Ecotoxicological evaluation of tributyltin toxicity to the equilateral venus clam, Gomphina veneriformis (Bivalvia: Veneridae)

Park, K.,Kim, R.,Park, J.J.,Shin, H.C.,Lee, J.S.,Cho, H.S.,Lee, Y.G.,Kim, J.,Kwak, I.S. Academic Press 2012 Fish & shellfish immunology Vol.32 No.3

Tributyltin (TBT) is the most common pesticide in marine and freshwater environments. To evaluate the potential ecological risk posed by TBT, we measured biological responses such as growth rate, gonad index, sex ratio, the percentage of intersex gonads, filtration rate, and gill abnormalities in the equilateral venus clam (Gomphina veneriformis). Additionally, the biochemical and molecular responses were evaluated in G. veneriformis exposed to various concentrations of TBT. The growth of G. veneriformis was significantly delayed in a dose-dependent manner after exposure to all tested TBT concentrations. After TBT was administered to G. veneriformis, the gonad index decreased and the sex balance was altered. The percentage of intersex gonads also increased significantly in treated females, whereas no intersex gonads were detected in the solvent control group. Additionally, intersex gonads were detected in male G. veneriformis specimens exposed to relatively high TBT concentrations (20 μg L<SUP>-1</SUP>). The filtration rate was also reduced in a dose-dependent manner in TBT-exposed G. veneriformis. We also noted abnormal gill morphology in TBT-exposed G. veneriformis. Furthermore, increases in antioxidant enzyme activities were observed in TBT-exposed G. veneriformis clams, regardless of dosage. Vitellogenin gene expression also increased significantly in a dose-dependent manner in G. veneriformis exposed to TBT. These results provide valuable information regarding our understanding of the toxicology of TBT in G. veneriformis. Moreover, the responses of biological and molecular factors could be utilized as information for risk assessments and marine monitoring of TBT toxicity.

수온과 염분의 변화에 따른 연령별 대복 (Gomphina veneriformis: Bivalvia) 의 여과율 변동

신현출,이정호,정효진,이정식,박정준,김배훈,Shin, Hyun-Chool,Lee, Jung-Ho,Jeong, Hyo-Jin,Lee, Jung-Sick,Park, Jung-Jun,Kim, Bae-Hoon 한국패류학회 2009 The Korean Journal of Malacology Vol.25 No.2

동해안의 유용 이매패류인 대복 (Gomphina veneriformis; Bivalvia)을 대상으로 기초 생리, 생태적 특성을 파악하기 위해 수온과 염분에 따른 대복의 여과율 변동을 측정하였다. 실험에 사용된 먹이생물은 황색편모조강 Isochrysis galbana (KMCC H-002)를 f/2배지를 이용하여 고농도로 단일배양한 후 사용하였다. 여과율 측정은 간접 측정법을 이용하였고, 실험에 사용된 먹이생물은 광학현미경 하에서 혈구계산판을 이용하여 계수하였다. 수온별 대복의 여과율은 2, 3, 4년생 모두 저온에서 수온이 상승할수록 여과율이 급격히 증가하여 $15^{\circ}C$에서 최대로 나타났으며, 이 후 감소하는 동일한 경향을 보였다. 염분별 대복의 여과율은 2, 3, 4년생 모두 30-35 psu의 염분범위에서 최대로 나타났다. 연령별 대복의 여과율은 수온, 염분변화에 관계없이 2년생 대복이 가장 높은 여과율을 기록했고, 4년생 대복의 여과율이 가장 낮았다. 다항회귀분석 결과 수온별 대복의 여과율 곡선은 모든 연령군에서 대체적으로 저온 구간으로 치우친 경향을 나타내었고 온도계수 $Q_{10}$ 값 역시 저온에서 높게 나타나 저온의 영향을 많이 받는 것으로 나타났다. 염분별 여과율 곡선은 모든 연령군에서 대체적으로 고염 구간으로 치우친 경향이 나타나 저염분으로 갈수록 여과율이 감소한다는 것을 알 수 있었다. 본 연구의 결과로 조하대 부유섭식 이매패류인 대복은 비교적 저온에서 서식하며 넓지 않은 온도범위에서 서식하는 협온성생물이며, 좁은 염분범위에서 높은 여과율을 보이는 협염성종임을 확인할 수 있었다. The present study was performed to describe the influence of water temperature and salinity on filtration rates of the venus clam, Gomphina veneriformis, a suspension-feeding (filter-feeding) bivalve species. The calmswere collected from the eastern coastal area of Sokcho, Gangneung and Jumunjin at Kangwon-do, Korea, during December 2006 and May 2007. Isochrysis galbana (KMCC H-002) cells as food organisms were indoor-cultured by f/2 medium, and were used to measure the filtration rate of clam. Filtration rates of clam were measured by indirect method. Cell concentration of food organisms were determined by direct counting cells used the hemacytometer under the light microscope. The filtration rates of clams by water temperature sharply increased with temperatures up to $15^{\circ}C$ as optimum temperature and above this temperature, the filtration rates decreased exponentially. Venus clams showed very low filtration rates at low salinity (10-15 psu) and maximum values at high salinity (30-35 psu). Regardless of water temperature and salt change, 2-year class clams showed high filtration rates, but low in 4-year-class. Polynomial regression curves with water temperature were shifted to the left in low temperature region. Thermal coefficient $Q_{10}$ values showed much higher values at low temperature range than at high temperature range, too. These results indicate that the venus clam is more sensitive in cold water. Polynomial regression curves with salinity were shifted to the right in high saline region. According to this study, the venus clam Gomphina veneriformis, subtidal filter-feeding bivalve, was the stenothermal organism, inhabited mainly in low temperature and the stenohaline, in high saline waters.

TBTCl 독성에 의한 대복, Gomphina veneriformis 소화선의 미세구조적 변화 및 지방갈색소 축적

박정준,이정식 한국패류학회 2010 The Korean Journal of Malacology Vol.26 No.1

The purpose of this study was to investigate the effect of tributyltin (TBT) on histopathological and ultrastructural changes in the digestive gland structure of the equilateral venus, Gomphina veneriformis. Experimental period was 36 weeks. Experimental groups consist of control condition and 3 TBTCl exposure conditions (0.4, 0.6, 0.8 TBTCl μg L-1). Outer envelop of the visceral mass of G. veneriformis exposed to TBTCl was observed disappearance of microvilli and cilia, decrease of mucous cell and partially destruction of epithelium. In the digestive gland showed an increase of number of hemocyte and mucopolysaccaride near the digestive tubule at early time of the exposure. Especially, in 0.8 μg TBTCl L-1 group, collapse of digestive tubule with modification of epithelium was observed. TEM observation revealed the numerous glycogen granules in epithelium of the outer envelop and connective tissue. In the ciliated cell of the primary duct formed the cilia in cytoplasm. Basophilic cell was observed destruction of the rough endoplasmic reticulum and mitochondria. Also, nucleus in the epithelium of the digestive tubule was disappeared heterochromatin and nucleolus, and condense. As the concentration of TBTCl increased,the accumulation of lipofucin increased in the digestive gland, but the collapse of digestive tubule induced a decrease of accumulation of lipofuscin.

대복, Gomphina veneriformis의 성장과 외투막 구조에 미치는 TBTCl의 독성

박정준,이정식 한국패류학회 2008 The Korean Journal of Malacology Vol.24 No.3

The results of the study were confirmed that tribultyltin chloride (TBTCl) induce reduction of growth and histopathological features in the mantle structure of the equilateral venus, Gomphina veneriformis. The experiments were taken for 36 weeks, commencing on October 25, 2005. Exposure concentrations consisted of 4 TBTCl exposure groups (0, 0.4, 0.6, 0.8 μg L-1). In this study, a regression analysis by power function on SPSS was applied to the shell length data in all experimental groups. For histological analysis, mantle tissues were characterized using H-E stain, AB-PAS (pH 2.5) reaction and Masson's trichrome stain and epidermal layer thickness and mucous cell distribution were analysed using the image analyser. The shell length growth was significantly different the control and TBTCl exposure groups from 12 weeks (P<0.05). The mantle had 4-folds (inner-inner, inner-outer, middle, outer) which epidermal layer consisted simple epithlia. A periostracum was originated in the periostracal groove where between the middle fold and the outer fold. The inner epidermal layer consisted simple ciliated columnar epithelia, but the outer epidermal layer consisted simple non-ciliated columnar epithelia. The mucous cell was observed alcian blue positive (7462c, 653c) and violet (2583c) in folds and alcian blue positive (647c, 7455c) in inner epidermal layer. After 12 weeks, mantle showed extension of hemolymph sinus, and increased blue (7455c) and violet (2587c) mucous cells in inner plica of the middle fold. Cilia and striated border was disappeared and mucous cell in inner epidermal layer was reduced. Especially, at 36 weeks, a serous histopathological problem in middle and outer fold near the periostracum was observed. Moreover, epidermal layer thickness and mucous cell distribution illustrated an a tendency to be decreased as TBTCl exposure time increased. Results of this study suggest that TBTCl induce growth disorder with histopathological changes.

대복, Gomphina veneriformis의 생존 및 저질 잠입성에 미치는 TBTCl의 독성

박정준,진영국,이정식 한국어병학회 2008 한국어병학회지 Vol.21 No.1

This study was conducted to find out biological responses of bivalves exposed to organotin compound. The results of the study confirmed that tribultyltin chloride (TBTCl) induce reduction of survival rate and burrowing activity, and histopathological feature in the foot structure of the equilateral venus, Gomphina veneriformis. The experimental period was 36 weeks. The experimental groups consisted of a control and 3 TBTCl exposure groups (0.4 ㎛ TBTCl L-¹, 0.6 ㎛ TBTCl L-¹, 0.8 ㎛ TBTCl L-¹). The survival rate and activity were recorded daily. For histological analysis, foot tissues were fixed in Bouin’s fluid and then stained H-E stain and AB-PAS (pH 2.5) reaction after having serially sectioned the tissue by paraffin method at thickness of 4~6 ㎛. The survival rate was not significantly different among the control and exposure groups for 20 weeks, but in 0.8 ㎛ TBTCl L-¹, it was on the decreased ever since the exposure. The burrowing activity was not significantly different in the exposure group compared to the control up to 12 weeks, but in 0.6, 0.8 ㎛ TBTCl L-¹, it measured the lowest level after 20 weeks. The foot is composed of the epidermal layer, connective tissue, and muscular layer. The epidermal layer is composed of simple columnar, cuboidal epithelium and mucous cells. The cilia were well developed on the apical surface of epithelium. Circular, longitudinal and transverse muscle bundle were well developed in the muscular layer. The majority mucous cells showed blue color (542c) when it subjected to AB-PAS (pH 2.5) reaction. No histopathological alterations in the foot were observed before 12 weeks of exposure. After 20 weeks of exposure to 0.8 ㎛ TBTCl L-¹, the foot samples of exposed G. veneriformis showed disappearance of cilia and striated border partially and extension of hemolymph sinus. The mucous cell increased in the marginal of foot. At 28-week of exposure to 0.4 ㎛ TBTCl L-1, it observed weekly acid (564c), neutral (264c) and mixed mucous cell. At 36-week of exposure to 0.6 ㎛ TBTCl L-1, it showed fragmentation of the muscle and collagen fiber bundle, and also diappearance of cilia on epithelia and edema of epithelium in 0.8 ㎛ TBTCl L-¹.

대복, Gomphina veneriformis의 정자형성과정 및 정자 미세구조

박채규,박정준,이정용,이정식,Park, Chae-Kyu,Park, Jung-Jun,Lee, Jeong-Yong,Lee, Jung-Sick 한국현미경학회 2002 Applied microscopy Vol.32 No.4

우리나라 동해안에 가장 많이 서식하는 조개류인 대복의 정소구조와 정자형성과정을 광학현미경과 투과전자현미경으로 조사한 결과는 다음과 같다. 대복의 정소는 소성결합조직으로 구성된 다수의 정자형성 소낭을 가진다. 동일한 정자형성 소낭 내에서는 여러 단계의 생식세포들이 관찰되었다. 정원세포들은 정자형성 소낭벽에 부착되어 있으며, 커다란 핵과 뚜렷한 인을 가진다. 정모세포에서는 연접사복합체와 골기체의 발달을 확인할 수 있었다. 정세포의 핵은 전자밀도가 높은 과립상의 염색질로 구성되며, 정자변태과정 동안에 핵의 응축 및 첨체와 편모의 형성을 관찰할 수 있었다. 정소 내에서 완숙 정자들은 다발을 형성하고 있으며, 두부, 중편, 미부로 구성되어 있었다. 두부의 길이는 약 $8.5{\mu}m$로, 첨체부와 핵 부위로 구분된다. 첨체는 길이 약 $1.1{\mu}m$의 총알형태였다. 두부와 첨체 사이에서는 미세섬유로 구성된 첨체기둥이 확인되었다. 중편에는 4개의 미토콘드리아를 가지며, 꼬리의 횡단면은 "9+2"의 구조를 나타냈다. Spermatogenesis and sperm ultrastructure are investigated by means of light and transmission electron microscopy in the equilateral venus, Gomphina veneriformis which is dominant bivalve in the east coast of Korea. In the active spermatogenic season, testis consists of numerous spermatogenic follicles which is contains germ cells in the different developmental stage. The spermatogonia attached to spermatogenic follicle wall and has a large nucleus with electron-dense nucleolus. The spermatocytes are characterized by appearance of synaptonemal complex and well-developed Golgi complex. Nucleus of spermatid consists of numerous heterogeneous granules with high electron density. Karyoplasmic condensation, acrosome and flagellum formations are observed during spermiogenesis. Testicular matured sperms of sperm bundle consists of head, midpiece and tail. The head is about $8.5{\mu}m$ long and comprises a long nucleus and a bullet-like acrosome ($8.5{\mu}m$ in length). Acrosomal rod of microfilaments is observed in the lumen between nucleus and acrosome. The midpiece has four mitochondria. And tail has the typical '9+2' microtubule system.

대복, Gomphina veneriformis 아가미의 조직학적 변화와 산소소비율에 미치는 TBTCl의 독성

박정준,이정식 한국어병학회 2008 한국어병학회지 Vol.21 No.1

This study was conducted to find out biological response of bivalves exposed to tributyltin chloride (TBTCl). The results of the study confirmed that TBTCl induce the reduction of oxygen consumption rate and histopathological feature in the gill structure of equilateral venus, Gomphina veneriformis. The experimental groups consisted of a control and 3 TBTCl exposure groups (0.4 ㎛ TBTCl L-¹, 0.6 ㎛ TBTCl L-¹, 0.8 ㎛ TBTCl L-¹) and the experimental period was 36 weeks. For histological analysis, gill tissues were fixed in Bouin's fluid and then stained H-E stain, AB-PAS (pH 2.5) reaction and Masson's trichrome stain after having serially sectioned the tissue by paraffin method at thickness of 4~6 ㎛. The oxygen consumption rate was not significantly different between the control and exposure groups at 4 weeks, but in all exposure groups at 28 weeks, it was significantly different to the control. Gill of G. veneriformis had demibranch that attached two sheets of lamellae and a lamella was composed of numerous filaments, numbering 25 on average. The frontal filament zone had three types of cilia; frontal, latero-frontal and lateral depending on locations while the lateral cilia were the longest and largest in number. The mucous cells observed in filaments were more abundant in the frontal filament zone than abfrontal filament zone and mucous substances were showed blue color (542c) in AB-PAS (pH 2.5) reaction. Gill exposed to TBTCl was extended hemolymph sinus and increased hemocytes at 4 weeks, and then it showed increases of mucous cells and partially disappearance of frontal cilia. In the group of 0.8 ㎍ TBTCl L-¹ at 12 weeks, hypertrophy of frontal and latero-frontal epithelia was observed. Also it observed m decrease of mucous cell containing weekly acid mucosubstance and appeared partially destruction muscle fiber bundle. In the groups of 0.4 and 0.6 ㎍ TBTCl L-¹ at 36 weeks, it appeared partially modification of epithelia and in 0.8 ㎍ TBTCl L-¹ group, observed filaments that come out chitinous rod from disappearance of frontal and latero-frontal epithelia.

Induction of Intersex and Masculinization of the Equilateral Venus, Gomphina veneriformis (Bivalvia: Veneridae) by Zinc

주선미,Jung Jun Park,Jung Sick Lee 한국통합생물학회 2009 Animal cells and systems Vol.13 No.3

This study aims to find out the effect of heavy metals, as is the case of EDCs (endocrine disrupting chemicals), on reproductive endocrine disruption of aquatic animals. In the present experiment zinc, which is a heavy metal well known for its androgenous activity, was used. The experimental period was 24 weeks, starting in November during the inactive stage of the clam’s reproductive cycle. The experimental groups were composed of one control condition and three zinc exposure conditions (0.64, 1.07, and 1.79 mg/L). The sex ratio (F:M) was 1:1.06 in the control group and 1:1.70 in all the exposed group, illustrating the tendency for higher proportion of males with increases in zinc concentration. Gonad maturity was higher in 1.07, and 1.79 mg/L groups compared to the control group, with higher maturity observed in males than females. Intersex individuals made up 24.7% of the exposed group, while females exhibited a higher ratio than the males with increasing zinc concentration. The results of this study indicate that zinc functions as an androgenic effector on the reproduction of Gomphina veneriformis. This study aims to find out the effect of heavy metals, as is the case of EDCs (endocrine disrupting chemicals), on reproductive endocrine disruption of aquatic animals. In the present experiment zinc, which is a heavy metal well known for its androgenous activity, was used. The experimental period was 24 weeks, starting in November during the inactive stage of the clam’s reproductive cycle. The experimental groups were composed of one control condition and three zinc exposure conditions (0.64, 1.07, and 1.79 mg/L). The sex ratio (F:M) was 1:1.06 in the control group and 1:1.70 in all the exposed group, illustrating the tendency for higher proportion of males with increases in zinc concentration. Gonad maturity was higher in 1.07, and 1.79 mg/L groups compared to the control group, with higher maturity observed in males than females. Intersex individuals made up 24.7% of the exposed group, while females exhibited a higher ratio than the males with increasing zinc concentration. The results of this study indicate that zinc functions as an androgenic effector on the reproduction of Gomphina veneriformis.

대복 Gomphina veneriformis의 성비, 성 성숙, intersex 및 지방갈색소 침적을 이용한 nonylphenol의 위해성 평가

이정식 ( Jung Sick Lee ),박정준 ( Jung Jun Park ) 한국수산과학회 2007 한국수산과학회지 Vol.40 No.1

대복, Gomphina veneriformis의 생존 및 저질 잠입성에 미치는 TBTCI의 독성

박정준 ( Jung Jun Park ),진영국 ( Young Guk Jin ),이정식 ( Jung Sick Lee ) 한국어병학회 2008 한국어병학회지 Vol.21 No.1