Journal of the Korea Academia-Industrial cooperation Society Vol. 18, No. 11 pp. 778-784, 2017 https://doi.org/10.5762/kais.2017.18.11.778 ISSN 1975-4701 / eissn 2288-4688 참굴, Crassostrea gigas 의패각운동을이용한유독와편모조 Alexandrium 속의모니터링연구 김윤정 1, 윤양호 2* 1 전남대학교대학원해양환경학과, 2 전남대학교해양기술학부 A Studies on the Bio-monitoring using Shell Valve Movements (SVMs) of Pacific Oyster Crassostrea gigas for Toxic Dinoflagellates, Genus Alexandrium Yoon Jeong Kim 1, Yang Ho Yoon 2* 1 Dept. of Enviromental Oceanography of Graduate school, Chonnam National University 2 Faculty of Marine Technology, Chonnam National University 요약참굴 (Crassostrea gigas) 의패각운동을이용하여 Alexandrium 속의조기출현모니터링가능성을고찰하였다. 지구규모에서패류독화를발생시키는 A. fundyense 와잠재적유독종으로알려진 A. affine 를이용하여홀소자센서 (Hall element sensor) 를사용하여참굴의패각운동 (SVMs) 을측정하였다. 참굴은 Isocrysis galbana 를먹이생물로하여안정화시킨다음 3 일간절식시킨후실험에제공하였다. 결과참굴은 A. fundyense 에대해세포밀도 20 cells/ml 에서 SVMs 횟수가 10.5 ± 1.2 times/hr 로증가하여민감하게반응하였고, 세포밀도 500 cells/ml 에서재차 14.1 ± 5.7 times/h, 5,000 cells/ml 에서 27.9 ± 11.1 times/hr 로 SVMs 가급격한증가를보였다. 그러나 A. affine 에대해서는세포밀도가 300 cells/ml 까지 6.7 ± 3.9 times/hr 로기준 SVMs 와유사하였고, 세포밀도 1,000 cells/ml 이상에서 15.3 ± 10.8 times/hr 로급격히증가하였다. 즉 A. fundyense 는 20 cells/ml 에서부터참굴의 SVMs 가민감하게반응하였지만, A. affine 는높은 1,000 cells/ml 의세포밀도에서 SVMs 가반응하였다. 이러한결과에서유독와편모조에대한참굴의 SVMs 는종에따른차이가있어, A. fundyense 의초기발생예보에는유용하게활용가능하지만, A. affine 에응용하는것은어렵다는결론을얻었다. Abstract We investigatedthe possibility of a bio-monitoring system for predicting toxic dinoflagellates (Genus Alexandrium) by the measuring shell valve movements(svms) of Pacific oyster, Crassostrea gigas (Mollusca: Bivalvia) using the Hall element sensor. We then described the SVMs of Pacific oyster exposed to the toxic algae under laboratory conditions. Pacific oyster used for experiment were fed Isochrysis galbana until they stabilized and kept under hunger conditions for three days to prevent the influence of food before the experiment. Pacific oyster were exposed to the toxic dinoflagellate, A. fundyense, and the potentially toxic dinoflagellate, A. affine. When Pacific oyster were exposed to A. fundyense, SVMs increased over 10 times/hr at low cell densities of 20 cells/ml. SVMs increased again at 14.1 ± 5.7 times/hr at 500 cells/ml, and 27.9 ± 11.1 times/hr at the high cell density of 5,000 cells/ml. However, in the presence of A. affine, SVMs increased at 6.7 ± 3.9 times/hr until 300 cells/ml, while they increased greatly to 15.3 ± 10.8 times/hr at 1,000 cells/ml. The SVMs of Pacific oyster indicated differences depending on species for toxic dinoflagellates. Therefore, the SVMs of Pacific oyster could be useful for A. fundyense, but would bedifficult to apply for A. affine. Keywords : Bio-monitoring, Genus Alexandrium, Hall element sensor, Pacific oyster, Shell valve movements(svms), Toxic dinoflagellates 이논문은해양수산부 ( 해양과학기술진흥원 ) 지원 2015년도미래해양산업기술개발사업인 이매패류바이오센서를활용한연안환경모니터링시스템개발 ( 과제번호 20150129) 지원으로수행되었음. * Corresponding Author : Yang Ho Yoon (Chonnam National Univ.) Tel: +82-61-659-7142 email: yoonyh@jnu.ac.kr Received August 31, 2017 Revised (1st September 27, 2017, 2nd October 13, 2017) Accepted November 3, 2017 Published November 30, 2017 778
참굴, Crassostrea gigas 의패각운동을이용한유독와편모조 Alexandrium 속의모니터링연구 1. 서론지구규모의연안 / 내만해역에발생하는유독와편모조에의한적조발생및패류독화는수권생태계를넘어인류의공중보건을위협할뿐만아니라막대한수산피해에따른사회문제로확산되면서 [1], 원인생물의거동및조기발생탐지등은범지구적인관심사가되고있다 [2]. 현재유독와편모조의출현및대량발생의예보는해역의물리 화학적환경인자측정과지속적인원인생물의거동을모니터링하거나인공위성및항공촬영에기반을둔해양관측에의존하는바가크지만, 최근이들원인생물에민감한반응을보이는검지소자로활용한바이오모니터링을이용한연구가진행되고있다 [3,4]. 즉생물은내부의생리요인및외적환경요인변화에민감한반응을보이기에 [5,6], 지속적인생물의행동관찰로서다양한인자의변화를감지할수있다 [4]. 이러한해양생물을이용한모니터링은민감도가높고, 정확한생물반응을관찰할수있는장점으로기존의화학 물리학적모니터링방법에비해높은효율성을나타낸다 [7]. 이매패인참굴은한국양식생산량의 14% 로, 전복다음으로높은생산을보이는패류로서국내전체패류생산량의 25% 를차지한다 ( 통계청, http://kostat.go.kr). 참굴과같은이매패류는이동성이거의없는저서생물로환경변화에수동적으로반응하기에해역에서환경지표생물로많이이용된다 [8,9]. 특히이매패류는내적생리변화및외적환경변화에패각의개폐활동인패각운동 (SVMs:shell valve movements) 으로반응한다 [10,11]. 이러한이매패류의 SVMs는처음패류의건강도를판단하는지표로이용되었다 [12]. 그러나 SVMs가생물의체내및외부환경변화에대한방어반응으로인지되면서 [13], 환경변화를탐지하는바이오모니터링수단으로활발하게사용되었다 [3,14,15,16]. 즉이매패류의 SVMs는호흡, 배설, 심박수, 먹이활동, 생물학적리듬등의내부적요인과급격한외부의환경자극, 그리고포식자로부터의회피등에의해변화를받는다 [17,18]. 특히유독, 유해플랑크톤등먹이생물의종에따른 SVMs 변화 [14,19] 를이용하여유해적조의조기탐지에유용하게활용할수있다는것이보고된다 [3]. 따라서이연구에서도참굴을대상이매패류로하여많은마비성패류독화원인종을포함하는유독와편모조인 Alexandrium 속의두종을대상으로세포밀도변화에따른참굴의 SVMs의변화를고찰하여, 이들유독플 랑크톤이패류양식장조기출현모니터링여부를평가하였다. 2. 재료및방법참굴은통영과여수돌산도굴양식장에서월동한 2 년생각굴을대상으로하였다. 실험에제공한참굴의각장, 각고및각폭은각각 93.0 ± 6.4mm, 27.7 ± 7.1mm, 및 53.8 ± 8.7mm의범위였고, 참굴의습중량은 71.7 ± 24.6g으로크기에는변화가없었지만, 무게에서산지에따라개체사이에다소차이를보였다. 유독플랑크톤은마비성패독을생산하는 Alexandrium fundyense 및잠재적유독종으로알려지는 A. affine를대상으로하였다. A. fundyense는주로온대해역에서저수온기에발견되며, 우리나라남해안에서일반적으로출현하는종이며, A. affine 또한 18 ~ 25 의수온대인온대해역에서봄과여름에주로출현한다 [20,21]. 이들유독플랑크톤은수온 20, 염분 30.0 psu, 광조건 100 μmol/ m2 /sec, 명암주기 (L:D) 12h:12h의조건에서배양하였다. 배양은 f/2 배지를사용하여 [22] 대수성장기까지증식시킨다음실험에제공하였다. SVMs 측정은참굴의왼쪽패각에홀소자센서 (Allegro MicroSystems, A1369EUA-24-T), 오른쪽패각에자석을산호접착제로부착하여홀센서와자석사이의거리에따른전압차이를이용하는측정장치 (Oceantech Co., OT-SVML -001) 를사용하였다. 홀센서는약 2g으로 15 ~ 1,000 mv의전압을 0.5 ~ 2.0초간격으로연속측정이가능하였다. 참굴은실험수조에서하루에 2번국립수산과학원굴양식매뉴얼에서참굴유생의먹이로권장하는침편모조류 Isocrysis galbana를먹이생물 [23] 로공급하여안정화시켰다. 실험은시작전 3일간절식상태로순치시킨다음유독와편모조의세포밀도가각각 0, 20, 50, 70, 100, 300, 500, 700, 1,000, 5,000 cells/ml가되도록단계별로조절하여각실험구간에서 1시간씩폭로시키면서연속적으로 SVMs를측정하였다. SVMs는 24시간명조건및수온 16.3 ± 0.35 의조건에서측정하였다. 수온을 20 이하로한것은참굴이 20 이상수온에서는산란하여체력이급격하게감소하기때문에이러한현상을방지하기위한조치였다 [24]. 또외부자극에대한스트레스및굴개체사이의간섭을최소화하기위해독립된공간을제공할수있도 779
한국산학기술학회논문지제 18 권제 11 호, 2017 Fig. 2. Standard pattern of SVMs on the Pacific oyster. Fig. 1. Schematic diagram for the SVMs(shell valve movements) measurement. 록, 특수수조에서 8개체의실험참굴의 SVMs를동시에연속측정하였다 (Fig. 1). 실험참굴에대한생산지별개체사이에차이는있었지만모든실험은동일개체에대해유해생물의세포밀도를변화시켜 SVMs를측정하였기에개채간의차이에따른오차는그다지문제가되지않을것으로판단되었다. 그리고모든측정된 SVMs는데이터로거를통해직접노트북에저장되게고안되어, 저장된자료를분석하였다. 3. 결과 1.1 참굴의기준 SVMs 참굴생체내부및외부환경자극에대한최적조건에서의 SVMs 측정및결과를객관화시킨다는것은매우어렵다. 때문에이연구에서각실험구의결과비교를하기위한기준 SVMs의파형과횟수는실험에제공한참굴을 3일동안절식한후먹이생물이없는여과해수에서 1 시간연속측정한 SVMs 결과를사용하였다 (Fig. 2). 참굴의기준 SVMs은개체에따라차이는있지만약 5 ~ 10 times/hr 범위에서 7 times/hr 전후의 SVMs 횟수를보였고 (Fig. 2A), SVMs 패턴은패각이닫히는것 ( 폐각 ) 은매우빠르게진행되는것에반해, 패각이열리는것 ( 개각 ) 은천천히진행되어, 개각이후일정시간개각상태를유지하였다 (Fig. 2B). 또한참굴은천천히개각할때배설하는것이관찰되었다. 1.2 유독와편모조 Alexandrium 속에대한 SVMs 변화마비성패류독화의대표적원인종인 A. fundyense에노출되었을때, 참굴의 SVMs 패턴은세포밀도가 20 cells/ml에서 SVMs 횟수가 10회를초과하여, 20 ~ 300 cells/ml의범위에서 10.5 ± 1.2 times/hr의 SVMs 횟수를보였고, 500 cells/ml에서재차 14.1 ± 5.7 times/hr 로급격하게증가하였다. 더욱이 5,000 cells/ml의세포밀도에서는 27.9 ± 11.1 times/hr를나타내었다 (Fig. 3A & 4A). 그러나잠재적유독플랑크톤인 A. affine에대해서는 50 cells/ml의세포밀도에서일시적으로폐각하 Fig. 3. The Fluctuations of SVMs of Pacific oyster on the variations of cell density for toxic dinoflagellates, Alexandrium fundyense (A) and A. affine (B). 780
참굴, Crassostrea gigas 의패각운동을이용한유독와편모조 Alexandrium 속의모니터링연구 Fig. 4. The fluctuations of SVMs times of Pacific oyster on the variations of cell density for toxic dinoflagellate, Alexandrium fundyense (A) and A. affine (B). 는현상이관찰되기도하였지만, 세포밀도가 300 cells/ml까지는 6.7 ± 3.9 times/hr로기준 SVMs와유사하였지만, 500 cells/ml에서 11.7 ± 4.6 times/hr, 1,000 cells/ml에서 15.3 ± 10.8 times/hr으로급격하게증가하였다 (Fig. 3B & 4B). 4. 고찰이매패류의 SVMs을이용한바이오모니터링방법은담수권에서독성물질을감지하기위해시작되었으나 [25], 지금은담수는물론해양환경에이루는수권전체에서다양한환경인자를검지하는방법으로독일 [26], 프랑스 [27] 등의유럽과, 일본 [3], 한국 [16], 대만 [28] 등북서태평양연안의패류양식장등에서광범위하게활용되고있다. 즉프랑스는굴양식장의패류독화원인종의출현을감시하기위해서 [27], 일본은진주조개양식장의대량폐사를발생시키는유해적조원인종인 Heterocapsa circularisquama의조기출현탐지 [3] 에이용되고있다. 그리고독일이나대만등은수권의유해물질탐지를위한방법 [26,28,29] 으로사용되고있으며, 국내에서도이상수온이나염분의변화 [7,16] 및해저빈산소및황화수소의발생 [15] 과같은환경인자의변화를탐지하는방법으로사용되고있다. 그러나국내에서는아직까지유해적조및패류독화원인종의조기출현을탐지하기위한모니터링방법으로사용된적은없다. SVMs의측정대상이매패류도패각이매끄럽고, 센서부착이쉬운담치류 [11-13,25] 를시작으로진주조개 [3], 바지락 [14], 가리비 [30], 재첩 [28] 등매우다양하지만, 동ㆍ서양을막론하고식용가치가높은굴 [7,19] 을대 상으로최근많은연구가진행되고있다. 먹이생물로제공한와편모조 Alexandrium 속은현재 41종이보고되지만 [31], 많은종에서마비성패류독을생산하여, 지구규모에서굴이나담치류등이매패류를독화시켜수산생물생산에막대한경제적손실을발생시키는것은물론인류의공중위생에도커다란위협으로작용한다 [1]. Alexandrium 속에서특히 A. fundyense는지구규모의발생과생산되는독성분및패류독의분포등이잘파악되어있다 [20,32]. 이연구에서도 A. fundyense 에대한참굴의 SVMs는낮은세포밀도에서부터민감하게반응하였다. 이러한반응은유해적조생물인 H. circularisquama에대한진주조개의반응에서도유사한결과를보였다 [3]. 이와같은유독와편모조에대한이매패류의반응은유독플랑크톤에대한내부방어행동으로내부적요인에의한결과이다 [18,19]. 그러나유독플랑크톤에대한이매패류의행동은독성분에민감하게반응하지않은진주담치와같은비저항성패류에서는여과율이나 SVMs 변화가거의발생하지않기에, 유독플랑크톤을먹이생물로서다량섭취하여빠른속도로체내에독소를축척하는것이알려진다 [33]. 그러나가리비와같이독성에민감한저항성패류는유독플랑크톤출현이감지되면먹이섭취행위인여과율을감소시켜, 체내에독소가축적되지않게하는행동을하는것이알려진다 [34]. 유독플랑크톤독성에대한참굴의감수성이담치류와가리비의중간정도로독성분에의해폐사되지않지만 SVMs 등생리활성에크게영향을받아 [19], 여과율이나생물발육속도가감소하는것이알려진다 [35-38]. 유독플랑크톤에대한이매패류의 SVMs가종에따라다른특성을보이는것과마찬가지로 Alexandrium 속에대해서도종에따라참굴의 SVMs가다른특이성, 즉유 781
한국산학기술학회논문지제 18 권제 11 호, 2017 독플랑크톤종특이성을보인다. 그리고 A. fundyense에대한참굴의 SVMs는낮은세포밀도에서특이성이관찰되지만, A. affine에대해서는 500 cells/ml 이상의매우높은세포밀도에서특이성이관찰되었다. 이러한결과는바이오모니터링을대상으로하는생물종은물론유독플랑크톤종에따른특이성을표현하는것으로, 이매패류의 SVMs를이용한바이오모니터링응용에는사전에현장에서대상으로하고자하는환경인자는물론유해생물의종을대상으로한 SVMs의패턴확보가필요한것이된다. 즉각바이오모니터링대상종에대한환경인자또는유해플랑크톤의 SVMs의결과물 (dictionary) 를정리하여, 상황에적합한대상생물을선택하여야한다는것을시사한다. 또한바이오모니터링에의한유해물질이나유해플랑크톤의출현탐지는낮은농도의물질유입이나유해플랑크톤의군성장이전단계의낮은세포밀도에서반응하여야만모니터링의유효성이확보될수있다. 때문에낮은세포밀도에서반응을보이는마비성패류독화원인종인 A. fundyense와는달리높은세포밀도에서반응을보이는잠재적유독종인 A. affine와같은종에대해서는참굴을이용한유해생물조기탐지모니터링이적합하지않기에모니터링이가능한유사패류종탐색이필요할것으로판단되었다. 최근진주조개양식으로유명한일본이세만에서유해적조발생으로진주조개가대량폐사되는것을방지하기위해, 유해적조 H. circularisquama의조기출현탐색을위한진주조개의 SVMs 측정기기를현장에설치하여적조발생에대한피해경감에크게기여하는것으로전해진다 [3, T. Honjo, personal community]. References [1] Y. H. Yoon, Red Tides The uprising from the sea-, Gipmundang, Seoul, pp. 1-531, 2010. [2] V. M. Bricelj, S. E. Shumway, Paralytic shellfish toxins in bivalve mollusks: occurrence, transfer kinetics, and biotrans- formation, Reviews in Fisheries Science, vol. 6, no. 4, pp. 315-383. 1998. DOI: http://dx.doi.org/10.1080/10641269891314294 [3] K. Nagai, T. Honjo, J. Go, H. Yamashita, S. J. Oh, Detecting the shellfish killer Heterocapsa circularisquama (Dinophyceae) by measuring bivalve valve activity with a hall element sensor, Aquaculture, vol. 255, no. 1-4, pp. 395-401. 2006 DOI: https://doi.org/10.1016/j.aquaculture.2005.12.018 [4] M. J. Bae, J. S. Kim, Y. S. Park, Y. S., 2012, Evaluation of changes in effluent quality from industrial complexes on the Korean nationwide scale using a self-organizing map, International Journal of Environmental Research and Public Health, vol. 9, no. 4, pp. 1182-1200. 2012. DOI: http://doi.org/10.3390/ijerph9041182 [5] A. Gerhardt, Aquatic behavioral ecotoxicology -prospects and limitations, Human and Ecological Risk Assessment: An International Journal, vol. 13, no. 3, pp. 481-491. 2007. DOI: http://dx.doi.org/10.1080/10807030701340839 [6] N. B. Davies, J.R. Krebs, S. A. West, An introduction to behavioural ecology (4rd eds). Wiley Blackwell Scientific, London, pp. 1-520, 2012. [7] S. J. Oh, J. H. Lee, S. Y. Kim, S. Y., 2013, Bio-monitoring system using shell valve movements of Pacific oyster (Crassostrea gigas) - I. detecting abnormal shell valve movements under low salinity using a hall element sensor, Journal of the Korean Society for Marine Environment and Energy, vol. 16, no. 2. pp. 138-142, 2013. DOI: http://dx.doi.org/10.7846/jkosmee.2013.16.2.138 [8] M. S. Jeng, W. L. Jeng, T.C. Hung, C. Y. Yeh, R. J. Tseng, P. J. Meng, B. C. Han, Mussel watch: a review of Cu and other metals in various marine organisms in Taiwan, 1991 98, Environmental Pollution, vol. 110, no. 2, pp. 207-215, 2000. DOI: http://dx.doi.org/10.1016/s0269-7491(99)00304-8 [9] J. Moroishi, I. J. Kang, K. Nagafuchi, T. Honjo, Y. Shimasaki and Y. Oshima, Biological monitoring to detect both water pollution and water quality recovery based on valve movements of freshwater bivalves (Corbicula japonica), Journal- Faculty of Agriculture Kyushu University, vol. 54, no. 2, pp. 413-420, 2009. [10] A. R. Manley, J. Davenport, Behavioural responses of some marine bivalves to heightened seawater copper concentrations, Bulletin of Environmental Contamination and Toxicology, vol. 22, no. pp. 6739-744, 1979. DOI: http://dx.doi.org/10.1007/bf02027017 [11] K. J. M. Kramer, H. A. Jenner, D. D. Zwart, The valve movement response of mussels: a tool in biological monitoring, Hydrobiologia, vol. 188, no. 1, pp. 433-443, 1989. DOI: https://doi.org/10.1007/bf00027811 [12] T. Fujii, S. Toda, Open and close shell-movement of the mussel, Mytilus edulis L. under natural conditions, Bulletin of National Research Institute of Aquaculture, vol. 20, pp. 33-40, 1991. [13] S. Rajagopal, G.V.D. Velde, H. A. Jenner, Shell valve movement response of dark false mussel, Mytilopsis leucophaeta, to chlorination, Water Research, vol. 31, no. 12, pp. 3187-3190, 1997. DOI: https://doi.org/10.1016/s0043-1354(97)00163-2 [14] L. Basti, K. Nagai, Y. Shimasaki, Y. Oshima, T. Honjo and S. Segawa, Effects of the toxic dinoflagellate Heterocapsa circularisquama on the valve movement behavior of the Manila clam Ruditapes philippinarum, Aquaculture, vol. 291, no. 1-2, pp. 41-47, 2009. DOI: https://doi.org/10.1016/j.aquaculture.2009.02.029 [15] J. Y. Jeon, S. Y. Moon, S. J. Oh, Bio-monitoring system using shell valve movements of Pacific oyster (Crassostrea gigas) (detecting abnormal shell valve 782
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한국산학기술학회논문지제 18 권제 11 호, 2017 김윤정 (Yoon Jeong Kim) [ 준회원 ] 2017 년 8 월 : 전남대학교대학원환경해양학과 ( 이학석사 ) 2017 년 11 월 ~ 현재 : 한국수산자원관리공단남해지사 < 관심분야 > 연안해양생태및해양환경관리, 바다목장및바다숲조성과관리 윤양호 (Yang Ho Yoon) [ 정회원 ] 1984 년 3 월 : Nagasaki 대학대학원수산학연구과 ( 수산학석사 ) 1989 년 3 월 : Hiroshima 대학대학원생물권과학연구과 ( 학술박사 ) 1990 년 3 월 ~ 2006 년 2 월 : 여수대학교교수 2006 년 3 월 ~ 현재 : 전남대학교교수 < 관심분야 > 식물플랑크톤생리ㆍ생태, 연안환경생태, 미세조류의산업이용, 해역이용및관리, 그리고환경보전 784