Poong Ho Kim

Seasonal Variability of Lipophilic Shellfish Toxins in Bivalves and Waters, and Abundance of Dinophysis spp. in Jinhae Bay, Korea

ABSTRACT Lipophilic shellfish toxins (LSTs) produced by dinoflagellates, including Dinophysis spp., can accumulate in bivalves. The seasonal variability of LSTs in bivalves and waters, and the abundance of Dinophysis spp. were investigated in Jinhae Bay, Republic of Korea, in 2007. Diarrhetic shellfish poisoning (DSP) such as okadaic acid and dinophysistoxin-1, and other lipophilic toxins such as pectenotoxin-2 (PTX2) and yessotoxin were analyzed by liquid chromatography—tandem mass spectrometry (LC-MS/MS). The toxin concentration in size-fractionated plankton material (20–100 µm in size) concentrated from a specific volume of water was converted into the toxin contents per unit volume of water. Among the genus Dinophysis, only D. acuminata was observed. The seasonal variability of okadaic acid, dinophysistoxin-1, and PTX2 per unit volume of water paralleled that of the abundance of D. acuminata. The cellular content and profile of toxins in D. acuminata varied, but PTX2 was mostly the dominant toxin. The toxins in the hepatopancreas of mussels (Mytilus galloprovincialis) were found from spring to autumn, and relatively high levels of toxins were detected in summer (June to August). Variation of the toxins in the hepatopancreas of mussels was remarkably consistent with both abundance of D. acuminata and toxin concentration in the water. Our results suggest that LC-MS/MS analysis of toxin in plankton concentrates has the possibility of becoming an alternative to or complement of the conventional microscopic method in toxic phytoplankton monitoring.

The first detection of okadaic acid and its derivatives in Korean bivalves by liquid chromatography-mass spectrometry

Lipophilic toxin profiles associated with diarrhetic shellfish poisoning in Korean bivalves were investigated by liquid chromatography-mass spectrometry (LC-MS). Okadaic acid (OA), dinophysistoxins (DTX), pectenotoxins (PTX) and yessotoxin (YTX) in bivalves were quantified by LC-MS. Thirty-eight bivalve samples were collected from five different areas on the south coast of Korea during January to June 2006. Dinophysistoxin-1 (DTX1) and OA were found in some samples of both mussel Mytilus edulis and oyster Crassostrea gigas. The levels of DTX1 and OA found in the hepatopancreas of the samples were 0.04–0.23 μg/g and 0.03–0.08 μg/g, respectively. Dinophysistoxin-3 was found in the hepatopancreas of the short-necked clam Ruditapes philippinarum at a concentration of 0.06 μg/g. PTX and YTX were not detected from all of the samples examined. This is the first report of the unambiguous identification of OA and its derivatives, by LC-MS, in Korean bivalves.

Organic Acid Extraction of Fluoride from Antarctic Krill Euphausia superba

The amount of fluoride removed from Antarctic krill via organic acid extraction depends on the extraction time, concentration, extraction volume, and the nature of the acid itself. The fluoride content in Euphausia superba was determined by measuring the concentration of fluoride ion in acidic extracts using an ion-selective electrode. The best results were obtained by adding 50 mL of 0.01 M citric acid to 0.3-0.5 g of krill and extracting for 5 min. Under these conditions, recoveries of fluoride from frozen whole krill and krill meat were 95.6-99.5% and 97.5-101.3%, respectively. The dry basis fluoride contents of krill by-product, krill meat and the boiled krill were 705, 298, and 575 ppm, respectively. These levels were significantly reduced by citric acid extraction.

Bioaccumulation of Heavy Metals in the Mussel Mytilus galloprovincialis in the Changseon area, Korea, and Assessment of Potential Risk to Human Health

From 2008 to 2013, the mussel Mytilus galloprovincialis were collected from a major area of mussel production (Changseon area), which is a designated shellfish-cultivating area for export, located on the southern coast of Korea. The samples were analyzed for mercury (Hg) using a direct Hg analyzer and for other metals, such as cadmium (Cd), lead (Pb), arsenic (As), chromium, copper, nickel, and zinc, using inductively coupled plasma mass spectrometry. The concentrations and bioaccumulation of the heavy met- als were determined, and a potential risk assessment was conducted to evaluate their hazards towards human consumption. The concentration and bioaccumulation ratio of Cd were the highest of the three hazardous metals (Cd, Pb, and Hg). The concentrations of hazardous metals in all samples were within the limits set by Korea and other countries. The estimated dietary intake (EDI) was compared to the provisional tolerable daily intake (PTDI) adopted by the Joint FAO/WHO Expert Committee on Food Additives and the U.S. Environmental Protection Agency. The EDIs of all heavy metals tested for mussel samples ranged from 0.01 to 4.99% of the PTDI; the highest value was measured for As. The hazard index (HI) can be used to assess the risk of heavy metal consump- tion associated with contaminated food. The HI for all samples was far less than 1.0, which indicates that the mussels produced in the Changseon area do not represent an appreciable hazard to humans and are fit for consumption.

Bacteriological quality evaluation of seawater and oysters from the Jaranman-Saryangdo area, a designated shellfish growing area in Korea: Impact of inland pollution sources

From 2011 to 2013, we conducted a full sanitary survey of pollution sources in proximity to a designated shellfish growing area in Korea, and their impact on the sea area therein. From this area, 836 seawater samples and 93 oyster samples were examined to evaluate their bacteriological quality. There were 483 potential pollution sources in the drainage area of the Jaranman-Saryangdo area, including 38 sources discharging water. It demonstrates that while many pollution sources have been identified, no significant impact occurred within the designated shellfish growing area. Variations in fecal coliform (FC) levels in seawater were closely related to rainfall. The FC levels of seawater and oysters from the designated area met the regulation limits set by various countries. Our study indicates that the oysters produced in this area are apparently safe for raw consumption based on their bacterial quality.

Evaluation of the Influence of Inland Pollution Sources on Shellfish Growing Areas after Rainfall Events in Geoje Bay, Korea

The influences of inland pollution sources because of rainfall events on the bacteriological water quality in Geoje Bay, a major shellfish production area in Korea, were investigated. The sanitary status of sea water and shellfish after rainfall events was also evaluated. The flow rates of 13 streams around Geoje Bay showed 6 to 7-fold increases after 15 to 21 mm of rainfall. Peak pollution was observed in the Naegan Stream, the Gandeok Stream and the Seojeong Stream. The calculated impact area of inland pollution sources was 3.1 ㎢ immediately after 15 mm of rainfall and expanded to 3.5 ㎢ after 24 hours. These calculations of impacted area matched results from fecal coliform analyses with sea water. The distance between the major pollution source in the bay (the Gandeok Stream) and the station with the worst bacteriological water quality immediately after 15 mm of rainfall, which was below the Korean standard, was 0.8 km in a straight line; this distance increased to 2.0 km after a period of 24 hours. The area impacted by inland pollution sources after a 15 mm rainfall event was wider than after a 21 mm rainfall. Although the flow rate from in-land pollution sources was higher, the concentration of fecal coliform in the discharged water was lower after higher rainfall events. These observations corresponded with the results of fecal coliform analyses with sea water samples. According to the evaluation of the influences of inland pollution sources and fecal coliform analyses on sea water and shellfish samples in Geoje Bay, pollutants from inland sources did not reach the boundary line of the shellfish growing area after rainfall events of 15 or 22 mm. The bacteriological water quality of the shellfish growing area in Geoje Bay met the Korean standard and US NSSP requirements for approved shellfish growing areas.Key words: Geoje Bay, Oyster, Fecal coliform, Shellfish growing area, Inland pollution source

Antimicrobial Resistance and Distribution of Virulence Factors of Vibrio parahaemolyticus Isolated from Shellfish Farms on the Southern Coast of Korea

장염비브리오균(Vibrio parahaemolyticus)는 해수 및 기수지 역에 분포하고 있는 저도 호염성 해양세균으로 계절에 따라 분 포가 바뀌는데 일반적으로 여름에서 초가을에 가장 많이 검출 된다고 알려져 있으며, 이 균에 오염된 어패류를 생식하거나 덜 익혀 섭취하여 나타나는 식중독 원인세균이다(Sakazaki et al., 1968; Honda and Iida, 1993). 최근 우리나라 식품 위생 수준이 향상되고 식품 안전 관리 기 술이 크게 발전하고 있지만, 미생물에 의한 식중독 사고는 발생 빈도수나 환자 발생수가 줄어들지 않고 있는 실정 이어서 과학 적이고 효과적인 식중독 예방 기술 개발이 절실히 필요하다. 현 재 식품 안전 관리의 과학화는 전세계가 지향하고 있으나, 식중 독 원인 미생물에 대한 위해도 평가는 대단히 어려운 과제로서 선진국에서도 아직 완전히 장착되지 않은 분야로 이에 대한 광 범위한 연구가 진행 중에 있다(KFDA, 2005). 2002년부터 2013년까지 우리나라에서 발생한 장염비브리 오균에 의한 식중독 사고는 주로 여름철에 집중적으로 발생하 남해안 패류양식장에서 분리한 Vibrio parahaemolyticus의 병원인자 분포 및 항균제 내성 박용수·박큰바위·권지영·유홍식·이희정·김지회·이태식·김풍호*

The Effects of Inland Pollution Sources around the Port of Jeokyang and Jangpo after Rainfall Events on Bacteriological Water Quality in the Changseon Area, Korea

The influences of inland pollution sources nearJeokyang and Jangpoports following rainfall events on the bacteriological water quality and safety of commercial shellfish were investigated in the Changseon area, Korea. Stream flow rates exhibited 1.9- to 5.7-fold increases after rainfall events and then fell to 68~81% of that level after 24 h. The calculated impact area of inland pollution sources was 0.47 in the Jeokyangport area and 0.27 in the Jangpoport area at 24 h following 11 mm of rainfall. When the flow rate of inland pollution increased, the level of male-specific bacteriophage (MSB) decreased, and no MSB could be detected in seawater samples, whereas 30 PFU/100 g was detected in shellfish samples. Fecal coliform levels in seawater and shellfish samples did not exceed their respective tolerance levels (4.5 MPN/100 mL) and 130 MPN/100 g, respectively and thus complied with the standard for approved shellfish growing area. The sanitary conditions of areas adjacent to Jeokyang and Jangpoports under conditions of rainfall below 29 mm met the criteria for approved growing area under the United States National Shellfish Sanitation Program and the EC Regulations.

Antimicrobial Resistance in Escherichia coli Isolated from Shellfish Farms on the West Coast of Korea

우리나라에서 패류는 연간 400천 톤 이상이 생산되고, 총 수산물 생산량의 14.3%를 차지하고 있으며 수천 년 동안 균들이인류에게 없어서는 안 될 중요한 동물성 영향을단백질 공급원이며 식량자원이다(MOF, 2013). 연안해역에 주로 서식하는 (Chae et al., 2005; Lee et al., 2005; Jeong et al., 2010). 패류는 육상과 인접해 있어 배수유역으로부터 유입되는 오염물질의 영향을 쉽게 받을 수 있으며 , 또한 이동성이 거의 없고, 여과섭이 활동을 통하여 먹이를 항균제가섭취하는 특성 때문에 패류는 해수 중에 부유하는 병원성 70% 세균, 바이러스 등을 체내에 쉽게 축적하게 된다(Grimes, 1991; Feldhusen, 2000). 최근 인구의 증가와 산업화로 인하여 일부 생활폐수, 산업폐수 내에그리고 사람이나 가축으로부터 배설된 분변 등의 오염원이 하수구나 있기하천을 통하여 해상으로 유입됨으로써 연안 해역의 수질에 악 내성균을영향을 미치고 있다(Hill et al., 2006; Lee et al., 2010). 또한, 기후변화의 영향으로 있다폭우의 빈도와 성에태풍의 강도 증가는 많은 강우를 발생시켜 분변성 오염물질들이 일시에 패류생산해역으로 유입되어 병원성세균 및 바이러스에 의해 쉽게 오염될 수 있다(Park et al., 2011; Park et al., 2012). 이러한분변성오염물질중에는항균제내성함유되어있어패류의위생학적안전성뿐만아니라해양상재세균의항균제내성획득에 미칠수있을것이다특히항균제를투여한 축산 동물이나 사람의 분변으로부터약제내성균이자연환경으로방출되기도하며사람이나동물에게투여한 완전히소화흡수되지않고섭취된항균제의경우약 정도가성분이변하지않은상태로배설된다는보고도있다(Kummerer, 2009). 한편, 대장균은사람이나동물의장상재하는정상세균총으로빈번하게투여되는항균제에노출되어 때문에항균제에의한내성획득과정을이해하고 모니터링함에있어매우유용한세균으로알려져(Levin et al., 1997). 이미국내에서도대장균의항균제내관한모니터링및연구가많이이루어지고있다(Chae et

Effect of Heat Treatment on Male specific Coliphage and Norovirus Concentrations in Norovirus Contaminated Oyster Crassostrea gigas

바이러스성 식중독의 대표적인 원인체인 노로바이러스는 Caliciviridae과에 속하며 외피(envelope)가 없는 단일가닥 RNA 바이러스(약 7.6 kb)로 3개의 open reading frame (ORF) 로 구성되어 있고, 변이가 쉬운 RNA 바이러스 특성상 노로바 이러스는 5개의 genogroup (GI-GV)으로 분류되고 있으며, 이들 중 GI 및 GII genogroup의 노로바이러스가 바이러스 성 식중독을 일으키는 원인체로 알려져 있다. 노로바이러스는 10~100개의 소량 입자로도 감염증을 유발시킬 수 있으며, 인체 감염시 많은 수의 바이러스가 분변으로 배출되는 특징이 있다 (Koopmans et al., 2002; Blackburn et al., 2004). 노로바이러스 식중독은 감염증 환자의 분변에 의해 식품이 오 염되고, 오염된 식품을 섭취할 경우 환자가 발생하는 순환고리 (fecal-oral route)를 형성하고 있다. 감염증 환자의 분변에 오 염된 해역에서 생산된 패류는 노로바이러스 식중독의 매개체 역할을 할 가능성이 크며, 패류 중에서도 주로 날 것으로 섭취 가 많은 굴에 의한 노로바이러스 식중독 사고는 호주, 뉴질랜 드, 일본 등 여러나라에서 보고 되고 있다(Webby et al., 2007; Simmons et al., 2007; Alfano-Sobsey et al., 2012; Iritani et al., 2014). 한편, male specific coliphage (MSC)는 처리되지 않은 폐수 또는 하수처리장에서 처리된 후 배출되는 방출수 중에 높은 농 도로 검출된다. MSC의 크기(head diameter)는 25 nm이고, 단 일 가닥 RNA 구조이며, 염소소독 및 환경스트레스에 대한 내성 정도가 노로바이러스 등 다른 장관계바이러스의 생화학적 특성 하고 유사하여 인간 분변 유래 장관계바이러스의 오염지표로써 주목 받고 있다(Burkhardt et al., 1992). 우리나라에서 굴 생산해역의 대부분이 연안해역에 위치하고 있어 육ᆞ해상오염원으로부터 유입되는 분변성 오염물질의 영 가열처리 조건에 따른 오염굴(Crassostrea gigas) 중의 Male Specific Coliphage와 노로바이러스 농도변화 박큰바위·박용수·권지영·유홍식·이희정·김지회·이태식·김풍호*