標準編號:	GB/T 31270.11-2014
中文名稱:	化學農藥環境安全評價試驗準則 第11部分：家蠶急性毒性試驗
英文名稱:	Test guidelines on environmental safety assessment for chemical pesticides. Part 11: Silkworm acute toxicity test
中標分類:	B13    肥料與土壤調理劑
行業分類:	GB    國家標準
ICS分類:	65.100 65.100    殺蟲劑和其他農用化工產品 65.100
發布機構:	中華人民共和國國家質量監督檢驗檢疫總局 中國國家標準化管理委員會
發布日期:	2014-10-10
實施日期:	2015-3-11
標準狀態:	valid
翻譯語言:	英文
文件格式:	PDF
中文字符數:	5000 字
翻譯價格(元):	290.0
交付時間:	付款后 1 個工作日

Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative. GB/T 31270 consists of 21 parts under the general title Test guidelines on environmental safety assessment for chemical pesticides: ——Part 1: Transformation in soils; ——Part 2: Hydrolysis; ——Part 3: Phototransformation; ——Part 4: Adsorption/desorption; ——Part 5: Leaching in soil; ——Part 6: Volatility; ——Part 7: Bioconcentration test; ——Part 8: Degradation in water-sediment systems; ——Part 9: Avian acute toxicity test; ——Part 10: Honeybee acute toxicity test; ——Part 11: Silkworm acute toxicity test; ——Part 12: Fish acute toxicity test; ——Part 13: Daphnia sp.acute immobilisation test; ——Part 14: Alga growth inhibition test; ——Part 15: Earthworm acute toxicity test; ——Part 16: Soil microorganism toxicity test; ——Part 17: Trichogramma acute toxicity test; ——Part 18: Amphibian acute toxicity test; ——Part 19: Effects on non-target plants; ——Part 20: Livestock short-term dietary toxicity test; ——Part 21: Macro-crustacean toxicity test. This is Part 11 of GB/T 31270. This part is developed in accordance with the rules given in GB/T 1.1-2009. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. The issuing body of this document shall not be held responsible for identifying any or all such patent rights. This standard was proposed by and is under the jurisdiction of the Ministry of Agriculture of the People's Republic of China. Test guidelines on environmental safety assessment for chemical pesticides - Part 11: Silkworm acute toxicity test 1 Scope This part of GB/T 31270 specifies the basic requirements for materials, conditions, operation, quality control, data processing, test reports, etc. for acute toxicity test of chemical pesticides on silkworm with leaf-dipping method and fumigation method. This part applies to silkworm acute toxicity tests with leaf-dipping method and fumigation method for the registration of chemical pesticides. It may be used as reference for other types of pesticides. This part does not apply to volatile and insoluble chemical pesticides. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 median lethal concentration concentration of the test substance, at which 50% of the test organisms die in the toxicity test with leaf-dipping method, expressed by LC50 Note: the unit is mg a.i./L. 2.2 test substance substance to be tested 2.3 chemical pesticide pesticide made of chemical substances through artificial synthesis, for some, the active substances in natural products are used as the mother body, and then subjected to imitation, structural transformation and innovation, they are bionic synthetic pesticides Synonym: synthetic organic pesticide. [Definition 2.3.1, NY/T 1667.1-2008] 2.4 technical material final product consisting of the active ingredients and impurities obtained during the manufacturing process, which shall not contain visible foreign substances or any additives, and if necessary, a small amount of stabilizer may be added [Definition 2.5.1, NY/T 1667.2-2008] 2.5 formulation product product stable in use and made of technical material (or technical concentrate) of pesticides and auxiliaries [Definition 2.4, NY/T 1667.2-2008] 2.6 active ingredient; a.i. specific chemical ingredient that is biologically active in a pesticide product [Definition 3.1, NY/T 1667.2-2008] 2.7 reference substances chemical substance or mixture used in a test to confirm or deny certain characteristics of a test substance or to judge the effectiveness of a test system 3 Test overview 3.1 Method overview The silkworm acute toxicity test is carried out by leaf-dipping method or fumigation method, and the relevant methods are selected according to the regulations of pesticide registration management and other regulations. 3.2 Leaf-dipping method The mulberry leaves are dipped into different concentrations of pesticide liquid, and dried to feed the silkworm. The treated mulberry leaves are used for feeding throughout the test, then the toxicity symptoms and death conditions of test silkworms are observed after 24h, 48h, 72h, and 96h; at the end of the test, the data shall be subjected to statistic analysis and median lethal concentration LC50 is calculated. 3.3 Fumigation method The test based on the pesticide application conditions in simulation chamber shall be carried out on the fumigation test device or fumigation chamber meeting the test requirements. The relevant parameters of fumigation test device or the fumigation chamber shall be designed according to the recommended dosage on the premise of meeting the test requirements. The test substance is quantitatively burned (or electrically heated) in the test device or the fumigation chamber. From the beginning of fumigation, the toxicity symptoms of the silkworm in the fumigation test device are observed at 0.5h, 2h, 4h, 6h and 8h. After 8h, the silkworms in the test device are taken out, and the silkworm deaths at 24h and 48h are observed under the normal feeding conditions of the silkworms. 4 Test methods 4.1 Materials and conditions 4.1.1 Test organism The test silkworm (Bombyx mori) species are selected from Jingsong × Haoyue, Chunlei × Zhenzhu, Suju × Minghu, or other representative lines. The second-instar silkworm is used as materials for toxicity test. 4.1.2 Test substance Pure pesticides, technical materials or formulation products shall be used as test substance. For insoluble pesticides, a small amount of organic solvents, emulsifiers or dispersing agents which are low in toxicity to silkworm may be used, and the amount thereof shall not exceed 0.1mL(g)/L. 4.1.3 Main apparatus and instruments Main apparatus and instruments are as follows: ——artificial climate chamber; ——electronic balance; ——culture dish; ——fumigation box, etc. 4.1.4 Test conditions Newly-hatched silkworm shall be fed at test temperature of (25±2)℃ and relative humidity of 70%～85%. 4.2 Test operations 4.2.1 Leaf-dipping method 4.2.1.1 Pre-test 3~5 concentration groups are set at a large difference according to the conditions of the formal test; the highest total survival concentration and the lowest total lethal concentration for the silkworm are calculated through pre-test. 4.2.1.2 Formal test 5~7 concentration groups are set based on certain proportional difference (geometrical difference shall be controlled within 2.2 times) within a certain range of concentration determined by the pre-test, each group consists 20 silkworms and blank control is set; if solvent cosolvent is used, a solvent control group shall be added. 3 replicates are set for the control group and each concentration group. The second-instar silkworms are cultured in a culture dish, and the mulberry leaves are completely dipped into different concentrations of the pesticide liquid for 10s, and dried for use by the silkworms. The treated mulberry leaves are used for feeding throughout the test, then the toxicity symptoms and death conditions of test silkworms are observed at 24h, 48h, 72h, and 96h. At the end of the test, the data shall be subjected to statistic analysis and median lethal concentration LC50 and 95% confidence limit are calculated. If the insect growth regulator is adopted as test substance, and the death rate of silkworm increases by more than 10% between 72h and 96h, the observation time shall be extended until the death rate increases by less than 10% within 24h. 4.2.1.3 Limit test The upper concentration limit is set as 2000mg a.i./L, that is, if the silkworm does not die when the concentration of the test substance reaches 2000mg a.i./L, it is not necessary to continue the test. If the solubility of the test substance is less than 2000mg a.i./L, the maximum solubility is used as the upper concentration limit. 4.2.2 Fumigation method The fumigation test can be carried out in a fumigation box or other satisfactory test device. The test substance is quantitatively burned (or electrically heated) in the test device. From the beginning of fumigation, the toxicity symptoms of the silkworm in the fumigation test device are observed at 0.5h, 2h, 4h, 6h and 8h. After 8h, the silkworms in the test device are taken out, and the silkworm deaths at 24h and 48h are observed under the normal feeding conditions of the silkworms. 9 replicates are set for each treatment group and a blank control (3 replicates) is set at the same time. The food intake situation of silkworm (reduced or refusal), discomfort symptoms (escape, head-up, shaking head, headbang, twisting, struggling, emesis, etc.) and death conditions are observed and recorded. 4.3 Data processing 4.3.1 Selection of statistical analysis method Karber method, linear interpolation method or probit graphical method may be adopted to calculate the median lethal concentration LC50 of pesticide toxicity on silkworm in leaf-dipping method at each observation time (24h, 48h, 72h and 96h); data statistics software may also be adopted for analysis and calculation.

Foreword i 1 Scope 2 Terms and definitions 3 Test overview 4 Test methods 5 Test report Annex A (Informative) Classification of toxicity levels of pesticides to silkworm Bibliography

ICS 65.100 B 13 中華人民共和國國家標準 GB/T 31270.11—2014 化學農藥環境安全評價試驗準則 第11部分：家蠶急性毒性試驗 Test guidelines on environmental safety assessment for chemical pesticides—Part 11：Silkworm acute toxicity test 2014—10—10發布 2015—03—11實施 中華人民共和國國家質量監督檢驗檢疫總局 中國國家標準化管理委員會 發布 前言 GB/T 31270《化學農藥環境安全評價試驗準則》分為21個部分： ——第1部分：土壤降解試驗； ——第2部分：水解試驗； ——第3部分：光解試驗； ——第4部分：土壤吸附/解吸試驗； ——第5部分：土壤淋溶試驗； ——第6部分：揮發性試驗； ——第7部分：生物富集試驗； ——第8部分：水-沉積物系統降解試驗； ——第9部分：鳥類急性毒性試驗； ——第10部分：蜜蜂急性毒性試驗； ——第11部分：家蠶急性毒性試驗； ——第12部分：魚類急性毒性試驗； ——第13部分：溞類急性活動抑制試驗； ——第14部分：藻類生長抑制試驗； ——第15部分：蚯蚓急性毒性試驗； ——第16部分：土壤微生物毒性試驗； ——第17部分：天敵赤眼蜂急性毒性試驗； ——第18部分：天敵兩棲類急性毒性試驗； ——第19部分：非靶標植物影響試驗； ——第20部分：家畜短期飼喂毒性試驗； ——第21部分：大型甲殼類生物毒性試驗。 本部分是GB/T 31270的第11部分。 本部分按照GB/T 1.1—2009給出的規則起草。 請注意本文件的某些內容可能涉及專利。本文件的發布機構不承擔識別這些專利的責任。 本部分由中華人民共和國農業部提出并歸口。 化學農藥環境安全評價試驗準則 第11部分：家蠶急性毒性試驗 1 范圍 GB/T 31270的本部分規定了浸葉法和熏蒸法測定化學農藥對家蠶急性毒性試驗的材料、條件、操作、質量控制、數據處理、試驗報告等的基本要求。 本部分適用于為化學農藥登記而進行的家蠶浸葉法毒性試驗和熏蒸法毒性試驗，其他類型的農藥可參照使用。 本部分不適用于易揮發和難溶解的化學農藥。 2術語和定義 下列術語和定義適用于本文件。 2.1 半致死濃度median lethal concentration 在浸葉法毒性試驗中，引起50％供試生物死亡時的供試物濃度，用LC50表示。 注：單位為mg a.i./L。 2.2 供試物test substance 試驗中需要測試的物質。 2.3 化學農藥 chemical pesticide 利用化學物質人工合成的農藥。其中有些以天然產品中的活性物質為母體，進行仿制、結構改造，創新而成，為仿生合成農藥。 同義詞：有機合成農藥synthetic organic pesticide。 [NY/T 1667.1—2008，定義2.3.1] 2.4 原藥technical material 在制造過程中得到的有效成分及雜質組成的最終產品，不能含有可見的外來物質和任何添加物，必要時可加入少量的穩定劑。 [NY/T 1667.2—2008，定義2.5.1] 2.5 制劑 formulation product 由農藥原藥(或母藥)和助劑制成使用狀態穩定的產品。 [NY/T 1667.2—2008，定義2.4] 2.6 有效成分 active ingredient；a.i. 農藥產品中具有生物活性的特定化學結構成分。 [NY/T 1667.2—2008，定義3.1] Z.7 參比物質 reference substances 在測試中為證實或否定供試物的某種特性或判斷測試系統有效性而使用的化學物質或混合物。 3試驗概述 3.1 方法概述 家蠶急性毒性試驗包括浸葉法毒性試驗方法和熏蒸法毒性試驗方法，根據農藥登記管理法規及其他規定選擇相關方法進行試驗。 3.2浸葉法毒性試驗 采用不同濃度的藥液浸漬桑葉，晾干后飼喂家蠶。整個試驗期間飼喂處理桑葉，觀察24 h、48 h、72 h、96 h后受試家蠶的中毒癥狀及死亡情況，試驗結束后對數據進行統計分析，并計算半致死濃度LC50值。 3.3熏蒸法毒性試驗 針對衛生用藥模擬室內施藥條件進行的試驗，應在滿足試驗要求的熏蒸試驗裝置或熏蒸室內進行。熏蒸試驗裝置或熏蒸室應在滿足試驗要求的前提下，按照推薦用藥量設計相關參數。供試物在試驗裝置或熏蒸室中定量燃燒(或電加熱)，從熏蒸開始，按0.5 h、2 h、4 h、6 h、8 h觀察記錄熏蒸試驗裝置內家蠶的毒性反應癥狀，8 h后將試驗裝置內的家蠶取出，在家蠶常規飼養條件下繼續觀察24 h及48 h的家蠶死亡情況。 4試驗方法 4.1材料和條件 4.1.1供試生物 試驗用家蠶(Bombyx mori)品種選用菁松×皓月，春蕾×鎮珠，蘇菊×明虎，或其他有代表性的品系。以二齡起蠶為毒性試驗材料。 4.1.2供試物 供試物應使用農藥純品、原藥或制劑。難溶于水的可用少量對家蠶毒性小的有機溶劑、乳化劑或分散劑助溶，其用量不得超過0.1 mL(g)/L。 4.1.3主要儀器設備 主要儀器設備如下： ——人工氣候室； ——電子天平； ——培養皿； ——熏蒸箱等。 4.1.4試驗條件 蟻蠶飼養和試驗溫度為(25±2)℃，相對濕度為70％～85％。 4.2試驗操作 4.2.1 浸葉法 4.2.1.1 預試驗 按正式試驗的條件，以較大的間距設置3個～5個濃度組，通過預試驗求出家蠶最高全存活濃度和最低全致死濃度。 4.2.1.2正式試驗 根據預試驗確定的濃度范圍按一定比例間距(幾何級差應控制在2.2以內)設置5個～7個濃度組，每組20頭蠶，并設空白對照，加溶劑助溶的還需設溶劑對照。對照組和每一濃度組均設3個重復。在培養皿內飼養二齡起蠶，用不同濃度的藥液完全浸漬桑葉10 s，晾干后供蠶食用。整個試驗期間飼喂處理桑葉，觀察并記錄24 h、48 h、72 h和96 h試驗用家蠶的中毒癥狀及死亡情況。試驗結束后對數據進行數理統計，計算半致死濃度LC50值及95％置信限。 若供試物為昆蟲生長調節劑，且試驗72 h～96 h之間家蠶的死亡率增加10％以上，應延長觀察時間，直至24 h內死亡率增加小于10％。 4.2.1.3 限度試驗 設置上限濃度2 000 mg a.i./L，即在供試物達2 000 mg a.i./L時仍未見家蠶死亡，則無需繼續進行試驗。若供試物溶解度小于2 000 mg a.i./L，則采用其最大溶解度作為上限濃度。 4.2.2 熏蒸法 熏蒸試驗可在熏蒸箱或其他可滿足試驗要求的試驗裝置內進行。供試物在試驗裝置內定量燃燒(或電熱片加熱)，從熏蒸開始，按0.5 h、2 h、4 h、6 h、8 h觀察記錄熏蒸試驗裝置內家蠶的毒性反應癥狀，8 h后，將熏蒸試驗裝置內的家蠶取出，在家蠶常規飼養條件下繼續觀察24 h及48 h的家蠶死亡情況。每個處理組設置9個重復，同時設置空白對照(設3個重復)。觀察記錄家蠶攝食情況(減少或拒食)、不適癥狀(逃避、昂頭、晃頭、甩頭、扭曲掙扎、吐水等)及死亡情況等。 4.3數據處理 4.3.1 統計分析方法的選擇 可采用寇氏法、直線內插法或概率單位圖解法計算每一觀察時間(24 h、48 h、72 h、96 h)的家蠶浸葉法毒性的半致死濃度LC50，也可采用數據統計軟件進行分析和計算。 4.3.2 寇氏法 用寇氏法可求出家蠶在24 h、48 h、72 h和96 h的LC50值及95％置信限。 LC50的計算見式(1)： logLC50=Xm-i(∑P-0.5) (1) 式中： Xm——最高濃度的對數； i——相鄰濃度比值的對數； ∑P——各組死亡率的總和(以小數表示)。 95％置信限的計算見式(2)： 95％置信限=logLC50±1.96SlogLC50 (2) 標準誤的計算見式(3)： (3) 式中： p——1個組的死亡率； q——1-p； n——各濃度組家蠶的數量。 4.3.3直線內插法 采用線性刻度坐標，繪制死亡百分率對試驗物質濃度的曲線，求出50％死亡時的LC50值。 4.3.4概率單位圖解法 用半對數紙，以濃度對數為橫坐標、死亡百分率對應的概率單位為縱坐標繪圖。將各實測值在圖上用目測法畫一條相關直線，從直線中讀出致死50％的濃度對數，估算出LC50值。 4.4質量控制 質量控制條件包括： ——對照組死亡率不超過10％； ——實驗室內應定期(蠶卵每批一次，同批蠶卵至少每兩個月一次)進行參比物質試驗，推薦參比物質為樂果。 5試驗報告 試驗報告應包括下列內容： ——供試物的信息，包括供試農藥的通用名、化學名稱、結構式、CAS號、純度、基本理化性質、來源等； ——供試生物的名稱、來源、大小及飼養情況； ——試驗條件，包括試驗溫度、試驗方法等； ——試驗藥液的濃度，24 h、48 h、72 h及96 h的LC50值(浸葉法)或8 h、24 h、48 h的死亡率(熏蒸法)和95％置信限，并給出所采用的計算方法； ——對照組家蠶是否出現死亡及異常反應； ——觀察到的毒性效應，如受試家蠶的任何不正常的行為、中毒癥狀等； ——對家蠶的毒性等級劃分參見附錄A。 附 錄 A (資料性附錄) 農藥對家蠶毒性等級劃分 按農藥對家蠶急性浸葉法的毒性半致死濃度LC50(96 h)，將農藥對家蠶的急性毒性分為四級，見表A.1。 表A.1 農藥對家蠶的毒性等級劃分 毒性等級 LC50(96 h)/(mg a.i./L) 劇毒 LC50≤0.5 高毒 0.5200 熏蒸試驗主要是針對衛生用藥模擬室內施藥條件下進行的試驗，如果家蠶的死亡率大于10％以上時，即視為對家蠶高風險。 參考文獻 [1] NY/T 1667.1—2008農藥登記管理術語 第1部分：基本術語 [2] NY/T 1667.2—2008農藥登記管理術語 第2部分：產品化學 [3] OECD(1998a).Guideline 213：Honeybees，acute oral toxicity test，OECD Guidelines for the Testing of chemicals. [4] OECD(1998b).Guideline 214：Honeybees，acute contact toxicity test，OECD Guidelines for the Testing of chemicals. [5] OECD(1992).Guidelines.203：Fish，acute toxicity test，OECD Guidelines for the Testing of Chemicals. [6] 蔡道基.農藥環境毒理學研究.北京：中國環境科學出版社，1999. [7] NY/T 1154.14—2008農藥室內生物測定試驗準則 殺蟲劑 第14部分：浸葉法 [8]GB/T 13917.10—2009農藥登記用衛生殺蟲劑室內藥效試驗及評價 第10部分：模擬現場