한수지 50(6), , 2017 Original Article Korean J Fish Aquat Sci 50(6), ,2017 마비성패류독소검출을위한분석법비교 이가정 * 권순재 정연중 손광태 하광수 1 목종수 1 김지회 2 국립수산과학원남동해수

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1 한수지 50(6), , 2017 Original Article Korean J Fish Aquat Sci 50(6), ,2017 마비성패류독소검출을위한분석법비교 이가정 * 권순재 정연중 손광태 하광수 1 목종수 1 김지회 2 국립수산과학원남동해수산연구소, 1 국립수산과학원식품위생가공과, 2 국립수산과학원연구기획과 Comparison of Analytical Methods for the Detection of Paralytic Shellfish Toxins (PSTs) Ka Jeong Lee*, Soon Jae Kwon, Yeoun Joong Jung, Kwang Tae Son, Kwang Soo Ha 1, Jong Soo Mok 1 and Ji Hoe Kim 2 Southeast Sea Fisheries Research Institute, National Institute of Fisheries Science, Tongyeong 53085, Korea 1 Food Safety and Processing Research Division, National Institute of Fisheries Science, Busan 46083, Korea 2 Research and Development Planning Division, National Institute of Fisheries Science, Busan 46083, Korea Paralytic shellfish toxins (PSTs) are produced by marine dinoflagellate phytoplankton Alexandrium spp. and Gymnodinium spp. These toxins accumulate in filter feeding organisms such as bivalves and the ingestion of contaminated shellfish can cause illness in humans. The mouse bioassay (MBA) has been the preferred PST testing method worldwide for more than 50 years. However, this assay has several disadvantages, such as detection limits, non-toxicprofiles, and the ethical issues of using animals. The aim of this study was to establish an alternative to the MBA method for testing for PSTs. We optimized the analysis conditions of a post-column oxidation-high performance liquid chromatography (PCOX-HPLC) method and the Scotia Rapid Test Kit, and then compared the accuracy of these methods to the MBA method. The results demonstrated a strong correlation between the PCOX-HPLC method and the MBA, although the PCOX-HPLC method required expensive equipment and standard material, and was time consuming. The Scotia Rapid Test Kit promises to be a useful tool, as it provided rapid and qualitative results, although the method sometimes gave a false positive result that could not be explained by toxin profiles. Key words: Paralytic shellfish toxins (PSTs), Mouse bioassay (MBA), Scotia Rapid Test Kit, Post-column oxidation- HPLC (PCOX-HPLC) 서론 (Paralytic Shellfish Toxins, PSTs) Alexandrium spp., Gymnodinium spp. (filter feeding) (Thomas et al., 2006).,, (NIFS, 2016) (Fig. 1).,. AOAC official method (OM) (mouse bioassay; AOAC MBA) (AOAC, 2005), 40 µg/100 g STX equivalent (equiv.) 50 (Schantz et al., 1958)., (Hess et al., 2006), (Park et al., 1986).,. AOAC (Association of analytical communities) HPLC Korean J Fish Aquat Sci 50(6) , December 2017 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 31 October 2017; Revised 16 November 2017; Accepted 8 December 2017 *Corresponding author: Tel: Fax: address: kajlee@korea.kr Copyright 2017 The Korean Society of Fisheries and Aquatic Science 669 pissn: , eissn:

2 670 이가정ㆍ권순재ㆍ정연중ㆍ손광태ㆍ하광수ㆍ목종수ㆍ김지회 pre-column oxidation-hplc postcolumn oxidation-hplc (PCOX-HPLC) (AOAC Official method) (AOAC, 2011)., (US National shellfish sanitation program, NSSP) PCOX-HPLC Scotia rapid test (NSSP, 2005)., (Mytilus galloprovincialis), (Crassostrea gigas), (Ruditapes philippinarum) (Styela clava), PCOX-HPLC (Scotia Rapid Test Kit). 재료및방법 시료채취및운반 (Mytilus galloprovincialis), (Crassostrea gigas), (Ruditapes philippinarum) (Styela clava), , , psu. 시약및표준물질 (analytical grade, Merck, Darmstadt, Germany) 0.1 N,, PCOX-HPLC. PCOX-HPLC Song et al. (2013), NEO (neosaxitoxin), STX (saxitoxin), dcstx (decarbamoylsaxitoxin), GTX1 (gonyautoxin-1), GTX2 (gonyautoxin-2), GTX3 (gonyautoxin-3), GTX4 (gonyautoxin-4), GTX5 (gonyautoxin-5), dcgtx2 (decarbamoylgonyautoxin-2), dcgtx3 (decarbamoylgonyautoxin-3), C1 (N-sulfocarbamoylgonyautoxin-C1) C2 (N-sulfocarbamoylgonyautoxin-C2) NRC (Halifax, Canada)., Scotia rapid testing Ltd. (Halifax, Canada). 마우스시험 (KMFDS, 2015) 1 ml g ICR Sommer s Table. No. of analytes (n) Fig. 1. Number of analytes for paralytic shellfish toxins ( ). PCOX-HPLC 시험 PCOX-HPLC AOAC (2011) Song et al. (2013),., HPLC fluorescence detector e FLR Detector (Waters, MA, USA), detector excitation wavelength 330 nm, emission wavelength 390 nm. Post-column reaction system postcolumn derivatizer (Waters, MA, USA) 80. GTXs/STXs column % A 0.8 ml/ min, C1 C2 column % C 0.8 ml/min. column GTX STX ZorbaxBonus-RP (Agilent, CA, USA, T, mm, 3.5 µm), C1 C2 Phenosphere-NEXT, (Phenomenex, CA, USA, 00G-4307-E0, mm, 5 µm). single point calibration peak saxitoxin µg/100 g STX equiv.. 면역시험키트시험 Scotia buffer positive ( ) negative ( ). 결과및고찰 마우스시험법 Year 4

마비성패류독소분석법비교 671 PCOX-HPLC. 52 10, 6 7,. 7 85-627 µg/100 g STX equiv.., (Lee et al., 2006). 6 42-57 µg/100 g STX equiv., 7 42-94 µg/100 g STX equiv.. (filter feeding) (Kim et al., 1998),.

3 마비성패류독소분석법비교 671 PCOX-HPLC , 6 7, µg/100 g STX equiv.., (Lee et al., 2006) µg/100 g STX equiv., µg/100 g STX equiv.. (filter feeding) (Kim et al., 1998),. PCOX-HPLC 법 Post column oxidation HPLC (PCOX-HPLC). 52,,, GTX2, %, GTX1,4 1-48%, STX 18-39%, (Table 1)., C1 C2. PCOX-HPLC (r 2 ) (Fig. 3). 40 µg/100 g STX equiv. PCOX- HPLC. PCOX-HPLC,, (Rourke et al., 2008).,. 면역시험키트법, PCOX-HPLC. Scotia rapid test Jellet rapid test. polyclonal PSP toxin antibodies (lateral flow immunochromatography) (A) (B) Fig. 2. Results of Scotia rapid test strips. C, control line; T, test line; A, Negative (not detected); B, Positive(detected). MBA (μg/100 g STX equiv.) y = x R 2 = MBA HPLC PCOX-HPLC (μg/100 g STX equiv.) Fig. 3. Correlation between mouse bioassay and post-column oxidation-high performance liquid chromatography (HPLC) method µg/100 g STX equiv.., running buffer 35. test line ( T line) control line ( C ) (, positive) (, negative). Fig. 2, test line, (Cefas, 2007). table 1.,, false positive ( ) false negative ( ).,

4 672 이가정ㆍ권순재ㆍ정연중ㆍ손광태ㆍ하광수ㆍ목종수ㆍ김지회 Table 1. Comparison of MBA, PCOX-HPLC methods and Scotia rapid kit for the detection of paralytic shellfish toxins Sample Mussel Oyster Short neck Clam PSTs Toxicity by MBA 1 (µg/100 g STX equiv.) Scotia Rapid Kit (µg/100 g STX equiv.) PSTs concentration by PCOX-HPLC % Molar fraction Total STX 3 dc-stx 4 NEO 5 GTX1,4 6 GTX2,3 7 GTX5 8 dc-gtx2,3 9 1 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

5 마비성패류독소분석법비교 673 Table 1. Continued Sample Warty Sea Squirt PSTs Toxicity by MBA 1 (µg/100 g STX equiv.) Scotia Rapid Kit (µg/100 g STX equiv.) PSTs concentration by PCOX-HPLC % Molar fraction Total STX 3 dc-stx 4 NEO 5 GTX1,4 6 GTX2,3 7 GTX5 8 dc-gtx2,3 9 1 ND ND ND ND ND ND ND ND ND ND ND ND ND MBA, Mouse Bioassay; 2 ND, Not detected; 3 STX, Saxitoxin; 4 dc-stx, Decarbamoylsaxitoxin; 5 NEO, Neosaxitoxin; 6 GTX1,4, Gonyautoxin 1,4; 7 GTX2,3, Gonyautoxin 2,3; 8 GTX5, Gonyautoxin 5; 9 dc-gtx2,3, decarbamoylgonyautoxin 2,3; 10 -, negative (not detected); 11 +, positive (detected). false positive, µg/100 g STX equiv. 2 false negative., false positive false negative. false positive false negative negative. STX, GTX2,3, C1,2, GTX5 dcstx, NEO GTX1,4 (Laycock et al., 2001), STX (100%), dcstx (30%), GTX2,3 (60%), GTX5 (40%), C1,2 (33%), dcgtx2,3 (7%), NEO (20%) GTX1,4 (2%) (Jellet et al., 2002)., GTX2, GTX µg/100 g STX equiv., STX 19.9 µg/100 g STX equiv., GTX1, µg/100 g STX equiv. (Cefas, 2007). 2 false negative GTX2,3 (53-55%) 23.3 µg/100 g STX equiv.., positive negative. 분석방법의비교,, PCOX-HPLC. PCOX-HPLC, PCOX- HPLC,,.,, 80 µg/100 g STX equiv., positive negative., false positive, 40 µg/100 g STX equiv. false positive (Cost et al., 2009; Oshiro et al., 2009).,, PCOX-HPLC (FAO/WHO, 2016)., PCOX-HPLC,.,.

6 674 이가정ㆍ권순재ㆍ정연중ㆍ손광태ㆍ하광수ㆍ목종수ㆍ김지회 사사 2017 (R ). References AOAC (Association of Official Analytical Chemists) AOAC Official Method Paralytic Shellfish Poison. Biological method. Final action. In: AOAC Official methods for analysis, 18 th Edition Chapter 49: Natural toxins (chapter ed. Truckses MW), AOAC International, Gaithersburg, MD, U.S.A., AOAC (Association of Official Analytical Chemists) AOAC Official Method Post column oxidation method(pcox) AOAC International, Gaithersburg, MD, U.S.A., 12. CEFAS (The Centre for Environment, Fisheries and Aquaculture Science) The use of the Jellett rapid testing kit for PSP toxins detection in the UK statutory monitoring programme for marine biotoxins. CEFAS, Lowestoft, UK, 7-8. Costa PR, Baugh KA, Bruce W, Raymond R, Shelly LN, Natália T, Stacey ME and Kathi AL Comparative determination of paralytic shellfish toxins (PSTs) using five defferent toxin detection methods in shellfish species collected in the Aleutian Islands, Alaska. Toxicon 54, org/ /j.toxicon FAO/WHO (Food and Agriculture Organization of the United Nations /World Health Organization) Technical report. Toxicity equivalency factors for marine biotoxin associated with bivalve molluscs. FAO/WHO, Rome, Italy, 9-13, 27. Hess P, Grune B, Anderson DB, Aune T, Botana LM, Caricato P, van Egmond HP, Halder M, Hall S. Lawrence JF, Moffat C, Poletti R, Richmond J, Rossini GP, Seamer C and Vilageliu JS Three Rs approaches in marine biotoxin testing: the report and recommendations of a Joint ECVAM/DG SANCO Workshop (ECVAM Workshop 54). Altem Lab Aanim 34, Jellet JF, Roberts RL, Laycock MV, Quilliam MA and Barrett RE Detection of paralytic shellfish poisoning (PSP) toxins in shellfish tissue using MIST Alert TM, a new rapid test, in parallel with the regulatory AOAC mouse bioassay. Toxicon 40, (02) Kim YS and Moon TS Filtering rate with effect of water temperature and size of two farming ascidians Styela clava and S. plicata, and a farming mussel Mytilus edulis, J Korean Fish Soc 31, KMFDS (Korea Ministry of Food and Drug Safety) Korean Food Standard. Chapter 6.3. Shellfish toxins Paralytic shellfish toxins. KMFDS, Cheongju, Korea, Laycock MV, Jellet JF, Belland ER, Bishop PC, Thérault BL, Russel-Tattrie AL, Quilliam MA, Cembella AD and Richards RC MIST Alert TM : a rapid assay for paralytic shellfish poisoning toxins. In: Hallegraff GM, Blackburn SI, Bolch CJ and Lewis RJ. (Eds.), Harmful Algal Blooms International Oceanographic Commission, Paris, France, Lee TS, Mok JS, Son KT, Oh EG, Kim PH, Lee KJ, Lee HJ and Kim JH Paralytic shellfish poisoning in mussel from coastal area of Korea. National Institute of Fisheries Science, Busan, Korea NIFS (National Institute of Fisheries Science) Annual report of National Fisheries Research and Development Institute. NIFS, Busan, Korea, 63. Oshiro M, Pham L, Csuti D, Dodd M, Inami GB and Brenden RA Paralytic shellfish poisoning surveillance in California using the Jellet Rapid PSP test. Harmful Algae 5, Park DL, Adams WN, Graham SL and Jackson RC Variability of mouse bioassay for determination of paralytic shellfish poisoning toxins. J Assoc Off Anal Chem 69, Rourke WA, Murphy CJ, Pitcher G, Van de Riet JM, Burns G, Thomas KM and Quilliam MA Rapid postcolumn methodology for determination of paralytic shellfish toxins in shellfish tissue. J AOAC Int 91, Schantz EJ, McFarren EF, Schafer ML and Lewis KH Purified shellfish poison for bioassay standardization. J Assoc Off Anal Chem 41, Song KC, Lee KJ, Yu HS, Mok JS, Kim JH, Lim KS, Lee MA and Kim MH Intra-laboratory validation of an HPLC Post-column oxidation method for the analysis of PSP toxins in oysters and mussels. Korean J Food Sci and Tech 45, Thomas K, Chung S, Ku J, Reeves K and Quilliam MA Analysis of PSP toxins by liquid chromatography with post column oxidation and fluorescence detection. In: Molluscan Shellfish Safety. Henshilwood K, Deegan B, McMahon T, Cusack C, Keaveney S, Silke J, O Cinneide M, Lyons D, Hess p (ed). Marine Institute, Galway, Ireland NSSP (US National Shellfish Sanitation Program) NSSP Guidance Document. Proposal , Rapid Screening Method for PSP, NSSP, AL, U.S.A.,

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03-서연옥.hwp 농업생명과학연구 49(4) pp.31-37 Journal of Agriculture & Life Science 49(4) pp.31-37 Print ISSN 1598-5504 Online ISSN 2383-8272 http://dx.doi.org/10.14397/jals.2015.49.4.31 국가산림자원조사 자료를 적용한 충남지역 사유림경영율 추정 서연옥