내분비계 장애물질 (Endocrine Disruptors: EDs)이란 ‘내분비 기능에 변화를 일으켜, 생체 또는 그 자손의 건강에 위해한 영향을 미치는 외인성 물질’로서 세계야생생물보호기금 (World Wildife Fund: WWF)의 목록에는 67 여종, 일본 후생성에서는 142 여종의 물질을 내분비계 장애물질로 분류하고 있다. 이렇게 분류된 내분비계장애물질 가운데에는 많은 종류의 농약이 포함되어 있으며, 이들이 자연상태계에 미치는 영향에 대한 많은 보고가 있다. 양서류는 먹이연쇄의 중위포식자로 내분비계 장애물질의 순환 및 생채축적 회로에서 중요한 위치를 갖는다. 또한 농경지나 계류 등에 서식하며 산란하는 습성이 있다. 이러한 서식지는 농약 등에 노출받기 쉬우므로 환경오염평가 대상동물로 중요하다. 본 연구에서는 국내에서 사용되고 있는 제초제, 살충제 등이 양서류 발생에 미치는 영향을 평가하고, 이들 물질에 의한 노출을 평가할 수 있는 biomarker 유전자를 발굴하고자 하였다. 우리나라 전역에 많이 분포하는 무당개구리(Bombina orientalis)의 초기 배아발생 과정에서 이들 농약을 처리하였을 때, 물질의 종류에 따라 정도의 차이는 있으나 농도 의존적으로 배아 및 올챙이의 치사율이 높게 나타났다. 또한 농약은 배아 및 올챙이의 기형을 유발하였다. 기형의 종류는 농약에 따라 서로 상이한 형태로도 나타났으나, 특히 몸통휨 또는 꼬리휨등의 척추골 기형이 많이 나타났다. 환경 위해성 평가 biomarker 유전자로서 골격계 형성에 관여하는 Sox9 유전자를 선택하였다. 무당개구리에서 처음으로 Sox9 유전자를 동정하였으며, 농약을 처리한 실험군에서의 Sox9 유전자의 발현 양상은 대조군에 비하여 높게 나타났다. 본 연구결과 생태계에서 내분비계 장애물질의 위해성 평가를 위해 양서류 초기배아의 치사 및 기형의 정도를 관찰하는 방법이 유효할 것으로 사료된다. 또한 야생동물의 biomarker 유전자를 발굴과 이를 이용한 위해성 평가는 더욱 정밀한 평가법으로 유용할 것이다.

The egg bags of Korean salamander(Hynobius leechii) were collected from farmlands in Gyeongsangnam-do area. The assumed breeding time, numerical variation of embryos in each egg bag, mortality and the rates of abnormalities were investigated. The toxicity of benomyl, the metabolite carbendazim and BIC which were frequently spread in agricultural area and caused spontaneous embryonic malformation was investigated. The assumed breeding time between the end of February and the end of March has the difference about a month because of a habitat and it takes about 2 or 3 weeks from laying eggs to hatching. The length of each egg bag and the number of embryos were very varied in each area. It is due to geographical variation. Among egg bags in total study area, only 406 of egg bags(17.70% of total egg bags) developed all of embryos to normal larvae, and 78.49% of total embryos were normally developed. The patterns of spontaneous embryonic malformation were 26 species from A to Z and the abnormal patterns in individual were 8 species and above. the geographical differences about the abnormal pattern were identified and 11 habitats categorized 4 groups. The most frequent abnormality in Gyeongsangnam-do area is the dysplasia of external gill. The caudal dysplasia, abdominal blister and dysplasia of fin were also frequently observed. Individuals showing severe external defect were histologically studied and they showed retinal hypo-pigmentation, thyroid carcinoma, somatic muscular dysplasia, degeneration of cephalic neuron and various organ dysplasia. Benomyl and carbendazim were treated by 10pM$^{\sim}$10uM and BIC was treated by 1$^{\sim}$40ppm to know the effect of toxicity about toxic substance of salamander. After benomyl was treated, a survival rate was sharply dropped from 2 to 8 days. $LC_{100}$ identified in $1{\mu}M$, $LC_{50}$ identified between 100nM and $1{\mu}M$. $EC_{50}$ was assumed between 10nM and 100nM. The prevalent external malformation was abdomen swelled abnormally and histo-pathological effects were abdomen, neural tube and lens hernia. This suggests that benomyl is the toxicitic substance which inhibits the development of digestive system and nervous system. The result of treated carbendazim was similar to that of the treated benomyl. The survival rate is sharply dropped between 2 and 6 days. $LC_{100}$ was identified $1{\mu}M$ and $LC_{50}$ was identified between 10nM and 100nM. This shows that cabendazim has stronger lethal toxicity than benomyl. Ventral blister, eye dysplasia and cephalic dysplasia in the individual of external malformation mean that cabendazim affected nervous system much more than benomyl. Because the toxicity of BIC affected less in the beginning but affected more in the near hatching period, the period causing toxicity is somewhat different. $LC_{100}$ identified near 40ppm and $LC_{50}$ identified near 25ppm. The external defect shows mainly ventral blister and histo-pathological results show intestinal deformities. This result suggests the BIC inhibited strongly the development of digestive system. These abnormal developments may be caused by antimitotic action, inhibition of tubulin complex, destruction of microtubule, inhibitions of neurulation and closing of neural fold, and by the inhibition of movement of neural crest cells of benomyl. These abnormal developments may be caused by the rupture of epithelium, the loss of microtubule, the reduction of spindle size, the inhibition of spindle assembly formation, the destruction of spindle poles of carbendazim. These abnormal developments may be caused cytotoxicity by inhibition of the synthesis of a number of macromolecules and similar reaction the inhibition of benomyl.