STATUS OF CIGUATERA IN THE PHILIPPINES NATIONAL FISHERIES RESEARCH AND DEVELOPMENT INSTITUTE, QUEZON CITY, PHILIPPINES

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1 STATUS OF CIGUATERA IN THE PHILIPPINES NATIONAL FISHERIES RESEARCH AND DEVELOPMENT INSTITUTE, QUEZON CITY, PHILIPPINES by BRYAN E. TANYAG, FLORDELIZA D. CAMBIA & DR. ULYSSES M. MONTOJO Seafood Quality and Safety Section Fisheries Post Harvest Research and Development Division

2 WHAT IS CIGUATERA? Sea Food Poisoning Ciguatoxins Heat stable and lipid soluble polyether compounds Gambierdiscus toxicus

3 EPIDEMIOLOGY Globally 50, ,000 cases per year In the Pacific Islands, there are 40,000 reported CFP cases in seventeen (17) countries

4 PHILIPPINES Archipelagic Belongs to the so called Pacific Coral Triangle Gambierdiscus sp. Prorocentrum sp. Ostreopsis sp.

5 PHILIPPINES Experienced in three major islands of the country (Luzon, Visayas and Mindanao): Palawan in 2011 Iloilo in Panay Island in 2011 (Mendoza, 2013) Basilan in 1989 Month/Year Province No. of Cases Implicated fishes 1989 Basilan, Mindanao 19 Lethrinus sp., Pagellus sp., Gymnocamius sp. Spratilloides, Scarus sp. Sept-Nov 1997,1998 June 2001 Romblon, Luzon 32 Lutjanus sp., Lethrinus sp., Scarus sp., Stolephorus sp., Pagellus sp. Navotas, Metro Manila 50 Barracuda Antique, Visayas 33 Barracuda June 2006 Ilo-ilo, Visayas 5 Lutjanus sp. October 2008 Masbate, Luzon 30 Tanguigue, Rumpe, Malasugi June 2010 Ilo-ilo, Visayas 22 Lutjanus sp. June 2011 Ilo-ilo, Visayas 4 Lutjanus sp. July 2011 Ilo-ilo, Visayas 11 Lutjanus sp. Off waters of Palawan, Luzon xx Barracuda August 2011 Aklan, Visayas 21 Barracuda September 2011 Ilo-ilo, Visayas 3 Lutjanus sp. Sources: DA-BFAR, DOH; as reported in local news

6 SCREENING OF POSSIBLE CIGUATERA AND CIGUATERA- LIKE TOXINS IN SIX GENERA OF REEF FISHES FROM WEST PHILIPPINE SEA AND SULU SEA TANYAG, B.E., OPINION, A.G.R., PERELONIA, K.B.S., CAMBIA, F.D., & MONTOJO, U.M. Followed the method described by Lewis (1995) in the detection of ciguatoxins. Reported the presence of assumed P-CTX-1 from the area. Parrot fish have the highest toxicity followed by rabbit fish and snapper. Prorocentrum spp. have the highest cell density count among the benthic dinoflagellates observed (Gambierdiscus sp. & Ostreopsis spp.)

7 PHILIPPINES Affected the development, growth, and stability of reef fish trade Banning of grouper (1998) and barracuda (2001) No regulatory limit for Ciguatoxin US-FDA proposed 0.01 ppb safety level for CTX

8 HOW TOXINS ARE DETECTED? LC/MS-MS Cell Based Assay Receptor Binding Assay (RBA) MOUSE BIOASSAY

9 COMPARISON OF METHODS IN THE DETECTION OF CIGUATOXINS USING ANIMAL ASSAY TANYAG, B.E., PERELONIA, K.B.S., CAMBIA, F.D., MONTOJO, U.M. Objectives: Compare the two methods described by Lewis (1995) and Oshiro et al. (2010) in the detection of Ciguatoxins Determine the most accurate and reliable method in the analysis of Ciguatoxins Optimize the method used in the detection of Ciguatoxins

10 SAMPLE COLLECTION Palawan Island in Luzon Sphyraena sp. and Gymnothorax sp.

Diethyl ether (1:1 v:v) Ether solubles (contains the CTXs) 2x water solubles Acetone solubles Acetone insolubles

11 EXTRACTION PROCEDURE Fish Tissue Sample (>50g) LEWIS (1995) X 70 C water bath (cook ~15mins) Methanol-water solubles N-Hexane solubles Acetone[3:1 v:w (ml:g)] Vacuum filter the acetone slurry 2x Ethanol-water (1:3) at ~50mL per 100g tissue sample + Diethyl ether (1:1 v:v) Ether solubles (contains the CTXs) 2x water solubles Acetone solubles Acetone insolubles Chloroform-methanol (97:3) Methanol-water (9:1) at ~50mL per 100g tissue sample + hexane (1:1 v:v) 2x 5% Tween 60/0.9% saline solution X MBA

12 EXTRACTION PROCEDURE OSHIRO, et al. (2010) Fish Tissue Sample (240g) Homogenized with 350mL acetone 2x X Hexane (50mL) + Methanolwater (9:1, 25mL) Acetone insolubles Acetone solubles Methanol-water solubles Extracted with 100mL of diethyl ether 2x 1% Tween 60/0.9% saline solution MBA Water solubles Ether solubles X

13 MOUSE BIOASSAY LEWIS, 1995 Inject 0.5 ml (40g/ml) of suspension prepared to mice (intraperitoneal route) = NON-TOXIC = NON-TOXIC = TOXIC Note: Alive Dead If two (2) or three (3) mice died, the sample is considered toxic

14 MOUSE BIOASSAY OSHIRO ET AL., 2010 Inject 1ml (20g/ml) of suspension prepared to mice (intraperitoneal route) = NON-TOXIC Inject the remaining 1ml of the suspension Non-toxic = TOXIC Toxic Note: Alive Dead

15 DIFFERENCES BETWEEN THE TWO METHODS LEWIS (1995) OSHIRO et al. (2010) Cooking of samples Ethanol-water Higher amount of fish tissues required Higher amount of solvents required Chloroform-methanol

16 FINDINGS Out of forty seven (47) specimens examined, no positive samples were detected using the method described by Lewis (1995). Nine (9) positive specimens were detected using the method described by Oshiro (2010).

17 FINDINGS Twenty three percent (23%) of the total Sphyraena sp. (barracuda) tested positive with ciguatoxins. On the other hand, twelve percent (12%) of the Gymnothorax sp. (moray eel) were detected to have toxins.

18 FINDINGS The toxic manifestations exhibited by the dying mice were same as to what the Food and Agriculture Organization (2004) has stated namely body weakness, gasping or dyspnoea, diarrhoea, convulsion and slow motility. However, negative specimens also manifested the same symptoms, implying that reef fish samples may contain low concentration of the toxin that is not enough to kill a mice to be considered as toxic.

19 FINDINGS The method described by Oshiro et al. (2010) is more sensitive in detecting ciguatoxins than the method described by Lewis (1995). All positive specimens were detected by this method. Most negative specimens still manifested the symptoms described by FAO (2004) implying that the method used may not be suitable in detecting low concentrations of this type of toxin. The method may be reliable in the initial detection/screening of the toxin in fish species but limited to semi-quantification of CTX.

20 ON-GOING & FUTURE ACTIVITIES Establish baseline information of Ciguatera on five commercially important reef fishes such as barracuda, grouper, parrot fish, rabbit fish, and snapper. Enhance the method of detection of ciguatoxins using LC/MS- MS (equipment is for delivery by fourth quarter of 2018) Propose a regulatory limit of ciguatoxin in the country (hopefully).

21 THANK YOU FOR LISTENING!

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