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Molecular Phylogenetic Study of the Endangered Land Snail Satsuma myomphala Based on Metallothionein Gene. (Metallothionein 유전자를 기초로 한 멸종위기 육상 달팽이 Satsuma myomphala (거제외줄달팽이) 의 분자계통학적 연구)

  • Sang, Min Kyu;Kang, Se Won;Hwang, Hee-Ju;Chung, Jong Min;Song, Dae Kwon;Min, Hye Rin;Park, Jie Eun;Ha, Hee Cheol;Lee, Hyun Jun;Hong, Chan Eui;Ahn, Young Mo;Park, So Young;Park, Young-Su;Park, Hong Seog;Han, Yeon Soo;Lee, Jun Sang;Lee, Yong Seok
    • The Korean Journal of Malacology
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    • v.32 no.4
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    • pp.263-268
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    • 2016
  • Metallothionein (MT) family of metal-binding proteins are involved in maintaining homeostasis and heavy metal poisoning. Recently, MT has been considered as a biomarker that can identify a particular species, very similar to the use of cytochrome oxidase I (COI) gene. Satsuma myomphala species of land snails have been reported from North-East Asia, including South Korea and Japan. In particular, the land snail species have been known from only a limited area of Geoje Island, Gyeongsangnam-do province of South Korea. Genetic studies of S. myomphala has been limited with only 6 nucleotide, 2 protein registered on the NCBI server. For elucidating the genetic information of S. myomphala, we conducted RNA sequencing analysis using Illumina HiSeq 2500 next-generation platform. We screened the MT gene from the RNA-Seq database to confirm the molecular phylogenetic relationship. After sequencing, the de novo analysis and clustering generated 103,774 unigenes. After annotation against PANM database using BLAST program, we obtained MT sequence of 74 amino acid residues containing the coding region of 222 bp. Based on this sequence, we found about 53 sequences using the BLAST program in NCBI nr database. Using ClustalX alignment, Maximum-Likehood Tree of MEGA program, we confirmed the molecular phylogenetic relationships that showed similarity with mollusks such as Helix pomatia and H. aspersa, Megathura crenulata.

Silica and Iron Oxide Recovery and Mineral Carbonation from Serpentine Minerals Using Acid Dissolution and pH Swing Processes (산 처리와 pH 조절을 이용한 사문석군 광물로부터 규소와 철산화물 회수 및 광물 탄산화 연구)

  • Baek, Jiyeon;Jo, Yeonu;Lee, Jeongheon;Kwon, Nayoon;Kim, Yeram;Choi, Suk;Kim, Sunghee;Roh, Yul
    • Economic and Environmental Geology
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    • v.49 no.1
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    • pp.13-22
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    • 2016
  • The objectives of this study were to recover silica and iron oxides and $CO_2$ sequestration using serpentine via various acid dissolution and pH swing processes. Serpentine collected from Guhang-myeon in S. Korea were mainly composed of antigorite and magnetite consisting of $SiO_2$ (45.3 wt.%), MgO (41.3 wt.%), $Fe_2O_3$ (12.2 wt.%). Serpentine pulverized ($${\leq_-}75{\mu}m$$) and then dissolved in 3 different acids, HCl, $H_2SO_4$, $HNO_3$. Residues treated with acidic solution were recovered from the solution (step 1). And then the residual solution containing dissolved serpentine was titrated using $NH_4OH$. And pH of the solution increased up to pH=8.6 to obtain reddish precipitates (step 2). After recovery of the precipitates, the residual solution reacted with $CO_2$ and then pH increased up to pH=9.5 to precipitate white materials (step 3). The mineralogical characteristics of the original sample and harvested precipitates were examined by XRD, and TEM-EDS analyses. ICP-AES analysis was also used to investigate solution chemistry. The dissolved ions were Mg, Si, and Fe. The antigorite became noncrystralline silica after acid treatment (step 1). The precipitate at pH=8.6 was mainly amorphous iron oxide, of which size ranged from 2 to 10 nm and mainly consisting of Fe, O, and Si (step 2). At pH=9.5, nesquehonite [$Mg(HCO_3)(OH){\cdot}2(H_2O)$] and lasfordite [$MgCO_3{\cdot}H_2O$] were formed after reaction with $CO_2$ (step 3). The size of carbonated minerals was ranged from 1 to $6{\mu}m$. These results indicated that the acid treatment of serpentine and pH swing processes for the serpentine can be used for synthesis of other materials such as silica, iron oxides and magnesium carbonate. Also, This process may be useful for the precursor synthesis and $CO_2$ sequestration via mineral carbonation.

Exposure Assessment of Apple Orchard Workers to the Insecticide Imidacloprid Using Whole Body Dosimetry During Mixing/Loading and Application (전신복장법을 이용한 농약 조제 및 살포 과정 중 살충제 Imidacloprid에 대한 사과 과수원 농작업자의 노출 평가)

  • Lee, Jae Yun;Noh, Hyun Ho;Park, Hyo Kyoung;Jeong, Hye Rim;Jin, Me Jee;Park, Kyung-Hun;Kim, Jeong-Han;Kyung, Kee Sung
    • The Korean Journal of Pesticide Science
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    • v.20 no.3
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    • pp.271-279
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    • 2016
  • To evaluate exposure characteristics of the insecticide imidacloprid to apple orchard workers during treatment on orchard fields and evaluate its potential risk using a whole body dosimetry (WBD) method, 1,000-time diluted acephate+imidacloprid 25(20+5)% solutions were sprayed on 10 apple orchard fields in Cheongju with a speed sprayer at a rate of 3,000 L/ha/person, after put on clothes such as inner/outer clothes, personal air pump with a IOM sampler, nitrile glove and mask. Exposure test included mixing, loading and application steps. The test pesticide imidacloprid residues in the collected samples were analyzed with a HPLC-DAD. Recoveries ranged from 81.5 to 108.6% for analytical method validation and from 73.8 to 86.7% for field recovery. Total exposed amounts to mixer/loader and applicator were found to be 0.0014-0.0279% of total applied active ingredient of imidacloprid. Glove exposure of both mixer/loader and applicator was higher than the other parts. Margins of safety of mixer/loader and applicator were calculated to be 97-355 and 46-196, respectively, indicating that exposure risk of imidacloprid to apple orchard workers by spraying with a speed sprayer was very low.

Development of Multi-residue Analysis and Monitoring of Persistent Organic Pollutants (POPs) - Used Organochlorine Pesticides in Korea (국내에 사용되어 금지된 유기염소계 농약류의 다중분석법과 모니터링)

  • Lim, Sung-Jin;Oh, Young-Tak;Yang, Ji-Yeon;Ro, Jin-Ho;Choi, Geun-Hyoung;Ryu, Song-Hee;Moon, Byeong-Chul;Park, Byung-Jun
    • The Korean Journal of Pesticide Science
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    • v.20 no.4
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    • pp.319-325
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    • 2016
  • This study was conducted to investigate residual organochlorine pesticides in green house soil and oriental melon, green pepper, and lettuce. The majority of them were designated as persistent organic pollutants (POPs) by the international community at the Stockholm Convention on Persistent Organic Pollutant. Extraction and clean-up method were developed using the QuEChERS method for residual organochlorine pesticides (OCPs) in soil and oriental melon, green pepper and lettuce. Recovery of OCPs in greenhouse soil and oriental melon, green pepper, and lettuce ranged from 73.3-110.6%. Limit of detection (LOD) of OCPs in soil and 3 crops were 0.01-0.08 and $0.11-0.17{\mu}g/kg$. The residues of OCPs in oriental melon, green pepper and lettuce greenhouse soil were analyzed by the developed method, and dieldrin, ${\beta}-endosulfan$ and endosulfan sulfate were detected at 1.4-72.5, 0.1-78.7 and $0.0-214.1{\mu}g/kg$, respectively. The detection frequency of 3 compounds in soils were 52 (29.7%), 34 (19.4%) and 57 (32.6%) among 175 samples, respectively. However, these compounds were not detected in all crop samples. The residue level in 3 crops were lower than 1/58.8 of maximum residue level of them. These results showed that the OCPs residue in oriental melon, green pepper, and lettuce greenhouse soil were not as high as crop safety threatening.

Monitoring of Pesticides in the Yeongsan and Seomjin River Basin (영산강 및 섬진강 수계 중 농약 분포 조사)

  • Lee, Young-Jun;Choi, Jeong-Heui;Kim, Sang Don;Jung, Hee-Jung;Lee, Hyung-Jin;Shim, Jae-Han
    • Korean Journal of Environmental Agriculture
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    • v.34 no.4
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    • pp.274-281
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    • 2015
  • BACKGROUND: A lasting release of low levels of persistence chemicals including pesticides and pharmaceuticals into river has a bad influence on aquatic ecosystems and humans. The present study monitored pesticide residues in the Yeongsan and Seomjin river basins and their tributaries as a fundamental study for water quality standard of pesticides.METHODS AND RESULTS: Nine pesticides(aldicarb, carbaryl, carbofuran, chlorpyrifos, 2,4-D, MCPA, methomyl, metolachlor, and molinate) were determined from water samples using SPE-Oasis HLB(pH 2) and LC/MS/MS. Validation of the method was conducted through matrix-matched internal calibration curve, method detection limit(MDL), limit of quantification(LOQ), accuracy, precision, and recovery. MDLs of all pesticides satisfied the GV/10 values. Linearity(r2) was 0.9965- 0.9999, and a percentage of accuracy, precision, and recovery was 89.4-113.6%, 3.1-14.0%, and 90.8-106.2%, respectively. All pesticides exclusive of aldicarb were determined in the river samples, and there was a connection between the positive monitoring results and agricultural use of the pesticides.CONCLUSION: Monitoring outcomes of the present study implied that pesticides were a possible non-point pollutant source in the Yeongsan and Seomjin river basins and tributaries. Therefore, it is required to produce and accumulate more monitoring results on pesticides in river waters to set water quality standards, finally to preserve aquatic ecosystems.

Gas Chromatographic Method for Multiresidue Analysis of Unregistered Pesticides in Imported Agricultural Commodities (가스 크로마토그래피를 이용한 수입농산물 중 국내 미등록 농약의 다성분 동시분석법 적용)

  • Lee, Ji-Won;Kang, Jeong-Kyun;Kwon, Hyeyoung;Ro, Jin-Ho;Jin, Yong-Duk;Lee, Je Bong;Hong, Su-Myeoung;Kim, Teak-Kyum;Cho, Nam-Jun
    • The Korean Journal of Pesticide Science
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    • v.19 no.3
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    • pp.161-173
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    • 2015
  • The multiresidue method 4.1.2.2 in Korea Food Code was extended for the analysis of 24 unregistered pesticide residues. The method includes acetonitrile extraction, liquid-liquid partition, Florisil SPE clean-up and GC analysis. The limits of quantification (LOQ) range of the method was 0.02~0.05 mg/kg for orange, brown rice and banana. The linearity for targeted pesticides were $R^2$ > 0.99 at the level ranged from 0.05 to 5 mg/L. Recovery test was performed at two concentration levels of LOQ and 4~10 times of LOQ. Recoveries and relative standard deviations (RSDs) of target pesticides were acceptable, showing 70~120% range and less than 20%, respectively, except for ethiprole, picloram and sulcotrion. This method is effectively applicable to routine analysis of target pesticides in orange, brown rice and banana.

Evolutionary Explanation for Beauveria bassiana Being a Potent Biological Control Agent Against Agricultural Pests

  • Han, Jae-Gu
    • 한국균학회소식:학술대회논문집
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    • pp.27-28
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    • 2014
  • Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.

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Expression Profiling of MLO Family Genes under Podosphaera xanthii Infection and Exogenous Application of Phytohormones in Cucumis melo L. (멜론 흰가루병균 및 식물 호르몬 처리하에서 MLO 유전자군의 발현검정)

  • Howlader, Jewel;Kim, Hoy-Taek;Park, Jong-In;Ahmed, Nasar Uddin;Robin, Arif Hasan Khan;Jung, Hee-Jeong;Nou, III-Sup
    • Journal of Life Science
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    • v.26 no.4
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    • pp.419-430
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    • 2016
  • Powdery mildew disease caused by Podosphaera xanthii is a major concern for Cucumis melo production worldwide. Knowledge on genetic behavior of the related genes and their modulating phytohormones often offer the most efficient approach to develop resistance against different diseases. Mildew Resistance Locus O (MLO) genes encode proteins with seven transmembrane domains that have significant function in plant resistance to powdery mildew fungus. We collected 14 MLO genes from ‘Melonomics’ database. Multiple sequence analysis of MLO proteins revealed the existence of both evolutionary conserved cysteine and proline residues. Moreover, natural genetic variation in conserved amino acids and their replacement by other amino acids are also observed. Real-time quantitative PCR expression analysis was conducted for the leaf samples of P. xanthii infected and phyto-hormones (methyl jasmonate and salicylic acid) treated plants in melon ‘SCNU1154’ line. Upon P. xanthii infection using 7 different races, the melon line showed variable disease reactions with respect to spread of infection symptoms and disease severity. Three out of 14 CmMLO genes were up-regulated and 7 were down-regulated in leaf samples in response to all races. The up- or down-regulation of the other 4 CmMLO genes was race-specific. The expression of 14 CmMLO genes under methyl jasmonate and salicylic acid application was also variable. Eleven CmMLO genes were up-regulated under salicylic acid treatment, and 7 were up-regulated under methyl jasmonate treatments in C. melo L. Taken together, these stress-responsive CmMLO genes might be useful resources for the development of powdery mildew disease resistant C. melo L.

The Effects of a Fermentation Product by Aspergillus oryzae on the in vitro Digestibilities of Dry Matter, Fiber and Protein and pH in the Fermentation Fluid of Diets for Dairy Cows (착유우 사료에 대한 Aspergillus oryzae 발효물질 첨가가 in vitro 건물, 섬유소 및 단백질 소화율과 발효액의 pH에 미치는 영향)

  • Myung, Yoon-Ah;Park, Duk-Sub;Lee, Soo-Kee;Park, Jong-Soo;Kim, Yong-Kook
    • Korean Journal of Agricultural Science
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    • v.29 no.2
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    • pp.20-34
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    • 2002
  • This study was conducted to examine the effects of an Aspergillus oryzae fermentation culture on the in vitro digestibilities of dry matter, crude fiber, acid detergent fiber (ADF), neutral detergent fiber (NDF), crude protein, and pH in in vitro experiment of diets for dairy cows. A fungal species, Aspergillus oryzae was supplied by Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea (KCTC 1229). The experimental diets were commercial compound feed (concentrate) and total mixed ration (TMR) for lactating cows, of which chemical analyses were determined at Research and Development Institute, Woosung Feed Co., Ltd., while the digestibilities were done at the laboratory of Chungnam National University. Aspergillus oryzae culture products were added to compound feed and TMR at the rate of 0, 1.0, 2.0, 3.0% respectively. The experimental diet with the rumen fluid sampled from Holstein fresian milking cows were used and digested for 24 hrs, 48hrs and 72hrs in the shaking incubator. The residues of the digesta were digested for 48hrs in the incubator in which put 30ml of 0.1N HCl with 0.2% pepsin at $39^{\circ}C$. The final precipitates were dried for 48hrs in the drier at $60^{\circ}C$. These experimental procedures were triplicated to determine the in vitro digestibility of dry matter, crude fiber, ADF, NDF, crude protein and pH. Compared to control diet, not added Aspergillus oryzae, the DM digestibility of fungal diets were improved 2.1%(63.1%), 9.7%(68.5%) and 9.0%(68.0%) for 24 hour fermentation in compound feed while 4.8%(60.0%), 6.4%(61.1%) and 2.9%(58.8%) in TMR. On the contrary, for 48 hour and 72 hour digestibilities, the effects of Aspergillus oryzae culture on the digestibility of dry matter were relatively lowered compared to 24 hour digestibility. Referring to the digestibility of dietary fiber, Aspergillus oryzae was believed to significantly improve digestibilities of crude fiber, ADF and NDF. Those were increased up to 13.3%(53.3%) for 24 hour fermentation, while 2.4%(54.6%) for 3.0% added for 72 hour fermentation in compound feed. However, there were no significant differences among the treatments for the inclusion rate of Aspergillus oryzae, even though the more inclusion rate, the better digestibility. The protein digestibilities were significantly improved from 0.4%(79.7%) to 9.4%(71.8%) by adding Aspergillus oryzae into compound feed. However, there were no significant differences between the two experimental diets, 2.0% and 3.0% Aspergillus oryzae included diets. In case of TMR, the protein digestibilities were significantly improved from 4.0%(70.4%) to 6.3%(65.1%) by adding Aspergillus oryzae. However, there were no significant differences between the two experimental diets, 2.0% and 3.0% Aspergillus oryzae included diets. In this study, there were no significant differences among the treatments in pH. On the contrary, there were slightly decrease in pH by adding Aspergillus oryzae into experimental diets but not significant. Summarizing the results of this examination, Aspergillus oryzae fermentation culture is believed to improve the digestibilities of dry matter, fiber and crude protein in cattle diets. However, more detailed research for the mechanism of the fungal culture is required to improve ruminal environment.

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The Risk Assessment of Butachlor for the Freshwater Aquatic Organisms (Butachlor의 수서생물에 대한 위해성 평가)

  • Park, Yeon-Ki;Bae, Chul-Han;Kim, Byung-Seok;Lee, Jea-Bong;You, Are-Sun;Hong, Soon-Sung;Park, Kyung-Hoon;Shin, Jin-Sup;Hong, Moo-Ki;Lee, Kyu-Seung;Lee, Jung-Ho
    • The Korean Journal of Pesticide Science
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    • v.13 no.1
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    • pp.1-12
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    • 2009
  • To assess the effect of butachlor on freshwater aquatic organisms, acute toxicity studies for algae, invertebrate and fishes were conducted. The algae grow inhibition studies were carried out to determine the growth inhibition effects of butachlor (Tech. 93.4%) in Pseudokirchneriella subcapitata (formerly knows as Selenastrum capriconutum), Desmodesmus subspicatus (formerly known as Scendusmus subspicatus), and Chlorella vulgaris during the exposure period of 72 hours. The toxicological responses of P. subcapitata, D. subspicatus, and C. vulgaris to butachlor, expressed in individual $ErC_{50}$ values were 0.002, 0.019, and $10.4mgL^{-1}$, respectively and NOEC values were 0.0008, 0.0016, and $5.34mg\;L^{-1}$, respectively. P. subcapitata was more sensitive than any other algae species. Butachlor has very high toxicity to the algae, such as P. subcapitata and D. subspicatu. In the acute immobilisation test for Daphnia magna, the 24 and $48h-EC_{50}$ values were 2.55 and $1.50mg\;L^{-1}$, respectively. As the results of the acute toxicity test on Cyprinus carpio, Oryzias latipes and Misgurnus anguillicaudatus, the $96h-LC_{50}s$ were 0.62, 0.41 and $0.24mg\;L^{-1}$, respectively. The following ecological risk assessment of butachlor was performed on the basis of the toxicological data of algae, invertebrate and fish and exposure concentrations in rice paddy, drain and river. When a butachlor formulation is applied in rice paddy field according to label recommendation, the measured concentration of butachlor in paddy water was $0.41mg\;L^{-1}$ and the predicted environmental concentration (PEC) of butachlor in drain water was $0.03 mg\;L^{-1}$. Residues of butachlor detected in major rivers between 1997 and 1998 were ranged from $0.0004mg\;L^{-1}$ to $0.0029mg\;L^{-1}$. Toxicity exposure ratios (TERs) of algae in rice paddy, drain and river were 0.004, 0.05 and 0.36, respectively and indicated that butachlor has a risk to algae in rice paddy, drain and river. On the other hand, TERs of invertebrate in rice paddy, drain and river were 3.6, 50 and 357, respectively, well above 2, indicating no risk to invertebrate. TERs of fish in rice paddy, drain and river were 0.58, 8 and 57, respectively. The TERs for fish indicated that butachlor poses a risk to fish in rice paddy but has no risk to fish in agricultural drain and river. In conclusion, butachlor has a minimal risk to algae in agricultural drain and river exposed from rice drainage but has no risk to invertebrate and fish.