J. Korean Soc. Environ. Eng., 38(4), 193~200, 2016 Original Paper http://dx.doi.org/10.4491/ksee.2016.38.4.193 ISSN 1225-5025, e-issn 2383-7810 생태독성실험을위한한국우점종유리물벼룩 (Daphnia galeata) 의특성및독성민감도 : 큰물벼룩 (Daphnia magna) 과의비교연구 Characteristics and Toxicity Sensitivity of Korean Dominant Species Daphnia galeata for Ecotoxicity Testing: Comparative Study with Daphnia magna 최영설 곽진일 안윤주 Rongxue Cui Jin Il Kwak Youn-Joo An 건국대학교환경과학과 Department of Environmental Science, Konkuk University (Received November 11, 2015; Revised February 25, 2016; Accepted March 14, 2016) Abstract : Water flea is a widely used test species in the aquatic ecotoxicity test. In Korea, D. magna is currently used as a standard test species, however that do not inhabit in the Korean aquatic ecosystem. In this study, Daphnia galeata, which is a dominant species in the Korean aquatic ecosystem, was collected from domestic lake and investigated to suggest the D. galeata for ecotoxicity assessment in Korea. We investigated the characteristics, life span, and toxicity sensitivity compared with D. magna. The life span test of D. galeata was performed in this study, and then the results were compared to the sensitivity with D. magna to confirm the applicability for ecotoxicity assessment. The 48h-L(E)C50 values for seven heavy metals (As, Cd, Cr, Cu, Hg, Ni, and Zn) of D. galeata and D. magna were collected and analyzed. As a results, shorter lifetime, less reproduction, smaller body size of D. galeata were observed compared with D. magna. D. galeata was similar or more sensitive than D. magna for seven heavy metals. Therefore, we propose that D. galeata is a suitable test species for ecotoxicity testing in Korea. Key Words : Daphnia galeata, Daphnia magna, Life Span, Ecotoxicity, Toxicity Sensitivity 요약 : 생태독성관리제도가국내에도입된이래 Daphnia magna( 큰물벼룩 ) 는시험종으로널리사용되고있다. 다만 D. magna 는국내비서식종으로국내의담수환경을반영한생태독성평가에는제한점이존재한다. 따라서국내서식물벼룩중적합한시험종을선정하여생태독성평가에대한활용가능성을평가할필요가있다. Daphnia galeata( 유리물벼룩 ) 는국내한강수계우점종이며대부분의수계에서식하고있는물벼룩이다. 본연구에서는 D. galeata 의국내고유시험종으로서의활용가능성을평가하고자 D. galeata 의특성및생활사를확인하여 D. magna 와비교하였다. 또한 D. galeata 의독성민감도를확인하고자 D. galeata 및 D. magna 에대한 7 종중금속 (As, Cd, Cr, Cu, Hg, Ni, Zn) 급성독성자료조사, 수집하여민감도를비교분석하였다. D. galeata 의생활사측정결과 D. galeata 의생존기간은 28 ± 8 일로 D. magna 의절반수준으로나타났으며생존기간동안의어미개체당새끼생산량도 D. magna 보다적었으며, 평균적인체구도 D. magna 보다 D. galeata 가작았다. 독성민감도는 7 종의중금속에대해 D. galeata 가 D. magna 보다더민감하거나유사한것으로확인되었다. 장단점을종합적으로고려한결과, D. galeata 는국내고유생태독성시험종으로적합하며차후 D. magna 를대체하거나병행하여사용할수있는시험종으로사료된다. 주제어 : 유리물벼룩, 큰물벼룩, 생활사, 생태독성, 독성민감도 1. 서론 물벼룩은담수생태계에서식하는대표적수서무척추동물로조류 (algae) 를섭취하는 1차소비자임과동시에어류의피식자로서먹이사슬에서매우중요한역할을하므로화학물질의생태독성평가에빈번하게사용되는생물종이다. 1,2) 현재경제협력개발기구 (OECD), 3,4) 국제표준화기구 (ISO) 5,6) 등국제기관에서는 Daphnia magna( 큰물벼룩 ) 를표준시험종으로추천하고있으며, 실제연구에서도 D. magna는가장많이사용되고있다. 우리나라환경부의 수질및수생태계보전에관한법률시행규칙 에근거하여수질오염물질의배출허용기준 7) 과폐수종말처리시설의방류수수질기준 8) 에서생태독성을평가하도록기준이마련되어있는데, 이때수질오염공정시험기준에따라 D. magna를독성시험에사용하도록제시하고있다. 9,10) 그러나, D. magna는미북반구에주로서식 10) 하고국내수계에는서식하지않는물벼룩 11) 으로국내담수생태계에대한영향을직접적으로평가하기에는한계가존재한다. 현재국내서식종인 Moina macrocopa( 모이나물벼록 ) 를대상으로한고유종시험종연구가진행되고있으나, 10) 국내에서식하는물벼룩표준시험종은지리적분포, 대표성, 인공배양가능성, 독성민감도등문제로인하여매우제한적이므로, 국내담수생태환경을반영할수있는국내시험종의도입방안에대한연구가필요하다. 국내수계에서식하는것으로알려진물벼룩은총 29속 53 종으로 Daphnia galeata, Daphnia pulex, Bosmina longirostris, Corresponding author E-mail: anyjoo@konkuk.ac.kr Tel: 02-2049-6090 Fax: 02-2201-6295
194 J. Korean Soc. Environ. Eng. 최영설 곽진일 안윤주 Diaphanosoma brochyurum, Moina macrocopa 등이있다. 12) 본연구에서는국내우점종이며 D. magna와동일한 Daphnia 속에속하는물벼룩인 D. galeata( 유리물벼룩 ) 를대상으로국내고유시험종으로서의적용가능성을평가하였다. D. galeata 는도감에의하면 몸은대체로긴달걀형이고꼬리길이가긴편이며각자는긴것이많다. 머리부와갑각사이는목홈에의한구별이명확하지않으며입이잘발달되어있다. 단안인것이보통이며꼬리발톱근처에 10~20개의미세한털이존재하고몸의뒤쪽등에는 1~4개의돌기가있으며수컷은긴제1촉각을가짐 으로보고되어있다. 13,14) D. galeata 는전형적인원양물벼룩 15) 이며습한바람이부는자연환경에서수면위 70-80 cm에서 10-50 cm 길이, 3-5 cm 넓이의무리를지어다니고한무리에는약 1,000-100,000마리의개체가있다. 16) Koivisto 15) 의연구에서는 D. galeata 성체의크기를 1.0-2.5 mm로보고하였으며, 국내연구 16) 결과에서는팔당호에서식하는 D. galeata의크기가 0.300-1.525 mm로측정된것으로확인되었다. D. galeata는전세계적으로신북구와구북구에분포되어있고, 18) 국내에서는한강수계의우점물벼룩이며, 팔당호, 17,19,20) 소양호, 21) 파로호, 20) 청평호, 22,23) 의암호, 23) 춘천호, 22,23) 충주호, 19,24,25) 주암호 14) 등대부분수계에서서식하고있는것으로확인되었다. 다수의문헌에서보고된바와같이 D. galeata는국내수계에우점하여서식하는종이지만, 국내에서 D. galeata를이용한생태독성연구는미비하며 12,26) 국외에서도많지않은것으로조사되었다. D. galeata를이용한독성연구는총 23건으로, 물질별로는중금속류가 10건, 유기화합물관련독성연구가 12건, 나노물질독성연구가 1건으로확인되었다 (Table 1). 따라서본연구에서는 D. galeata를국내고유생태독성시험종으로제안하고자 D. galeata를채집및배양을하였으며, 표준시험종인 D. magna와의특성, 생활사및독성민감도를비교하였다. 한편, 본연구결과는 국내수생생물보호를위한환경기준마련연구 27) 에서국내수생생물보호기준제안을위한기반자료로활용되었다. 2. 연구방법 2.1. Daphnia galeata 채집및배양 D. galeata는 2012년 5월에두차례에걸쳐건국대학교교내에위치한호수 ( 일감호 ) 에서동물플랑크톤채집망을이용하여채집하였다. 채집개체는육안, 현미경관찰, 기존문헌및도감분석을통해 D. galeata로추측되는개체를분리하였다. 채집 0일부터 5일째까지는 GF/F filter (Whatman FG/F filter, Whatman) 로여과한일감호호숫물에서 D. galeata로추측되는개체를배양하면서실험실환경에서의배양적합성을확인하였다. 채집후 6일째부터는일감호호숫물과 modified moderately hard water (MHW) 28) 을 4:1, 2:1, 1:1, 그리고 1:2의비율로혼합한배양액 (media) 에서순차적으로배양하여, 채집한 D. galeata로추측되는개체가실험실조 Table 1. 48h-E(L)C50 values for Daphnia galeata exposed to chemicals in the previous studies Heavy metal Test chemical Arsenic Cadmium Chromium 48h-E(L)C50 (95%) (µg/l) 610 (530-700); 730 (660-820) Test method OECD a) 26) 30 (30-30); 30 (30-30) OECD 26) 40-53) - - 12) 110 (100-130); 210 (200-220) OECD 26) - - 54) 30 (30-30); 30 (30-40) OECD 26) 22.6; 124 OECD 35) 22.6 OECD 55) Copper 3.2 (2.9-3.7); 6.9 (5.9-8.2); 10.1 (8.9-11.5); 10.3 (9.0-11.8) OECD 56) 1.5 OECD 57) - - 58) Copper 4.1, 7.4 SFS b) 15) Mercury 20 (10-20); 20 (10-20) OECD 26) Nickel 1400 (1330-1470); 1820 (1750-1890) OECD 26) Silver 1.71 (1.37-2.21) OECD 73) Zinc Fenvalerate 2,3,4,6-TeCP 140 (130-160); 280 (260-300) OECD 26) 1001 OECD 59) 1001 OECD 55) 730 (685-779) ISO c) 60) 0.16 (0.09-0.29); 0.29 (0.23-0.35) - 61-63) 330; 510-64) 590-65) 580-66) Organic chemical Nonylphenol LAS12-4600 - - 67) 68) p-nonylphenol 60.8; 67.1-69) λ-cyhalothrin 117 (86.6-157); 397 (267-590) - 70) Fluazinam 77 (66-91); 82 (51-132) - 71) Carbonyl 11.32 (10.90-11.90) - 71) Methomyl 11.99 (10.97-13.07) OECD 72) AgNP (15 nm) 13.9 (9.13-21.4) OECD 73) CuNP (25 nm) 12 Reference Nanoparticles CuNP (50 nm) CuNP (78 nm) 61 148-58) CuNP (100 nm) 40 CuNP (500 nm) 20 a) OECD guidelines for testing of chemicals, No. 202. Daphnia sp., acute immobilisation test b) SFS 5062 (1984). Water quality. Determination of the acute toxicity with water flea, Daphnia magna Straus. c) ISO 6341:1996. Water quality-determination of the imhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea)-Acute toxicity test. Journal of KSEE Vol.38, No.4 April, 2016
J. Korean Soc. Environ. Eng. 생태독성실험을위한한국우점종유리물벼룩 (Daphnia galeata) 의특성및독성민감도 : 큰물벼룩 (Daphnia magna) 과의비교연구 195 건과인공배양액인 MHW에순응할수있도록하였다. 이후인공배양액인 100% MHW에서의배양 유지가성공함에따라물벼룩분류전문가의도움으로동정평가를진행하여 D. galeata종임을검증받았다. 실험실내에서배양시온도는 21, 명암광주기는 16:8 시간으로하였으며, 먹이는담수녹조류인 Chlamydomonas reinhardtii, Chlorella vulgaris 및 Pseudokirchneriella subcapitata를공급하여지속적으로계대배양하였다. 2.2. 생활사 (Life span) 비교 100% MHW와실험실조건에서 D. galeata 의순응완료후 D. galeata의생활사를측정하였으며, 유사조건에서의 D. magna 생활사 29) 와비교분석하였다. 본연구에서는 D. galeata 의생활사를측정하기위해 35 ml glass vial (30(D) mm 75(H) mm) 에 20 ml의 MHW를첨가후 3배 (brood) 이후의 24시간미만의어린 (neonate) D. galeata 암컷개체를 1마리씩노출하여총 29반복을관찰하였다. D. galeata 수컷의경우암컷보다상대적으로긴제1촉각을갖고있으므로, 14) 현미경을이용하여제1촉각의길이를확인하여암컷을선별하였다 (Fig. 1). 생활사관찰동안온도 21, 명암광주기 16:8 시간조건으로유지하였으며, MHW는 2일에한번씩교체하였다. 먹이는반복수당매일 50 µl의 C. reinhardtii 를공급하였으며 24시간마다유영장애, 치사및새끼생산수를확인하였다. 한편, D. galeata의형태학적특성은기존문헌조사를통해분석하였다. 2.3. 독성민감도비교 D. galeata를국내고유생태독성시험종으로제안하기위해서는기존에사용되고있는 D. magna의독성민감도와비교가필요하다. 본연구에서는 D. galeata와 D. magna의독성민감도를비교하기위해수계주요오염물질인중금속 7종 (As 3+, Cd 2+, Cr 6+, Cu 2+, Hg 2+, Ni 2+, Zn 2+ ) 을선택하였으 며, 각물질에대한물벼룩의 48h-E(L)C50값을조사, 비교하였다. 각물질별 D. magna에대한 48h-E(L)C50값은 US EPA ECOTOX Database (http://cfpub.epa.gov/ecotox/) 에서수집하였으며, 수집된자료중중금속무기염을이용한독성값만민감도비교에활용하였다. 수집된자료중제시된 48h-E(L)C50는 As 3+, Cd 2+, Cr 6+, Cu 2+, Hg 2+, Ni 2+ 및 Zn 2+ 별로각각 16, 217, 57, 404, 30, 21 및 111개로확인되었다. 30) 반면, D. galeata를이용한 7종중금속에대한 48h-E(L)C50 독성자료는 Table 1에제시된바와같이매우제한적이다. 3. 결과및고찰 3.1. Daphnia galeata와 Daphnia magna의형태학적특성및생활사비교 Malone 16) 의연구에의하면, D. galeata의머리모양은두 가지형태로구분할수있다. 한종은머리가둥글고다른 한종은삼각형모양이나두종모두머리끝부분이뾰족한 것으로알려져있다. 본연구에서채집한어린 (neonate) D. galeata 또한두가지다른형태의머리모양을한 D. galeata 가모두관찰되었으나개체가성숙됨에따라머리가점차둥글어져뾰족한부분이사라지는것으로확인되었다 (Fig. 1). D. galeata는 D. galeata Sars, D. galeata mendotae, D. galeata galeata 31) 등여러아종이있는데, 머리의모양이다 른것이관찰된것은 D. galeata 의아종에따른특성이거나계절적, 북방적변이에의한것으로보고되었다. 32) 기존연 구의조사결과, D. galeata 성체 (adult) 의강모간격및필터 메쉬크기 (filter mesh size) 는각각 0.4-1.3 µm과 0.32-1.0 µm이며, D. magna의강모간격및필터메쉬크기는각각 0.3-0.8 µm, 0.24-0.64 µm 로 D. magna 의강모간격및필터 메쉬보다 D. galeata 가큰것으로나타났지만, 성체의크기 는 D. galeata 가 1.0-2.5 mm 로 D. magna (2.0-3.0 mm) 보다 Fig. 1. Images of female and male Daphnia galeata and Daphnia magna. (A) D. galeata female at 24 h; (B) D. galeata male at 24 h; (C) D. galeata female at 21 d, (D) D. magna female at 24 h; (E) D. magna male at 24 h; (F) D. magna female at 21 d. 대한환경공학회지제 38 권제 4 호 2016 년 4 월
196 J. Korean Soc. Environ. Eng. 최영설 곽진일 안윤주 작은것으로확인되었다. 15,33) 본연구에서도실험실에서배 양중인 D. galeata 와 D. magna 를대상으로부화한지 24 시 간및 21 일째되는암컷을현미경으로관찰한결과, Fig. 1 에서와같이 D. magna 가 D. galeata 보다큰체구를가지고 있음을확인하였다. 본연구에서 D. galeata의생활사를관찰한결과, MHW 에서평균 28 ± 8일생존하는것으로확인되었으며, 첫배 (brood) 시간은 9 ±2 일로나타났다. 또한생활사동안에어 미개체당총새끼생산수는 29 ± 23마리, 배 (brood) 당 4±1 마리씩 2±1일간격으로새끼를생산하였으며 (Table 2), 배 반복 (number of broods produced) 은최대 16 번으로확인되 었다 (Fig. 2). 본측정결과는, 기존연구결과 34) 와도유사한데, 임 34) 의연구에서는여과한현장시료를이용하여 D. galeata 생활사를확인한결과평균 20일, 최대 40일정도생존하였 다고보고하고있다. 한편, 최와임 11) 의연구에서는 MHW 에 서 D. galeata 가 7 일밖에생존하지못하였고, 짧은생존기 간동안생식도관찰되지않았지만, M4 및비료를이용한 배양액에서는각각 21, 34 일동안의생존과 29, 42 마리새 끼생산을보고한바있다. MHW 에서의 D. magna 생활사 의경우, 기존연구결과를통해분석하였으며, 평균생존은 50.1 ± 10.9 일, 첫배시간은 9.0 ± 0.0 일, 어미당총새끼생 Table 2. Life span, first brood, total number of offspring per adult, and the number of offspring per broods of Daphnia galeata and Daphnia magna. Daphnia galeata a) Daphnia magna 29) Life span (days) 28±8 50.1±10.9 First brood (days) 9±2 9.0±0.0 Total number of offspring per adult 29±23 357.8±104.7 Number of offspring per brood 4±2 13.3±3.7 a) Data from this study 산수는 357.8 ± 104.7 마리, 배반복은평균 13.3 ± 3.7 회로확 인되었다 (Table 2). 29) D. galeata 와 D. magna 의생활사를비 교하면두종의첫배시간은비슷하지만 D. galeata 의생존 기간은 D. magna 의절반수준으로따라서배반복과총새끼 생산수가훨씬적은것으로확인되었다. 3.2. Daphnia galeata와 Daphnia magna의독성민감도비교기존연구결과를이용하여 7종중금속 (As 3+, Cd 2+, Cr 6+, Cu 2+, Hg 2+, Ni 2+, Zn 2+ ) 에대한 D. galeata와 D. magna의 48h-E(L)C50 비교결과 (Fig. 3), 중간값 (median) 을기준으로 Cd 2+, Cr 6+ 및 Ni 2+ 에대해 D. galeata와 D. magna의독성민감도는유의한차이는없는것으로나타났다. 그러나 As 3+, Cu 2+ 및 Zn 2+ 에대해서는 D. galeata가 D. magna보다민감하였으며, Hg 2+ 에대해서는 D. galeata가 D. magna보다약간둔감한것으로확인되었다. 한편평균값 (mean) 을기준으로독성민감도를비교할경우, Hg 2+ 에대해 D. magna보다둔감한것으로분석되었으나, 그외 6종중금속은 D. magna 보다민감한것으로나타났는데, 이는생체량차이로인해민감도차이가발생한것으로사료된다. 이러한결과는기존연구결과와도부합되는바 Bossuyt 등 35) 의연구에서현장수 (field water) 에서직접채집한 D. magna와 D. galeata를이용하여현장수와 ISO 배양액에서의구리독성평가를진행하여 48h-EC50 비교결과, D. galeata가 D. magna보다민감한것을관찰한바있다. 다만물벼룩에대한중금속독성은시험수의기본물리적특성인 ph, 36~46) 경도, 39,40,42,43,45,47,48) 알칼리도, 43,48) DOC, 36,38~40,44~46,49) DOM 45,47,49~51) 등과화학적특성인 Na +, 36,37,41,44,46,48) K +, 37,41,44,46) Mg 2+, 37,41,48,52) 2+ 37,38,41,44,46,48,52) Ca 의영향을받으며, 본연구에서제시된독성민감도결과 (Fig. 3) 또한동일한조건에서의독성민감도비교결과가아니라는한계점이존재한다. 현재까지 D. galeata 관련독성연구는제한적이며 (Table 1), 따라서본연구에서 D. magna 와의독성민감도비교에활용된 D. galeata의독성자료도부족한실정으로, 향후 D. galeata를이용한독성자료생산이필요하며, 더많은독성자료를활용한 D. magna와의독성민감도비교가요구된다. 3.3. 국내생태독성시험활용성 Fig. 2. Number of offspring per adult at each number of broods produced. The italic number under the each box plot indicates number of survivors of adult Daphnia galeata. Life span test was conducted until the last adult was dead. 7종중금속에대한독성민감도비교분석결과, D. galeata 는표준시험종으로사용되고있는 D. magna보다민감하거나유사한것으로확인되었다. 그러나본연구에서 D. galeata 를채집, 배양, 유지하고독성시험을진행하는과정에서, 생태독성시험에적용하기에 D. galeata는개체특성상몇가지제약이존재하는것으로확인되었다. 첫째로 D. galeata 의 entrapment 현상이다. D. galeata의경우자연환경이아닌실험실조건에서 D. magna와달리 entrapment 현상이쉽게발생하는것으로관찰되었다. Entrapment 현상이발생하면개체는실험용액의표면장력으로인하여수면에갇히게되 Journal of KSEE Vol.38, No.4 April, 2016
J. Korean Soc. Environ. Eng. 생태독성실험을위한한국우점종유리물벼룩 (Daphnia galeata) 의특성및독성민감도 : 큰물벼룩 (Daphnia magna) 과의비교연구 197 Fig. 3. 48 h-e(l)c values distribution of (A) As, (B) Cd, (C) Cr, (D) Cu, (E) Hg, (F) Ni, and (G) Zn for Daphnia galeata and Daphnia magna. The Daphnia galeata data were collected from Cui et al. 26) and Daphnia magna data were from USEPA ECOTOX. 30) 는데자체의힘으로는수면아래로벗어나지못하여유영장애가발생하며수면에갇히는시간이점차증가함에따라최종치사에이르는것으로관찰되었다. 따라서 D. galeata 를생태독성시험에적용하려면 entrapment 현상을제어해야하는데, 이러한현상은 1 mg/l의 saponin을시험용액에 첨가하면충분히제어가되었으며, 적용된 saponin농도에의한영향은무시할수있는수준으로확인되었다. 26) 두번째로확인된제약은 D. galeata의생존기간및새끼생산성이 D. magna보다상대적으로짧고적기때문에실험에필요한개체확보가 D. magna보다어려울수있다는것이다. 그러 대한환경공학회지제 38 권제 4 호 2016 년 4 월
198 J. Korean Soc. Environ. Eng. 최영설 곽진일 안윤주 나이제약은배양및유지개체수를늘리는방법으로충분히해결가능한것으로확인되었다. 따라서국내수계에우점하는 D. galeata는국내생태독성표준시험종으로충분히활용가능할것으로판단된다. 4. 결론 현재우리나라는 수질및수생태계보전에관한법률시행규칙 상수질오염물질의배출허용기준 7) 과폐수종말처리시설의방류수수질기준 8) 평가시수질오염공정시험기준에따라 D. magna를독성평가에사용하고있다. 9,10) 그러나 D. magna는국내수계비서식종으로시험물질이국내수생태계에대한영향을직접적으로반영하기에는한계가있다. 따라서본연구에서는생활사관찰및독성민감도비교분석을통해국내담수생태계의우점종이며실험실조건에서계대배양이가능한 D. galeata를국내고유생태독성시험종및향후국내수생생물보호기준설립에활용가능한종으로제안하였다. 한편, D. galeata는 국내수생생물보호를위한환경기준마련연구 (I) 27) 에서국내서식종을이용한독성자료생산에서풍년새우 (Branchinella kugenumaensis), 미꾸리 (Misgurnus anguillicaudatus) 와함께연구된바있다. 우리나라생태독성관리제도가확대되기위해서는국내시험종발굴에대한연구가절실하며, 국내수계에우점하는생물종을대상으로실험실배양조건확립및국제표준시험종과의비교연구가앞으로필요할것이다. Acknowledgement This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF), which is funded by the Ministry of Science, ICT, and Future Planning (2014R1A2A1A11050513) and the Ministry of Education (2013R1A1A2061386). The authors acknowledge their gratitude to Prof. Seong Myeong Yoon in Chosun University, Korea for his species identification. References 1. Sarma, S. S. S. and Nandini, S., Review of recent ecotoxicological studies on cladocerans, J. Environ. Sci. Heal. B, 41(8), 1417~1430(2006). 2. An, Y.-J., Nam, S.-H. and Lee, W.-M., Fundamentals of ecotoxicity evaluation methods using domestic aquatic organisms in Korea: (II) Water flea, Korean J. Limnol., 40(3), 357~ 369(2007). 3. Organization for Economic Cooperation and Development (OECD), Guideline for testing of chemicals 202 Daphnia sp., Acute immobilisation Stest, (2004). 4. Organization for Economic Cooperation and Development (OECD), Guideline for testing of chemicals 211 Daphnia magna reproduction test, (2012). 5. International Organization for Standardization (ISO), ISO 10706:2000 Water quality-determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea), (2000). 6. International Organization for Standardization (ISO), ISO 6341:2012 Water quality-determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea), (2012). 7. Ministry of Environment, Enforcement regulations of the water quality and aquatic ecosystem conservation act. annexed Table 13-the standards for permissible discharge of water pollutants, (2013). 8. Ministry of Environment, Enforcement regulations of the water quality and aquatic ecosystem conservation act. annexed Table 10-the standards for the quality of discharged water of the wastewater treatment facilities (2013). 9. Ministry of Environment, Standard Method of Water pollution ES 04751.1 Acute toxicity test method of the Daphnia magna Straus (cladocera, crustacea), (2015). 10. Ministry of Environment, Whole effluent toxicity information system, https://www.biowet.or.kr/iwt/ko/test/egovtest rganism.do(2015). 11. Choe, S.-H. and Lim, B.-J., Reproduction of water flea by the culture conditions, Korean J. Limnol., 36, 208~214(2003). 12. National Institute of Environmental Research, Behavior assessment of water flea on toxicants, (2001). 13. Ministry of Environment, National Institute of Environmental Research, Han River Environment Research Center, Illustration of biota in the Lake Paldang, p. 158(2007). 14. Ministry of Environment, National Institute of Environmental Research, Yeongsan River Environment Research Center, It's nature! - Illustration of organisms in Yeongsan and Seomjingan River, p. 84(2009). 15. Koivisto, S., Ketola, M. and Walls, M., Comparison of five cladoceran species in short- and long-term copper exposure, Hydrobiologia, 248(2), 125~136(1992). 16. Malone, B. J. and McQueen, D. J., Horizontal patchiness in zooplankton populations in two Ontario kettle lakes, Hydrobiologia, 99(2), 101~124(1983). 17. National Institute of Environmental Research, Mechanism of algal occurrence and succession in Lake Paldang - Elucidation on structure and function of phyto- and zooplankton, (2003). 18. Kotov, A., Crustacea-cladocera checklist, FADA (Freshwater Animal Diversity Assessment)(2013). 19. National Institute of Environmental Research, Survey on the structure of ecosystem in middle-downstream of South-Han river and Lake Paldang, (2005). 20. National Institute of Environmental Research, Survey on the structure of ecosystem in middle-downstream of South-Han river and Lake Paldang, (2006). Journal of KSEE Vol.38, No.4 April, 2016
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