moderate Moderate 2. Presence of Stored/Fishy Odor Perceptible Perceptible Perceptible 3. Presence of Bumt-Llke Odor None None None 4. Pretence of Other Off- Odor None None None 167 C. Irradiation dose applied: 15kGy 1. Intensity of Characteristic Mussel Odor Code Panelist No. 3A 3B 3C 1 3 4 4 2 4 3 4 3 4 4 4 4 5 3 4 5 3 4 5 6 4 4 4 Sam of Scores: 23 22 25 Average Score: 3.8 3.7 4.2 Corresponding Descriptor: Moderate Moderate Moderate 2. Presence of StondMsby Odor Code Panelist No. 3A 3B 3C 1 3 4 5 2 4 4 4 3 3 3 3 4 4 4 4 5 3 3 5 6 5 5 4 Sum of Scores: 22 23 25 Average Score: 3.7 3.8 4.2 Corresponding Descriptor: Perceptible Perceptible Perceptible 168 3. Presence of Burnt-Like Odor Code Panelist No. 3A 3B 3C 1 5 5 5 2 4 4 5 3 4 4 5 4 5 5 5 5 5 5 5 6 4 4 5 Sran of Scores: 27 27 30 Average Score: 4.5 4,5 5.0 Corresponding Descriptor: None None None 4. Pretence of Other Off-Odor CODE Panelist No. 3A 3B 3C 2 5 5 5 2 5 5 5 3 5 5 5 4 5 5 5 5 5 5 5 6 5 5 5 Sum of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corresponding Descriptor: None None None Summary of Results: — -• INTENSITY RATING ODOR DESCRIPTOR CODS 3A 3B 3C 1. Intensity of Characteristic Maud Odor Moderate Moderate Moderate 2. Presence of Stored/Fishy Odor Perceptible Perceptible Perceptible 3. Presence of BurnMJke Odor None None None 4. Presence of Other Off- Odor None None None 169 0. Irradiation dose applied: 20 kGy 1. Intensity of Characteristic Mussel Odor Code Panelist No. 2A 213 2C 1 4 4 3 2 4 4 3 3 4 4 4 4 4 4 3 5 5 4 3 6 4 4 4 Sam of Scores: 25 •24 20 Average Score: 4.2 4.0 3.3 Corresponding Descriptor: Moderate Moderate SHfcht 2. Presence of Stored/Fishy Odor Code Panelist No. 2A 2B 2C 1 5 4 4 2 4 4 4 3 4 3 3 4 3 4 4 5 4 3 3 6 4 5 5 Sum of Scores: 24 23 23 Average Score: 4.0 3.8 3.8 Corresponding Boamptor. Perceptible Perceptible Perceptible 170 3. Presence of Burnt-Lfke Odor Code Panelist No. 2A 2B 2C 1 5 5 5 2 3 5 5 3 5 4 4 4 5 5 5 5 5 5 5 6 5 4 4 Sum of Scores: 28 28 28 Average Score: 4.7 4.7 4.7 Corresponding Descriptor: None None None 4. Presence of Other Off-Odor Code Panelist No. 2A 2B 2C 1 5 5 5 2 5 5 5 3 5 5 5 4 5 5 5 5 5 5 5 6 5 5 5 Sam of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corretrpondinj; Descriptor None None None Summary of Results: INTENSITY RATING ODOR DESCRIPTOR CODE 2A 2B 2C 1. Intensity of Characteristic Mussel Odor Moderate Moderate SKsht 2. Presence of Stored/Fishy Odor Perceptible Perceptible Perceptible 3. Presence of Bnmt-Uke Odor None None None 4. Presence of Other Off- Odor None None None 171 APPENDIX U SENSORY EVALUATION OF IRRADIATED SHELLSTOCKS OF GREEN MUSSELS WITH LOW INITIAL TOXIN LEVEL (TRIAL 2) A. Irradiation dose applied: 5 kGy 1. Intensity of Characteristic Massel Odor Code Panelist No. 1A IB 1C 1 3 3 4 2 4 4 4 3 4 4 4 4 4 4 4 5 4 3 4 6 4 4 4 Sum of Scores: 23 22 24 Average Score: 3.8 3.7 4.0 Corresponding Descriptor: Moderate Moderate Moderate 2. Presence of Stored/Fishy Odor Code Panelist No. 1A IB 1C 1 4 3 3 2 4 4 4 3 4 4 4 4 4 4 4 5 4 4 3 6 4 4 4 Sum of Scores: 24 23 22 Average Score: 4.0 3.8 3.7 Corresponding Descriptor: Perceptible Perceptible Perceptible 172 3. Presence of Burnt-Like Odor Code Panelist No. 1A 18 1C Sum of Scores: 29 27 30 4.8 4.5 5.0 Average Score: Corresponding Descriptor. None None None 4. Presence of Other Off-Odor Code Panelist No. 1A IB 1C Sum of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corresponding Descriptor: None None None Summary of Results: Intensity Rating Odor Descriptor Code 1A IB 1C 1. Intensity of Characteristic Mussels Odor Moderate Moderate Moderate 2. Presence of Stored/Fishy Odor Perceptible Perceptible Perceptible 3. Presence of Barnt-Uke Odor None None None 4. Presence of Other Off- Odor None None None 173 B. irradiation dose applied: 10 kGy 1. Intensity of Characteristic Mussel Odor Code Panelist No. 2A 2B 2C 1 3 4 3 2 4 4 4 3 4 4 4 4 4 4 4 5 4 4 4 6 4 4 4 Sam of Scores: 23 24 23 Average Score: 3.8 4.0 3.8 Corresponding Descriptor Moderate Moderate Moderate 2. Presence of Stored/Fishy Odor Code Panelist No. 2A 2B 2C 1 4 4 4 2 4 4 4 3 4 4 4 4 4 3 3 5 3 4 4 6 4 4 4 Snm of Scores: 23 23 23 Average Score: 3.8 3.8 3.8 Corresponding Descriptor: Perceptible Perceptible Perceptible 174 3. Pretence of Bnntt-IJke Odor Code Panelist No. 2A 28 2C 1 5 5 5 2 5 5 4 3 5 5 5 4 5 5 5 5 5 5 5 6 5 5 5 Sum of Scores: 30 30 29 5.0 5.0 4.8 Average Score: Corresponding Descriptor: None None None 4. Presence of Other Off-Odor Code Panelist No. 2A 2B 2C 1 5 5 5 2 5 5 5 3 5 5 5 4 5 5 5 5 5 5 5 6 5 5 5 Sum of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corresponding Descriptor: None None None Summary of Results: Intensity Rating Odor Descriptor Code 2A 2B 2C 1. Intensity of Characteristic Mussels Odor Moderate Moderate Moderate 2. Presence of Stored/Fishy Odor Perceptible Perceptible Perceptible 3. Presence of Bnrnt-Iike Odor None None None 4. Presence of Other Off- Odor None None None 175 C. irradiation dose applied: 15 kGy 1. Inimsity off Characteristic MnstdQdor Code Panelist Ne. 3A 3B 3C 1 3 3 4 2 4 4 4 3 4 4 4 4 4 4 4 5 4 3 3 6 4 4 4 Sam of Scores: 23 22 23 Average Scare: 3.8 3.7 3.8 Corresponding Descriptor: Moderate Moderate Moderate 2. Plregmce of Sto.sd/Ffofay Odor Code Panelist No. 3A 3B 3C 1 4 3 4 2 4 4 4 3 4 4 4 4 4 3 3 5 4 4 4 6 4 4 4 Sum of Sc©r*s: 24 22 23 Average Score: 4.0 3.7 3.8 Corresponding Descriptor: Perceptible Percept&le Perceptible 176 3. Presence of BmmMJke Odor Code Panelist No. 3A 3B 3C 1 5 5 5 2 5 5 5 3 5 5 4 4 5 5 5 5 5 5 4 6 5 5 5 Sum of Scores: 30 30 28 5.0 5.0 4.7 Average Score: Corresponding Descriptor: None None None 4. Presence of Other Off-Odor Code Panelist No. 3A 3B 3C Sum of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corresponding Descriptor: None None None Summary of Result*: RESULT SENSORY ATTRIBUTE 3A SB 3C Odor Intensity 1. ietmsKy of Characteristic Mussels Odor Moderate Moderate Moderate 2. Presence of Stored/Fishy Odor Perceptible Perceptible Percept&le 3. Presence of Burst-Like Odor None None None 4. Preseace of Other Off- Odor None None None 177 D. Irradiation dose applied: 20 kGy 1. Ihteraity of Characterise MosseS Odor Code Panelist No. 4A 4B 4C 1 3 3 3 2 4 3 3 3 4 4 3 4 4 4 4 5 3 4 4 6 4 3 3 Sam of Scores: 22 21 20 Average Score: 3.7 3.5 3.3 Corresponding Descriptor: Moderate Moderate Slight 1. Pmmce of Stored/Fbhy Odor Code PaneHstNo. 4A 4B 4C 1 4 3 3 2 4 4 4 3 4 4 4 4 4 4 4 5 4 4 4 4 4 4 Sam of Scores: 24 23 23 Average Score: 4.0 3.8 i_ 3.8 Corresponding Descriptor: Perceptible Perceptible Perceptible 178 3. Presence of Bnmt-Lfke Odor Code Panelist No. 4A 4C 1 5 5 5 2 5 5 5 3 5 5 4 4 5 4 5 5 4 5 5 6 5 4 4 Swa of Scores: 29 28 28 Average Score: 4.8 4.7 4.7 Corresponding Descriptor: None None None 4. Presence of Ofliar Off-Odor Code Panelist No. 4A 4® 4C 1 5 5 5 2 5 5 5 3 5 5 5 4 5 5 5 5 5 5 5 6 5 5 5 Sum of Scores: 30 30 30 Average Score: 5.0 5.0 5.0 Corresponding Descriptor: None None None of Retails: RESULT SENSORY ATTRIBUTE 4A 4B 4C Odor Intensity 1. Intensity of Characteristic Massels Odor Moderate Moderate sHefxt 2. Presence of Stored/Fishy Odor Perceptible Perceptible Perceptible 3. Presence of Bnmt-Uke Odor None None None 4. Presence of Other Off- Odor None None None 179