Acute toxicity of 十多种to zebrafish

DOI：10.16661/ki.1672-3791.2018.16.228六种不同剂型的噻虫嗪对斑马鱼急性毒性研究杨玲莉1,2(1.湖南农业大学动物医学院 湖南长沙 410128;2.北京依科世福 北京 100000)摘 要：噻虫嗪（Thiamethoxam）通常为白色结晶粉末，是一种全新结构的第二代烟碱类高效低毒杀虫剂，对害虫具有胃毒、触杀及内吸活性，用于叶面喷雾及土壤灌根处理。

因噻虫嗪的毒性一度受争议。

本文通过六种剂型噻虫嗪对斑马鱼急性毒性研究，探究不同噻虫嗪制成不同剂型是否对斑马鱼毒性有所差异，为噻虫嗪登记做参考。

研究发现，噻虫嗪原药配成水剂为低毒、噻虫嗪悬浮剂和噻虫嗪悬浮种衣剂均为微毒。

噻虫嗪原药配成水剂和悬浮剂可以有效减小噻虫嗪毒性。

从鱼类保护的眼光来看，较之于噻虫嗪颗粒剂，更建议使用噻虫嗪水剂和悬浮剂。

关键词：斑马鱼 剂型 噻虫嗪 杀虫剂 急性毒性实验中图分类号：X.826 文献标识码：A 文章编号：1672-3791(2018)06(a)-0228-04农药在现代社会被广泛使用，这不仅仅给陆地生态环境造成了一定破坏，许多农药通过地表渗流和地下径流从农田流入地下，更给生活在水下或者水面的水生动物造成了影响。

农药危害最大的就是水中的鱼类。

有一些农药鱼类对其富集能力很强，容易给鱼类带来污染与危害，比如使鱼类脊椎畸形、出现血斑、侧游甚至引起鱼类大量死亡。

更可怕的是，如今随着植物虫害病害的日益严重，作为方便快捷经济的除害方式农药正在被越来越广泛使用并且肆意追加。

因此农药对水生物毒性的研究及农药毒性科学控制刻不容缓。

现今，农药的投产过程主要是通过几种原药配制不同制剂制成成品，再测过毒性能够投入使用就登记投入使用推广。

因此同种原药制成不同制剂对同种生物的毒性是不相同的，因此，在不可避免使用某种原药的前提下，可以通过此原药制成不同剂型来减少毒性，达到间接减少环境污染的效果。

1 绪论1.1 噻虫嗪在国内外研究现状噻虫嗪作为第二代烟碱类杀虫剂是瑞士先正达公司的专利产品，但他饱受争议，尤其对蜜蜂健康影响很大。

双酚A及其类似物对斑马鱼胚胎及幼鱼的毒性效应任文娟;汪贞;王蕾;杨先海;刘济宁【摘要】为探讨双酚A及其类似物对鱼类早期生长发育的毒性效应,研究了双酚A 及其7种类似物对斑马鱼胚胎及仔鱼的毒性效应.通过对胚胎的孵化率、心率、仔鱼体长等指标进行测定分析,结果显示:(1)双酚A及其7种类似物都可使斑马鱼胚胎出现心包水肿、卵黄囊肿、脊柱弯曲和尾部弯曲等症状.(2)综合考虑斑马鱼78 hpf胚胎心率、120 hpf胚胎孵化率及7 dpf仔鱼体长抑制率等指标,8种受试物中双酚P(BPP)的毒性最大,其次是双酚AP(BPAP)、双酚AF(BPAF)、双酚Z(BPZ),然后是双酚A(BPA)、双酚B(BPB)、双酚F(BPF),双酚S(BPS)的毒性最小,每种受试物的浓度与受精卵的孵化率、仔鱼心率呈明显负相关关系,与体长抑制率呈明显正相关关系.8种受试物毒性与辛醇-水分配系数(log Kow)呈正相关关系,logKow越大毒性则越大.%We evaluated the toxic effect of bisphenol A (BPA) and its seven analogues on zebrafish embryos and larvae.Based on the results of the hatching rate (120 hpf),heart rate (78 hpf),body length of larvae (7 dpf),it is indicated that:(1) BPA and its analogues caused cardiac edema,cyst vitelline,spinal curvature and tail bending;(2)the order of the toxicity is bisphenol P (BPP)＞ bisphenol AP (BPAP)＞ bisphenol AF (BPAF)＞ bisphenol Z (BPZ)＞ bisphenol A (BPA)＞ bisphenol B (BPB)＞ bisphenol F (BPF)＞ bisphenol S (BPS).The concentrations of the test substances were negatively correlated with the embryo hatching rate and the heart rate of larvae,while positive correlation with inhibition rate of larval length.In addition,the toxicity of these eight kinds of BPA analogues and the values of octanol-water partition (log Kow) were positively correlated.【期刊名称】《生态毒理学报》【年(卷),期】2017(012)001【总页数】9页(P184-192)【关键词】双酚A;双酚A类似物;斑马鱼;胚胎;仔鱼;毒性【作者】任文娟;汪贞;王蕾;杨先海;刘济宁【作者单位】环境保护部南京环境科学研究所,南京 210042;南京工业大学生物与制药工程学院,南京 211816;环境保护部南京环境科学研究所,南京 210042;环境保护部南京环境科学研究所,南京 210042;环境保护部南京环境科学研究所,南京210042;环境保护部南京环境科学研究所,南京 210042【正文语种】中文【中图分类】X171.5双酚A(bisphenol A, BPA)是全球生产量最大的化学原料之一，是生产高分子材料如聚碳酸酯、环氧树脂、增塑剂等的前体物质，广泛应用于杀菌剂、染料、医疗器械、食品包装材料和饮料容器、餐具、婴儿奶瓶及某些家庭用具等的生产，还可作为牙齿密封剂、牙科填充剂使用[1]。

生态毒理学报Asian Journal of Ecotoxicology第16卷第5期2021年10月V ol.16,No.5Oct.2021㊀㊀基金项目:上海市自然科学基金资助项目(17ZR1424400);上海市技术性贸易措施应对专项(16TBT009)㊀㊀第一作者:王绿平(1986 ),女,硕士,工程师,研究方向为生态毒理学,E -mail:*************.cn;*******************㊀㊀*通讯作者(Corresponding author ),E -mail:*************.cn;*******************DOI:10.7524/AJE.1673-5897.20201105002王绿平,张京佶,赵华清.稀有鮈鲫作为鱼类胚胎急性毒性试验受试鱼种的敏感性研究[J].生态毒理学报,2021,16(5):102-112Wang L P,Zhang J J,Zhao H Q.Sensitivity of Chinese rare minnows (Gobiocypris rarus )for fish embryo acute toxicity test [J].Asian Journal of Ecotox -icology,2021,16(5):102-112(in Chinese)稀有鮈鲫作为鱼类胚胎急性毒性试验受试鱼种的敏感性研究王绿平*,张京佶,赵华清上海市检测中心生物与安全检测实验室,上海201203收稿日期:2020-11-05㊀㊀录用日期:2021-01-04摘要:通过验证稀有鮈鲫在鱼类胚胎急性毒性试验中的敏感性,评价其在鱼类替代试验中的应用潜力,为相关化学品测试国家标准的制定提供依据㊂选取3,4-二氯苯胺(3,4-DCA)㊁五水硫酸铜(CSP)㊁2,3,6-三甲基苯酚(2,3,6-TMP)㊁二甲基亚砜(DMSO)㊁七水硫酸锌(ZSH)㊁三甘醇(TEG)㊁氯化钠(SC)㊁十二烷基硫酸钠(SDS)㊁重铬酸钾(PD)㊁甲基异噻唑啉酮(MIT)㊁二苯甲酮(BP)㊁二氯苯氧氯酚(TCS)㊁1,2-苯并异噻唑啉-3-酮(BIT)㊁三唑酮(T)㊁多菌灵(C)和N -(4-氯苯基)-N -(3,4-二氯苯基)脲(三氯卡班,TCC)16种化学品,按照‘OECD 化学品测试准则No.236鱼类胚胎急性毒性试验“,分别进行稀有鮈鲫胚胎急性毒性试验,将结果与稀有鮈鲫成鱼急性毒性试验结果比较,评价稀有鮈鲫在不同生命周期对同一化学品的敏感性差异;同时与已有的或试验所得的斑马鱼胚胎急性毒性试验结果比较,评估稀有鮈鲫胚胎与国际标准试验鱼种胚胎间的敏感性差异㊂稀有鮈鲫胚胎急性毒性试验96h 半数致死浓度(96h -LC 50)值与其成鱼及斑马鱼相比,敏感性类似,毒性值差异均未超过一个数量级㊂稀有鮈鲫作为一种中国本土的标准试验鱼种,其胚胎生物学特征与斑马鱼类似,其胚胎敏感性不亚于成鱼和其他国际标准鱼种,适宜进行鱼类胚胎急性毒性试验的应用㊂关键词:稀有鮈鲫;胚胎;急性毒性;敏感性文章编号:1673-5897(2021)5-102-11㊀㊀中图分类号:X171.5㊀㊀文献标识码:ASensitivity of Chinese Rare Minnows (Gobiocypris rarus )for Fish Embryo Acute Toxicity TestWang Lvping *,Zhang Jingji,Zhao HuaqingBioassay and Safety Assessment Laboratory,Shanghai Academy of Public Measurement,Shanghai 201203,ChinaReceived 5November 2020㊀㊀accepted 4January 2021Abstract :This study aims to evaluate the sensitivity of Chinese rare minnow (Gobiocypris rarus )as test organisms used in fish embryo acute toxicity test,and to lay a foundation for the proposed national standards of chemical tes -ting which uses fish embryo as an alternative to fish itself in acute toxicity test.According to the procedures of OECD Guidelines for the Testing of Chemicals No.236:Fish Embryo Acute Toxicity Test ,newly fertilized eggs of rare minnow were exposed to sixteen chemicals,i.e.,3,4-dichloroaniline (3,4-DCA),copper sulfate pentahydrate (CSP),2,3,6-trimethylphenol (2,3,6-TMP),dimethyl sulfoxide (DMSO),zinc sulfate (ZSH),triethylene glycol (TEG),sodium chloride (SC),sodium dodecyl sulfate (SDS),potassium bichromate (PD),2-methyl -4-isothiazolin . All Rights Reserved.第5期王绿平等:稀有鮈鲫作为鱼类胚胎急性毒性试验受试鱼种的敏感性研究103㊀3-one(MIT),benzophenone(BP),triclosan(TCS),1,2-benzisothiazolin-3-one(BIT),triadimefon(T),carbendazim(C)and triclocarban(TCC),and then,the respective median lethal concentration of the96h exposure(96h-LC50) were obtained for each chemical.By comparing the rare minnow embryo test data with the corresponding data from adult fish tests as well as those resulted from the zebra fish embryo tests,it can be found that the differences in toxicity values of different test organisms are not more than an order of magnitude,implying that the sensitivity of rare minnow embryos to studied chemicals in acute toxicity test is similar to adult rare minnow and zebra fish embryos.In conclusion,the endemic fish species Gobiocypris rarus is one of the promising candidates of standard fish species,and its embryos can produce reliable data in fish embryo acute toxicity tests.Keywords:Gobiocypris rarus;embryo;acute toxicity;sensitivity㊀㊀水生生物毒性测试广泛应用于评估化学品的水生态环境安全,而鱼类作为水生生态环境体系中的顶级脊椎动物,具有不可替代的地位,其生态毒性数据为水生生态风险评估与风险管理提供基础,具有重要的作用和意义[1-4]㊂随着替代(replacement)㊁减少(reduction)㊁优化(refinement)(3Rs)原则[5]在哺乳动物毒性测试中的广泛认可,近年来,该理念在环境毒理学领域的延伸也逐渐被关注[6]㊂欧盟分别于2009年和2013年,禁止用化妆品原料进行脊椎动物试验以及禁止销售用动物试验检测的化妆品原料及成品㊂此外,欧洲‘化学品的注册㊁评估㊁授权和限制“(REACH)法规也高度关注化学品毒性测试中实验动物福利问题,提倡减少测试中脊椎动物的数量,推动非动物测试(如胚胎㊁细胞和组织等)和替代策略的开发和验证[7]㊂目前,国际上首选鱼类急性毒性试验的替代方法,在2012年完成验证[8-9],于2013年由国际经济合作与发展组织(OECD)颁布,鱼类胚胎急性毒性试验(Test No.236:Fish embryo acute toxic-ity(FET)test,简称OECD236)[10]方法中指定的实验生物为斑马鱼(Danio rerio)受精卵㊂采用胚胎进行毒性试验除了可满足现行动物福利法规的要求,还存在以下几个优势㊂(1)亲鱼体型小,易于饲养㊁管理方便㊂(2)产卵量大㊂通常情况下亲鱼每次可产卵几百枚至上千枚不等,对于单个试验而言,同一来源的大批量受精卵可满足统计学意义上的各类分析㊂(3)试验体系小㊂整个试验可在24孔细胞板中完成,节约了空间和成本的同时,还减少了样品的用量和废液的排放㊂(4)测试周期短,试验周期为96h㊂从受精卵开始,通过卵黄囊吸收营养,胚胎可在24孔细胞板中存活多天,完全可以满足试验需求㊂且与传统鱼类急性毒性试验一致,结果具有一定可比性㊂(5)斑马鱼胚胎具有光学透明性[11],可通过显微镜观察各发育阶段的形态特征,定期观察各种效应指标(致死或致畸),为毒理学研究提供依据㊂(6)运用一定的标记技术,可透过细胞膜,准确地观察某个基因在组织器官或个体中的表达㊂鱼类胚胎急性毒性试验在技术上操作方便,试验结果稳定性及重复性较高[12]㊂与传统的成鱼或幼鱼的急慢性试验相比,更为灵敏且能提供更多的观察效应指标㊂不仅可用于研究化学品的环境毒性,还可用于对污水㊁废水的监测和控制㊂在德国,斑马鱼胚胎已经作为水质监测的标准实验材料,取代了用成鱼进行的毒理学研究[13-14]㊂由于胚胎期对外源化学物质相对敏感,研究也表明,鱼类胚胎急性毒性数据与鱼类急性毒性数据之间有着高度相关性[15]㊂因此,很多欧美国家已逐渐避免使用成鱼或幼鱼作为毒性研究材料,越来越多改用胚胎试验获取毒性数据㊂目前全球大部分生态毒性测试,从方法到试验物种,都是在欧美国家的应用研究基础上提出来的,对保护我国生态环境安全没有针对性㊂就鱼类胚胎急性毒性试验而言,OECD236中指定的试验物种为斑马鱼(Danio rerio)受精卵,并没有涉及其他鱼种㊂然而生态环境保护是具有区域特异性的,研究化学品对特定区域环境的毒性效应需要有符合我国环境保护特点的试验物种进行测试㊂稀有鮈鲫(Go-biocypris rarus)是我国特有的一种小型鲤科鱼类[16],我国‘化学品测试方法“已将稀有鮈鲫列为推荐受试鱼种之一[17-19]㊂其亲鱼性成熟时间短,繁殖季节长,产卵量大,可常年人工繁殖,因此具有成为中国特有模式鱼种的潜能㊂由于其胚胎也具有光学透明性,可透过卵膜清晰观察胚胎发育,同时其胚胎的形态特征㊁发育过程及孵化时间也与多数淡水硬骨鱼类似[20]㊂因此,以稀有鮈鲫为实验材料,开展相关的胚胎毒理学研究有一定的基础依据㊂此外,稀有鮈鲫生物学特征研究已较为系统,研究工作涉及鱼病学㊁. All Rights Reserved.104㊀生态毒理学报第16卷遗传学㊁环境科学㊁胚胎学和生理学等领域[21-24]㊂在此基础上,开展稀有鮈鲫在胚胎领域的毒理学研究将推动我国特有标准试验鱼种用于生态毒性测试的可持续战略,拓展稀有鮈鲫在毒性测试中的使用范围,为其最终成为国际公认的标准试验物种创造条件㊂无论是科学研究㊁环境监测还是毒性检测,实验动物的质量是实验数据可重复性(repeatability)㊁再现性(reproducibility)和敏感性(sensibility)的基石㊂研究表明,稀有鮈鲫在鱼类胚胎急性毒性试验中的结果重复性良好[25],需评估稀有鮈鲫在鱼类胚胎急性毒性试验中的敏感性㊂本研究选取16种不同种类的化学品开展稀有鮈鲫胚胎急性毒性试验,通过试验和已有文献资料相结合的方式,分别将稀有鮈鲫胚胎与斑马鱼胚胎,稀有鮈鲫胚胎与其成鱼进行比较,研究稀有鮈鲫胚胎的敏感性和可比性㊂1㊀材料与方法(Materials and methods)1.1㊀材料1.1.1㊀实验动物本研究中所使用的稀有鮈鲫亲鱼源自中国科学院水生生物研究所,为野生型封闭群(Ihb:IHB),或引种后自行繁育㊂为保证良好的受精率,选择实验室驯养条件下无肉眼可观察到感染或疾病㊁未经任何药物处理且已繁殖至少2次以上的稀有鮈鲫㊂鱼类驯养室内昼夜由定时器控制,提供12h(光)ʒ12h (暗)的循环光照;水温维持在(23ʃ2)ħ,每日投喂冷冻水蚯蚓(Limnodrilus hoffmeisteri)2次(早晚各1次)㊂试验用鱼卵以分组产卵方式获得,即将6尾性成熟的稀有鮈鲫(雌雄比为1ʒ2),于试验当天早晨置于交配盒中,为防止亲鱼捕食鱼卵,在交配盒中放入网架(孔径大小约为(2ʃ0.5)mm)㊂为能够获得足够数量的鱼卵,平行准备6组㊂待灯光转暗后1~2h,取出亲鱼,收集鱼卵㊂斑马鱼亲鱼来源于国家斑马鱼资源中心,为野生型AB系㊂实验室驯养条件下的斑马鱼无肉眼可观察的感染或疾病,且未经任何药物处理㊂试验所使用的亲鱼已繁殖至少2次以上,以保证有良好的受精率㊂鱼类驯养室内昼夜由定时器控制,提供12 h(光)ʒ12h(暗)的循环光照;水温维持在(23ʃ2)ħ,每日投喂冷冻水蚯蚓(Limnodrilus hoffmeisteri)2次(早晚各1次)㊂试验用鱼卵以分组产卵方式获得,即将6尾性成熟的斑马鱼(雌雄比为1ʒ2),于试验前一天晚上置于交配盒中,为防止亲鱼捕食鱼卵,在交配盒中放入网架(孔径大小约为(2ʃ0.5)mm)㊂为能够获得足够数量的鱼卵,平行准备6组㊂第2天待灯光转亮后1~2h,取出亲鱼,收集鱼卵㊂采用体视显微镜观察,选择受精且无明显不规则分裂(如不对称㊁囊泡形成)或破损的鱼卵进行试验㊂如果试验用鱼卵为n,则取总数为2n的鱼卵进行镜检,挑选出合格受精卵(x)备用,并计算其受精百分率(x/2nˑ100%)㊂1.1.2㊀受试化学品参考OECD斑马鱼胚胎急性毒性试验验证报告[9]和实验室已有数据,选取16种不同类别的化学品(表1)进行鱼类胚胎急性毒性试验㊂同时,进行其中14种化学品的鱼类急性毒性试验㊂1.1.3㊀试验用水试验用水为标准稀释水(表2),并在恒温储水箱中连续曝气至氧饱和,恒温(23ʃ2)ħ㊂水硬度为100~300mg㊃L-1(以CaCO3计),pH为6.5~8.5㊂1.1.4㊀仪器设备和试验容器体式显微镜(Lumar V12,Carl Zeiss,德国);药品稳定性试验箱(MT-750B,施都凯仪器设备有限公司,中国);多参数水质分析仪(WTW3430,Thermo Fisher,美国);电子分析天平(AL204,Mettler Toledo,瑞士);精密型照度仪(KIMO HQ210,法国);总有机碳分析仪(Multi N/C3100,耶拿公司,德国);微电脑总硬度浓度测定仪(HI96735,HANNA仪器公司,德国)㊂灭菌24孔标准带盖板;5L玻璃圆缸㊂1.2㊀鱼类胚胎急性毒性试验方法按照‘OECD化学品测试准则No.236鱼类胚胎急性毒性试验“[10]进行试验设计并制定试验方案,选用3,4-二氯苯胺(3,4-DCA)㊁五水硫酸铜(CSP)㊁2,3,6-三甲基苯酚(2,3,6-TMP)㊁二甲基亚砜(DMSO)㊁七水硫酸锌(ZSH)㊁三甘醇(TEG)㊁氯化钠(SC)㊁十二烷基硫酸钠(SDS)㊁重铬酸钾(PD)㊁甲基异噻唑啉酮(MIT)㊁二苯甲酮(BP)㊁二氯苯氧氯酚(TCS)㊁1,2-苯并异噻唑啉-3-酮(BIT)㊁三唑酮(T)㊁多菌灵(C)和N-(4-氯苯基)-N -(3,4-二氯苯基)脲(三氯卡班,TCC)16种化学品分别进行稀有鮈鲫和斑马鱼胚胎急性毒性试验,评价稀有鮈鲫胚胎对化学品的敏感性㊂1.2.1㊀试验原理将新受精的鱼类胚胎暴露于不同浓度的样品水溶液中96h㊂期间每24h,观察并记录以下1~4个死亡表征:(1)卵凝结;(2)体节缺失;(3)尾部未分离;(4)无心跳㊂当暴露结束时,通过上述4个表征的阳性结果确定急性毒性,并计算半数致死浓度(LC50)值㊂. All Rights Reserved.第5期王绿平等:稀有鮈鲫作为鱼类胚胎急性毒性试验受试鱼种的敏感性研究105㊀表1㊀受试化学品基本信息Table1㊀The basic information of the test chemicals化学品名Chemical name分子式Molecular formulaCAS号CAS number纯度Purity批号Batch No生产商Manufacturer3,4-二氯苯胺(3,4-DCA)3,4-dichloroaniline(3,4-DCA)C6H5Cl295-76-1>98%FHJ01-AMNI TCI 五水硫酸铜(CSP)Copper sulfate pentahydrate(CSP)CuSO4㊃5H2O7758-99-899%C10000665Macklin 2,3,6-三甲基苯酚(2,3,6-TMP)2,3,6-trimethylphenol(2,3,6-TMP)C9H12O2416-94-6>98.0%N6BDM-IL TCI二甲基亚砜(DMSO)Dimethyl sulfoxide(DMSO)C2H6OS67-68-5ȡ99.5%BCBV2983Sigma-Aldrich 七水硫酸锌(ZSH)Zinc sulfate(ZSH)ZnSO4㊃7H2O7446-20-0ȡ99.5%BCBS9911Sigma-Aldrich 三甘醇(TEG)Triethylene glycol(TEG)C6H14O4112-27-699%STBG9386Sigma-Aldrich 氯化钠(SC)Sodium chloride(SC)NaCl7647-14-599.5%C10042430Macklin 十二烷基硫酸钠(SDS)Sodium dodecyl sulfate(SDS)C12H25SO4Na151-21-3ȡ98.5%SLBR9016V Sigma-Aldrich重铬酸钾(PD) Potassium dichromate(PD)K2Cr2O77778-50-9ȡ99.8%20140708国药集团化学试剂有限公司Sinopharm ChemicalReagent Co.LTD2-甲基-4-异噻唑啉-3-酮(MIT) 2-methyl-4-isothiazolin-3-one(MIT)C4H5NOS2682-20-495%L6C0S119百灵威J&K Chemical二苯甲酮(BP)Benzophenone(BP)C6H5COC6H5119-61-9>99.0%5VW2J-RO TCI二氯苯氧氯酚(TCS)Triclosan(TCS)C12H7Cl3O23380-34-5>98.0%5SP7D-EQ TCI 1,2-苯并异噻唑啉-3-酮(BIT)1,2-benzisothiazolin-3-one(BIT)C7H5NOS2634-33-5>98.0%EJQ4C-CC TCI 三唑酮(T)Triadimefon(T)C14H16ClN3O243121-43-398.0%5-HBN-152-1TRC(加拿大Canada)多菌灵(C)Carbendazim(C)C9H9N3O210605-21-798.0%LHA0R15百灵威J&K Chemical N-(4-氯苯基)-N -(3,4-二氯苯基)脲(三氯卡班,TCC)Triclocarban(TCC)C13H9Cl3N2O101-20-2>98.0%GF01-CCOJ TCI表2㊀标准稀释水的配制方法Table2㊀The preparation method of standard diluted water化学品名Chemical name分子式Molecular formula浓度/(g㊃L-1)Concentration/(g㊃L-1)贮备液a Stock solution a CaCl2㊃2H2O11.76贮备液b Stock solution b MgSO4㊃7H2O 4.93贮备液c Stock solution c KCl0.23贮备液d Stock solution d NaHCO3 2.59标准稀释水Standard diluted water 去离子水的电导率ɤ10⋈S㊃cm-1,将上述4种贮备液各25mL加以混合并用去离子水稀释至1L,使用前连续曝气24h以上待用The conductivity of deionized water isɤ10⋈S㊃cm-1.25mL of each stock solution were mixed and diluted to1L with deionized water,and continuously aerated for more than24h before use注:贮备液及标准稀释水均使用分析纯试剂和去离子水配制而成㊂Note:Both the stock solutions and standard diluted water were prepared with pure analytical reagent and deionized water. . All Rights Reserved.106㊀生态毒理学报第16卷1.2.2㊀试验条件试验用水为标准稀释水,并在储水箱中24h连续曝气;试验温度(26ʃ1)ħ;每天12h光照㊂暴露时间:96h㊂试验开始:最晚在16细胞期前暴露于试验溶液中㊂试验方式:更新式,更新频率为24h㊂试验溶液:在已预处理24h的24孔板中,每孔注入新鲜制备的试验溶液㊂1.2.3㊀试验操作母液制备:可溶化学品(CSP㊁DMSO㊁ZSH㊁TEG㊁SC㊁SDS㊁PD和MIT)均于试验当天,称取适量样品直接溶于一定体积的试验用水中配制成高浓度的样品母液,其中CSP和ZSH用去离子水配制㊂低水溶性化学品(3,4-DCA㊁2,3,6-TMP㊁BP㊁TCS㊁BIT㊁T 和C)于试验前3d,称取适量样品添加到一定体积的的试验用水中,避光连续磁力搅拌72h,经0.45μm硝酸纤维素膜过滤后配制成试验体系下的样品饱和溶液作为样品母液㊂难溶性化学品(TCC)用助溶剂DMSO配制系列浓度的样品母液㊂试验溶液制备:根据母液实测浓度,将适量的样品母液添加到试验用水中,配制成一定浓度的试验溶液㊂暴露浓度:根据文献数据和预试验结果,各化学品设置5或6个试验浓度组(表3),均以几何级数分布,浓度的间隔系数不超过2.2,同时,每组设1个空白对照组(试验用水),对于斑马鱼,另设置1个4mg㊃L-1的3,4-DCA阳性对照组,所有试验组均不设平行㊂胚胎分配:所有试验均在标准24孔板中完成,每孔1个受精卵㊂样品组各浓度20个/板;以试验用水为介质,在上述各板中另放入4个鱼卵作为板内质控;空白对照组24个/板;阳性对照组(3,4-DCA,4mg㊃L-1,斑马鱼适用)(图1)㊂观察与记录:按1.2.4描述的原则进行观察和记录㊂试验结束时,如板内质控死亡超过1个,则整板无效,该浓度组剔除㊂水质参数测定:在试验开始和结束时测定对照组和最高浓度样品组的硬度和电导率,试验期间每次更新前后测定空白对照组和最高浓度样品组的pH㊂在试验结束时,测定空白对照组和最高浓度样品存活胚胎组的溶解氧浓度㊂试验期间,每日测定1次温度,测定温度时,随机选取3个试验容器进行测定㊂1.2.4㊀死亡表征的观察和判定每个受试胚胎每24h观察以下几个终点:卵凝结㊁体节未形成㊁尾部未与卵黄囊分离㊁失去心跳,观察到上述表征之一即可判定死亡㊂此外,从48h开始,每日观察1次样品组和空白对照组的孵化数㊂表3㊀各化学品在鱼类胚胎急性毒性试验中的浓度设置Table3㊀Concentrations of each chemical in Fish Embryo Acute Toxicity(FET)test化学品Chemical稀有鮈鲫胚胎Gobiocypris rarus embryo斑马鱼胚胎Danio rerio embryo3,4-DCA/(mg㊃L-1)5㊁7㊁10㊁13㊁200.5㊁1㊁2㊁4㊁8*CSP/(mg㊃L-1)0.7㊁1㊁1.5㊁2.3㊁3.50.15㊁0.3㊁0.6㊁1.2㊁2.4*2,3,6-TMP/(mg㊃L-1)10㊁15㊁22㊁33㊁508㊁12㊁18㊁27㊁40.5*DMSO/(g㊃L-1)20㊁30㊁45㊁67㊁10010㊁17㊁28.9㊁49.13㊁83.521*ZSH/(mg㊃L-1)4㊁6㊁9㊁13.5㊁2093.6㊁148㊁222㊁333㊁500TEG/(g㊃L-1)15㊁24㊁39㊁63㊁10020㊁30㊁45㊁67.5㊁101.25*SC/(g㊃L-1)2㊁3.2㊁5.1㊁8.2㊁131㊁2㊁4㊁8㊁16*SDS/(mg㊃L-1)4㊁6.8㊁11.6㊁19.7㊁33.5 1.2㊁1.8㊁2.7㊁4㊁6PD/(mg㊃L-1)198㊁297㊁445㊁667㊁1000482㊁578㊁694㊁833㊁1000MIT/(mg㊃L-1)9.48㊁14.2㊁21.4㊁32㊁4814.2㊁21.4㊁32㊁48㊁72BP/(mg㊃L-1) 5.0㊁8.0㊁12㊁19㊁30 5.0㊁8.0㊁12㊁19㊁30TCS/(mg㊃L-1)0.0886㊁0.124㊁0.174㊁0.243㊁0.340㊁0.4760.0886㊁0.124㊁0.174㊁0.243㊁0.340BIT/(mg㊃L-1) 3.0㊁4.15㊁6.75㊁10.1㊁15.2 2.62㊁3.28㊁4.10㊁5.12㊁6.4㊁8.0T/(mg㊃L-1)14㊁18㊁24㊁31㊁4014㊁18㊁24㊁31㊁40C/(mg㊃L-1)0.370㊁0.555㊁0.833㊁1.25㊁1.87㊁2.800.370㊁0.555㊁0.833㊁1.25㊁1.87㊁2.80TCC/(mg㊃L-1)0.0709㊁0.130㊁0.230㊁0.414㊁0.7450.0443㊁0.0709㊁0.113㊁0.181㊁0.290㊁0.464注:*文献数据[12]㊂Note:*literature data[12].. All Rights Reserved.第5期王绿平等:稀有鮈鲫作为鱼类胚胎急性毒性试验受试鱼种的敏感性研究107㊀图1㊀鱼类胚胎急性毒性(FET )试验24孔板布局图注:1~5为5个试验浓度样品组;nC 为空白对照组(试验用水);iC 为板内质控(试验用水);pC 为阳性对照组(3,4-DCA ,4mg ㊃L -1,斑马鱼适用)㊂Fig.1㊀Layout of 24-well pate for Fish Embryo Acute Toxicity (FET)testNote:1~5represent five test concentration groups;nC represents blank control group (test water);iC represents quality control in test plate (test water);pC represents positive control group (3,4-DCA,4mg ㊃L -1,for Danio rerio ).1.2.5㊀试验有效性判断参照OECD TG 236,本试验有效性判断原则为:试验用胚胎总受精率ȡ70%;试验期间,试验容器中温度维持在(26ʃ1)ħ之间;试验结束时,空白对照组胚胎存活率ȡ90%,空白对照组胚胎孵化率ȡ80%,阳性对照组(3,4-DCA ,4mg ㊃L -1,斑马鱼适用)死亡率至少为30%,空白对照组和最高浓度样品组试验溶液的溶解氧含量ȡ80%空气饱和值㊂1.2.6㊀数据处理计算试验开始后24㊁48㊁72和96h 各试验组(各板)胚胎的累计死亡率,绘制浓度-死亡率曲线图㊂采用点估计法估算96h -LC 50值及95%置信限㊂分析用软件ToxCalc(v5.0.32)完成㊂1.3㊀鱼类急性毒性试验方法按照‘OECD 化学品测试准则No.203鱼类急性毒性试验“[12]进行试验设计并制定试验方案,选用CSP ㊁2,3,6-TMP ㊁DMSO ㊁ZSH ㊁TEG ㊁SC ㊁SDS ㊁MIT ㊁BP ㊁TCS ㊁BIT ㊁T ㊁C 和TCC 14种化学品进行鱼类急性毒性试验,评价稀有鮈鲫对化学品的敏感性㊂1.3.1㊀试验原理在规定条件下,将试验鱼暴露于不同浓度的样品水溶液中96h ㊂在24㊁48㊁72和96h 时分别记录试验鱼的死亡数,确定96h -LC 50㊂1.3.2㊀试验条件试验用水为标准稀释水,并在储水箱中24h 连续曝气;试验温度(23ʃ2)ħ;每天12h 光照㊂暴露时间96h ,试验方式为更新式,更新频率为24h ㊂1.3.3㊀试验操作母液制备:可溶化学品(CSP ㊁DMSO ㊁ZSH ㊁TEG ㊁SC ㊁SDS 和MIT)均于试验当天,称取适量样品直接溶于一定体积的试验用水中配制成高浓度的样品母液㊂低水溶性化学品(2,3,6-TMP ㊁BP ㊁TCS ㊁BIT ㊁T 和C)于试验前3d ,称取适量样品添加到一定体积的的试验用水中,避光连续磁力搅拌72h ,经0.45μm 硝酸纤维素膜过滤后配制成试验体系下的样品饱和溶液作为样品母液㊂难溶性化学品(TCC)用助溶剂DMSO 配制系列浓度的样品母液㊂试验溶液制备:根据母液实测浓度,将适量的样品母液添加到试验用水中,配制成一定浓度的试验溶液㊂暴露浓度:根据预试验结果,13种化学品设置5. All Rights Reserved.108㊀生态毒理学报第16卷或6个试验浓度组(表4),均以几何级数分布,浓度的间隔系数不超过2.2;化学品C设置1个试验浓度组进行限度试验,同时,每组设1个空白对照组(试验用水),对于使用助溶剂的样品,另设置1个助溶剂对照组,所有试验组均不设平行㊂试验鱼分配:所有试验均在盛有4L试验溶液的5L玻璃圆缸中完成,所有试验对照组和各浓度组均放入7尾鱼㊂试验开始前在驯化群随机选择10尾鱼进行体质量和总长的测定,并计算试验鱼的承载量㊂观察与记录:在试验开始后0㊁24㊁48㊁72和96h观察并记录各试验容器内试验鱼的死亡数和异常表征㊂水质参数测定:在试验开始前测定一次试验用水的硬度和总有机碳(TOC)㊂在试验开始㊁每一次更新前后及试验结束时,测定并记录所有试验溶液的pH值㊁溶解氧和温度㊂1.3.4㊀试验有效性判断参照OECD TG203,本试验有效性判断原则为:试验结束时,空白对照组试验鱼的死亡率ɤ10%,试验期间,试验容器中温度维持在(23ʃ2)ħ之间,溶解氧含量ȡ60%空气饱和值㊂1.3.5㊀数据处理计算试验开始后24㊁48㊁72和96h各试验组试验鱼的累计死亡率,绘制浓度-死亡率曲线图㊂采用点估计法估算96h-LC50值及95%置信限㊂分析用软件ToxCalc(v5.0.32)完成㊂2㊀结果与讨论(Results and discussion)2.1㊀化学品对2种鱼类胚胎急性毒性结果比较各次试验开始时胚胎受精率均超过70%,空白对照组存活率为100%,孵化率为91.7%~100%;斑马鱼试验中,阳性对照组(3,4-DCA,4mg㊃L-1)死亡率为95.0%~100%;样品组板内质控均无死亡发生㊂试验溶液温度维持在(26ʃ1)ħ之间,空白对照组和最高浓度样品组试验溶液的溶解氧含量均超过80%空气饱和值,根据1.2.5描述的原则,本试验有效㊂如表5所示,16种化学品对稀有鮈鲫胚胎和斑马鱼胚胎的96h-LC50值比较可知,稀有鮈鲫胚胎和斑马鱼胚胎对其中15种化学品的敏感性相近,其中TEG㊁PD㊁MIT和BIT等4种化学品,稀有鮈鲫胚胎的敏感性优于斑马鱼㊂ZSH对稀有鮈鲫胚胎的96h-LC50值为10.4mg㊃L-1,斑马鱼的为238mg㊃L-1,两者差异约22.9倍,可见稀有鮈鲫胚胎对ZSH异常表4㊀各化学品在鱼类急性毒性(FAT)试验中的浓度设置Table4㊀Concentrations of each chemical in FishAcute Toxicity(FAT)test化学品Chemical稀有鮈鲫成鱼Adult Gobiocypris rarus 3,4-DCA/(mg㊃L-1) 2.0㊁3.6㊁6.5㊁12㊁21*CSP/(mg㊃L-1)0.50㊁0.84㊁1.4㊁2.4㊁4.02,3,6-TMP/(mg㊃L-1)9.80㊁14.7㊁22㊁33㊁50DMSO/(g㊃L-1)20㊁30㊁45㊁67㊁100ZSH/(mg㊃L-1)10㊁20㊁39㊁76㊁150TEG/(g㊃L-1)30㊁45㊁67㊁100㊁150SC/(g㊃L-1)8.0㊁8.85㊁9.80㊁10.8㊁12SDS/(mg㊃L-1)5㊁6.2㊁7.7㊁9.5㊁12PD/(mg㊃L-1)50㊁85㊁145㊁250㊁425*MIT/(mg㊃L-1)8.19㊁10.2㊁12.8㊁16.0㊁20.0BP/(mg㊃L-1) 2.81㊁4.19㊁6.31㊁9.43㊁14.2㊁21.2TCS/(mg㊃L-1)0.184㊁0.239㊁0.311㊁0.404㊁0.525BIT/(mg㊃L-1) 2.62㊁3.28㊁4.10㊁5.12㊁6.40T/(mg㊃L-1)7.13㊁8.20㊁9.43㊁10.8㊁12.5㊁14.3C/(mg㊃L-1)100%饱和溶液100%saturated concentration TCC/(mg㊃L-1)0.046㊁0.065㊁0.091㊁0.128㊁0.179注:*文献数据[27]㊂Note:*literature data[27].表5㊀化学品对2种鱼类胚胎的96h半数致死浓度(96h-LC50)Table5㊀The median lethal concentration of chemicalsat96h(96h-LC50)to two kinds of fish embryos 化学品Chemical稀有鮈鲫胚胎Gobiocyprisrarus embryo斑马鱼胚胎Daniorerio embryo 3,4-DCA/(mg㊃L-1)12.8 2.7*CSP/(mg㊃L-1) 1.760.291*2,3,6-TMP/(mg㊃L-1)14.110.8*DMSO/(g㊃L-1)51.734.1*ZSH/(mg㊃L-1)10.4238TEG/(g㊃L-1)52.054.8*SC/(g㊃L-1)10.4 5.14*SDS/(mg㊃L-1)18.2 5.13PD/(mg㊃L-1)534815MIT/(mg㊃L-1)31.438.9BP/(mg㊃L-1)25.417.9TCS/(mg㊃L-1)0.2600.154BIT/(mg㊃L-1) 3.697.08T/(mg㊃L-1)23.222.7C/(mg㊃L-1) 1.280.904TCC/(mg㊃L-1)0.2950.0704注:*文献数据[12]㊂Note:*literature data[12].. All Rights Reserved.第5期王绿平等:稀有鮈鲫作为鱼类胚胎急性毒性试验受试鱼种的敏感性研究109㊀敏感㊂此外,3,4-DCA㊁CSP㊁2,3,6-TMP㊁DMSO㊁SC㊁SDS㊁BP㊁TCS㊁T㊁C和TCC等11种化学品,斑马鱼胚胎的敏感性略优于稀有鮈鲫胚胎,但毒性值差异在1.02倍~6.05倍之间,有很好的可比性㊂鉴于化学品理化性质的不同和生物物种间的差异,稀有鮈鲫胚胎的敏感性不亚于斑马鱼胚胎,适宜鱼类胚胎急性毒性试验的应用㊂2.2㊀化学品对稀有鮈鲫胚胎和成鱼急性毒性结果比较㊀㊀鱼类胚胎阶段作为鱼类全生命周期的最初期,通常其敏感性会优于或不亚于其他时期㊂如表6所示,16种化学品对稀有鮈鲫胚胎及其成鱼的96h-LC50值比较可知,稀有鮈鲫胚胎及其成鱼对其中15种化学品的敏感性相近,其中2,3,6-TMP㊁ZSH㊁TEG㊁TCS㊁BIT和C等6种化学品,稀有鮈鲫胚胎的敏感性优于稀有鮈鲫成鱼㊂对于3,4-DCA㊁CSP㊁DMSO㊁SC㊁SDS㊁PD㊁MIT㊁BP㊁T和TCC等10种化学品,稀有鮈鲫成鱼的敏感性略优于稀有鮈鲫胚胎,这可能是化学品试验溶液暴露条件的不同导致的[28],但毒性值差异在1.13倍~5.57倍之间,有很好的可比性㊂此外,通过分析软件SPSS Statistics17.0对稀有鮈鲫胚胎及其成鱼96h-LC50值进行相关性分析,得出两者具有较好的相关性(P=0.879)㊂由此可见,稀有鮈鲫胚胎与其成鱼相比,敏感性类似,具有成为成鱼急性毒性试验替代方法的应用潜力㊂Dang等[29]和Lammer等[30]的研究表明,部分化学品理化性质的差异会导致化学品对成鱼的敏感性优于胚胎,但胚胎和成鱼之间均具有较好的相关性,试验数据为鱼类胚胎急性毒性试验替代鱼类急性毒性试验提供了有力的科学支持,与本文结论一致㊂此外,关于胚胎急性毒性试验(FET)作为成鱼急性毒性试验(AFT)的替代测试,有研究通过985个斑马鱼胚胎试验和1531个成鱼试验,建立数学模型[15],借助FET数据预测AFT值,实现试验和非试验相结合,在尽可能减少动物试验的前提下,为化学品环境管理提供更多基础数据㊂研究将16种化学品的FET试验数据代入公式(log FET LC50=(0.989ˑlog AFT LC50)-0.195[15]),预测AFT值(表7)㊂结果可见,所有AFT预测值和试验值相比,差异均未超过一个数量级,表明2种数据间相互预测和使用具有一定的可操作性㊂如后续有更多数据累积,可通过相互关系,建立稀有鮈鲫特有的FET与AFT的推表6㊀化学品对稀有鮈鲫胚胎和成鱼的96h-LC50 Table6㊀The96h-LC50of chemicals to Gobiocypris rarus embryos and adult Gobiocypris rarus化学品Chemical胚胎Embryo成鱼Adult fish 3,4-DCA/(mg㊃L-1)12.8 6.52*CSP/(mg㊃L-1) 1.76 1.292,3,6-TMP/(mg㊃L-1)14.117.0DMSO/(g㊃L-1)51.724.5ZSH/(mg㊃L-1)10.437.5TEG/(g㊃L-1)52.065.2SC/(g㊃L-1)10.49.19SDS/(mg㊃L-1)18.28.81PD/(mg㊃L-1)534116*MIT/(mg㊃L-1)31.415.6BP/(mg㊃L-1)25.410.3TCS/(mg㊃L-1)0.2600.293BIT/(mg㊃L-1) 3.69 4.41T/(mg㊃L-1)23.29.48C/(mg㊃L-1) 1.28>6.51TCC/(mg㊃L-1)0.2950.053注:*文献数据[27]㊂Note:*literature data[27].表7㊀化学品对稀有鮈鲫的FET和成鱼急性毒性试验(AFT)与非试验结果Table7㊀The results of chemicals to Gobiocypris rarus in FET,adult fish acute toxicitytest(AFT)and non-test results化学品ChemicalFET LC50试验值TestedFET LC50AFT LC50预测值PredictedAFT LC50AFT LC50试验值TestedAFT LC50 3,4-DCA/(mg㊃L-1)12.820.4 6.59CSP/(mg㊃L-1) 1.76 2.79 1.292,3,6-TMP/(mg㊃L-1)14.122.917.0DMSO/(g㊃L-1)51.785.124.5ZSH/(mg㊃L-1)10.416.837.5TEG/(g㊃L-1)52.085.665.2SC/(g㊃L-1)10.416.89.19SDS/(mg㊃L-1)18.229.68.81PD/(mg㊃L-1)534902116MIT/(mg㊃L-1)31.451.415.6BP/(mg㊃L-1)25.441.510.3TCS/(mg㊃L-1)0.2600.4030.293BIT/(mg㊃L-1) 3.69 5.90 4.41T/(mg㊃L-1)23.237.89.48C/(mg㊃L-1) 1.28 2.02>100TCC/(mg㊃L-1)0.2950.4580.053. 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© Agricultural and Food Science Manuscript received February 2008Acute toxicity of birch tar oil on aquatic organismsMarleena Hagner 1, Olli-Pekka Penttinen 1, Tiina Pasanen 1, Kari Tiilikkala 2 and Heikki Setälä1*1Department of Ecological and Environmental Sciences, University of Helsinki, Niemenkatu 73, FI-15140 Lahti,Finland, *e-mail: heikki.setala@helsinki.fi2MTT Agrifood Research Finland, Plant Production Research, Rillitie 1, FI-31600 Jokioinen, Finland Birch tar oil (BTO) is a by-product of processing birch wood in a pyrolysis system. Accumulating evidence suggests the suitability of BTO as a biocide or repellent in terrestrial environments for the control of weeds, insects, molluscs and rodents. Once applied as biocide, BTO may end up, either through run-off or leaching, in aquatic systems and may have adverse effects on non-target organisms. As very little is known about the toxicity of BTO to aquatic organisms, the present study investigated acute toxicity (LC 50/EC 50) of BTO for eight aquatic organisms. Bioassays with the Asellus aquaticus (crustacean), Lumbriculus variegatus (oligochaeta worm), Daphnia magna (crustacean), Lymnea sp. (mollusc), Lemna minor (vascular plant), Danio rerio (fish), Scenedesmus gracilis (algae), and Vibrio fischeri (bacterium) were performed accord-ing to ISO, OECD or USEPA-guidelines. The results indicated that BTO was practically nontoxic to most aquatic organisms as the median effective BTO concentrations against most organisms were > 150 mg l -1. In conclusion, our toxicity tests showed that aquatic organisms are to some extent, invariably sensitive to birch tar oil, but suggest that BTO does not pose a severe hazard to aquatic biota. We deduce that, unless BTOs are not applied in the immediate vicinity of water bodies, no special precaution is required.Key-words : acute toxicity test, aquatic organisms, birch tar oil, biocide, EC 50IntroductionBirch tar oil (BTO; CAS #8001-88-5, American Chemical Society 2007) is a crude by-product of the slow destructive distillation or pyrolysation, of wood and bark for processing into coal. Thereis anecdotal and scientific evidence suggesting the suitability of BTO as a biocide and/or repel-lent against molluscs, insects, weeds, and rodents (Hagner et al. unpublished, Hagner 2005, Hagner et al. 2010, Lindqvist et al. 2010, Salonen et al. 2008, Tiilikkala and Salonen 2008). Due to its novelty as a biocide/repellent/biological plant protection prod-uct, no comprehensive information on the effective compounds of BTO components is available and nothing is known about the toxicity of BTO against the aquatic organisms. Our preliminary analyses suggest phenols to be among the most interesting compounds of the BTO as biocontrol agents, but that also various other volatile compounds can play a role. Among the phenolic compounds alone, allylphenol, cresols, 4-ethyl guiaiacol, ethylvanil-lin, eugenol, guaiacol, isoeugenol, 4-methyl, and vanillin have been identified in biomass pyrolysis (Murwanashyaka et al. 2002).Two types of birch tar oils can be derived from the same pyrolysis process. BTO1 is the liquid ma-terial resulting from the early phase of the distilla-tion process when the temperature is below 380 o C. BTO2 is the more viscous component generated at the end of the process when the temperature rises to and above 400 o C. BTO2 has proven to be an effective snail and slug repellent when smeared on barrier fences or the walls of plants pots (Hagner 2005, Lindqvist et al. 2010), while BTO1, sprayed with a compressed air pump directly on soil sur-face, is effective against numerous weed and in-sect pest species, and as such, may be used as a herbicide/insecticide, for example, in potato fields (Hagner et al. unpublished; personal observations by the authors). An effective control of perennial weeds is likely to require high doses (1.36 dl m-2) of this substance (Hagner et a l. unpublished). When used for weed control of annual crops the required dose of BTO1 is about one-third, and for control-ling pest insects about a tenth of the dose applied for perennial grass control. In Finland, large scale field experiments using biological plant protection products can not be established if the ecological ef-fects of these products are not known. We therefore examined the general ecological effects of BTOs on target organisms in the laboratory.Chemical substances applied in terrestrial eco-systems are often detected in aquatic ecosystems (Accinelli et al. 2002, Larson et al. 1995, Shipi-talo and Owens 2003). Should the use of BTO as a plant protection product become common practice in horticultural or agricultural production, it is pos-sible that BTO compounds may leach to surface and ground waterways. As aquatic organisms are generally sensitive to various organic and inorganic pollutants (Connell et al. 1999), changes in the spe-cies composition of aquatic communities is likely to affect the function and structure of the whole ecosystem (Hanazato 1998). Consequently, accord-ing to international regulations (EC 1996) ecotoxi-cological effects of chemicals on the environment must be assessed before using in the field.To the best of our knowledge, no studies have been docu-mented with regard to the ecotoxicological effects of BTO on aquatic organisms. This study aims to assess the acute toxicity of BTO1 (EC50-values i.e. the concentration of BTO1 producing certain half-maximal effect) on an extensive group of aquatic organisms widely used in ecotoxicological studies. Of the organisms used, the water louse (Asellus aquaticus)and the oligochaeta worm (Lumbriculus variegates) are sediment dwelling benthic inver-tebrates, while the pond snail (Lymnea sp.) usu-ally harbours in aquatic plants (Olsen et al. 2005). Representing the pelagic and littoral organisms are the water flea (Daphnia magna), lesser duckweed (Lemna minor), zebrafish (Danio rerio), unicellular green algae (Scenedesmus gracilis), and fluores-cent bacteria (Vibrio fischeri).This study is part of a larger research program in which the effect of BTOs as a molluscicide (Hagner 2005, Lindqvist et al. 2010) and their eco-toxicological effects on both terrestrial (Hagner et al. 2010) and aquatic (the current study) organisms were investigated.Material and methodsBTO1 from pyrolysed birch wood and bark was kindly supplied by Charcoal Finland Ltd. The BTO1 concentrations used were chosen on the basis of preliminary tests, and prepared by add-ing a certain amount of BTO1 to the test solution and shaken vigorously for 10 seconds. To obtain acute toxicity within the duration of the laboratory protocol, it was necessary to test at concentrations greater than solubility of BTO1 in water by follow-ing the rationale given by Hatch and Burton 1998.Hagner, M. et al. Acute toxicity of birch tar oilThe pH level of test waters was routinely monitored during treatments. At least five different exposure concentrations were applied in a geometric series without adjusting pH after BTO1 application. The test organisms were added to test jars (n=3 per treat-ment) immediately after BTO1 application, except for D. rerio . In each test, control jars received no BTO1 (n=3). The following response variables were determined: 1) root length and leaf number of duckweed (IC 50, L . minor ), 2) mobility of the water flea (EC 50, D. magna ), 3) survival rate of the water louse (LC 50, A. aquaticus ), zebrafish (LC 50, D. rerio ), oligochaeta worm (LC 50, L. variegatus ) and pond snail (LC 50, Lymnea sp .), 4) inhibition of light emission capacity of the bacteria (IC 50, V. fisheri ) and 5) the number of cells of the algae S. gracilis . The organisms applied in the short-term toxicity test were not fed during the tests.Daphnia magna – water flea (Crustacea)A 48 h acute test with D. magna was performed according to standard procedure of ISO 6341 (1996) on up to 24h old newborns of daphnids obtained from the laboratory culture of the Fin-land’s Environmental Administration. Before using in the toxicity test, D. magna were kept in the test medium (reconstituted water (Elendt M7) prepared according to the OECD guideline (OECD 1997) for two months before the start of the experiment. Five newly hatched juveniles were carefully placed in each glass jar (20 ml vol.) containing 10 ml of solution. BTO1 concentrations ranged from 18 to 381 mg l -1 test medium, each with 3 replicates. The jars were covered with Parafilm ® M and kept in complete darkness at 22 o C. At time 24 and 48 h, surviving animals were counted.Lemna minor – lesser duckweed (Araceae)Growth inhibition test was carried out according to the ISO/WD standard 20079 (2005). Whole, healthy duckweed was collected from a pond in the city of Lahti, Southern Finland and kept in the pond water until the start of the test. Thetest medium was a modification of the Swedish standard (SIS) Lemna growth medium with pH adjusted to 6.5. The exposure concentrations ranged from 30 to 4900 mg l -1 test medium, each with 3 replicates. The test was performed in 50 ml glass Erlenmeyer flasks. Seven double-fronded healthy L. minor individuals, were transferred to each test flask. Before transformation, roots of the plants were removed. The flasks were covered with Parafilm ® M to minimize evaporation and kept in continuous light (20000 lx) at 22 o C . After seven days, the number of fronds and the length of roots were measured.Asellus aquaticus – water louse (Crustacea)A mortality test was performed following the common guidelines by USEPA (2002). Before the test, Asellus individuals − collected from the field − were kept in jars filled with lake water containing sand and decomposing litter of Alnus glutinosa L. (Betulaceae). Asellus were not acclimated to the test water prior to exposure because acclimation may result in differentiation between individuals. Availability of Asellus was rather limited and ac-climation of animals may therefore prove to be use-less if animals exposed to changing environmental conditions are likely to die within the acclimation period. Because some BTO compounds can bind to humus in lake water, artificial freshwater, prepared according the SFS 5062 (1984) -standard, was used as a test solution. Three replicates were prepared for each test concentration that ranged from 229 to 4900 mg BTO1 l -1. The test was performed in 125 ml glass jars containing 100 ml test solution. Three adult Asellus individuals were placed in each jar which was covered with Parafilm ® M. The jars were kept at 22 o C with a light:dark cycle 12:12h. Survival rate was recorded at time 24 and 48 h.Danio rerio – zebrafish (Cyprinidae)The mortality test for D . rerio was performed ac-cording to the standard procedure from the OECD Guideline 203 (1992). Fish, with an average lengthVol. 19(2010): 24–33.of 2 ±1 cm, originated from a commercial fish stock. The fish were held in the laboratory for 14 days before testing and fed once a day with commercially available fish (flake) food. Tap water was used as the test medium with TetraAqua® AquaSafe neu-tralizer (5 ml/10l test water) to improve the water quality for the fish. The test medium was allowed to stabilize for two days before BTO1 addition. The BTO1 concentrations ranged between 120 and 510 mg l-1 water (n=2). At 24 h after addition of BTO1, seven fish individuals were added to each tank (7000 ml capacity), containing 6000 ml water, and covered with plastic film. Tanks were kept at 22 o C with a light:dark cycle of 12:12h. During the test, tanks were aerated continuously ( > 6 % saturation) and the oxygen concentration and pH were measured every 24 h. Surviving fish were recorded at 24, 48, 72 and 96 h after the start of the test.Lumbriculus variegatus – worm(Lumbriculidae)The mortality test using L. variegatus worms fol-lowed the common guidelines by USEPA (2002) using worms originating from cultures maintained at the Department of Ecological and Environmental Sciences, University of Helsinki. Artificial fresh-water, prepared according to the SFS 5062 (1984) –standard, was used as the test medium. The BTO1 test concentrations ranged from 30 to 4900 mg l-1 test medium, each with 3 replicates. The test was performed in 125 ml glass jars, containing 100 ml test solution and covered with Parafilm® M. Five adult worms were carefully placed in each replicate jar which were kept at 22 o C with light:dark cycle of 12:12 h. The number of surviving worms was estimated at 24 and 48 h.Lymnea sp.– fresh water snail (Lymnaeidae)The mollusc mortality test with Lymnea sp. was performed following the guidelines described by USEPA (2002). Field-collected molluscs were kept in the laboratory in lake water one week before the start of the test. Artificial water was used as a test solution and prepared according to the SFS 5062 (1984) -standard. The experimental unit comprised 125 ml glass jars, each containing 100 ml test solution. The BTO1 test concentrations ranged between 80 and 8160 mg l-1 test medium, each with 3 replicates. Three snails were placed in each jar, covered with Parafilm® M, and kept at 22 o C with light:dark cycle of 12:12 h. Surviving molluscs were counted at 24 and 48 h.Vibrio fischeri – gram negative bacteria(Vibrionaceae)The BioTox method is a traditional and standard-ized way to measure the toxicity of chemicals using the photobacteria Vibrio fischeri. Briefly, the metabolic pathway responsible for light emission by the bacterium is intrinsically linked to cellular respiration and any disruption of normal cellular metabolism causes a decrease in light production. Toxicity assessment was performed using freeze-dried V. fischeri (strain NRRL B-11177, Aboatox Oy, Finland). The reagent was stored at -20 o C to preserve microbial activity. BTO test concentra-tions ranged between 30 and 4900 mg BTO1 l-1 test medium, each with 2 replicates. A 15 and 30 min luminescence inhibition test was performed according to the ISO 11348-3 (1998) -standard. Light emission was measured using a luminometer after 15 and 30 minutes exposure.Scenedesmus gracilis – green algae(Scenedesmaceae)Algae was obtained from a monospecies culture of S. gracilis and supplied by the Lammi Biological Station, University of Helsinki. A growth inhibi-tion test was performed according to the OECD Guideline 201 (1984). Algae were cultured in algal medium according to the guideline two months prior to the start of the test. BTO1 concentrationsHagner, M. et al. Acute toxicity of birch tar oilhighest concentrations tested (A. aquaticus > 1058 mg BTO1 l -1, L. variegatus > 381 mg BTO1 l -1, D. magna > 229 mg BTO1 l -1 and D. rerio > 370mg BTO1 l -1, Lymnaea sp. > 1760 mg BTO1 l -1), while the lowest concentrations tested (A. aquaticus <229 mg BTO1 l -1, L. variegatus <49 mg BTO1 l -1, D. magna <137 mg BTO1 l -1 and D. rerio <229 mg BTO1 l -1, Lymnaea sp. < 635 mg BTO1 l -1) had no observable effects on the mortality of the animals (Fig. 1, 2, 3, 4 & 5).Growth of L. minor in control cultures was rapid, although not exponential, during the test period. The number of fronds had quadrupled by the seventh day of the test and remained green and healthy throughout the test. Growth was complete-ly inhibited and the fronds turned white in color at the highest concentrations tested (381-4900 mg BTO1 l -1 growth medium). Interestingly, the low-est test concentrations (30-137 mg BTO1 l -1) had a positive effect on the number of fronds and the length of roots (Fig. 6). The IC 50 (Day 14) for root length in L. minor was 231 mg BTO1 l -1, and 229 mg BTO1 l -1 for the number of fronds.In the absence of BTO1, the growth of the alga S. gracilis was exponential, increasing from ~900 cells ml -1 to 22 000±2000 cells ml -1 after 72 h. Only the highest exposure concentration (381 mg BTO1 l -1) produced a slight inhibition of the cell growth, thereby preventing the calculation of the IC 50 value. An opposite pattern was observed in the luminescence inhibition test where the acute IC 50 value for V . fisheri could not be calculated because BTO1 caused 85% luminescence inhibition even at the lowest tested concentration (30 mg l -1). It is possible that a colouring effect of BTO1 in the samples may have had an affect on light inhibition.The sensitivity of different species to BTO1 was variable among the taxa, with the rank order being: V . fisheri (IC 50 < 30 mg l -1) > D . magna (EC - 155 mg l -1) < L . variegates (LC 50 176 mg l -1) < L . minor (IC 50 229-231 mg l -1) < S. gracilis < D. rerio (LC 50 320 mg l -1) < A. aquaticus (LC 50 397 mg l -1) < Lymnaea sp . (LC50 866 mg l -1) (Table 1.).ranged from 49 to 381 mg l -1 test medium, each with 3 replicates. The initial cell concentration of S . gracilis in test flasks was ~900 cells ml -1. The test was performed in 250 ml glass Erlenmeyer flasks, containing 100 ml test solution and covered with a plug of cotton wool to allow gas exchange during the test. The algae were incubated in the suspension under constant shaking (100 rpm) and continuous illumination (10 000 lx) at 22±1 o C. The cell concentrations and pH of the solutions were determined at 24, 48 and 72 hours after the start of the test. Cell concentrations were determined by microscopic counting. pH of the solvents was measured in the beginning and at the end of the experiment.Data analysisEC 50 values and 95% confidence limits of BTO1 for L . variegatus , D . rerio , A . aguaticus , and Lymnaea sp . were calculated by regression using the Probit analysis (SPSS 1999). EC 50 values for V . fisheri could not be calculated due to the 85% luminescence inhibition taking place already under the most diluted BTO1 concentration (30 mg l -1). The calculation of IC 50 values for L. minor , D . magna , and S. gracilis was carried out according to the following logistic model by Haanstra et al. (1985): Y = Ymax / (1 + (conc/EC 50)^b)). Where Y = response (e.g. number of roots/fronds/juve-niles); Ymax = maximum response in the untreated controls;EC 50 = concentration at which response is 50% of that in the controls; and b = slope of the dose-response curve.ResultsAs the mortality level in all control tests were below 10%, the toxicity tests for A. aquaticus , L. varie-gatus , D. magna , Lymnaea sp. and D. rerio were considered valid. Mortality rate was 100% at theVol. 19(2010): 24–33.DiscussionOur study demonstrates that aquatic organisms appear to be variably responsive to BTO1. The tests revealed the mollusc (Lymnaea sp .) and the crustacean (A. aquaticus ) to be more tolerant to BTO1 than the other tested organisms, whereas D. magna (small-sized crustacean) and L. variegates (Oligochaeta worm) were, in general, most sensi-tive to BTO1. Species-specific structural, as well as functional characteristics are often associated with the bioavailability of a chemical compound, which often explains the differences in sensitivity between species. For example, D. magna , a widely used species in toxicological testing (van der Ohe and Liess 2004), is exposed to various toxicants both through feeding and cuticula (Olsen et al.Fig. 5. Effect of BTO1 on survival rate (%) of Lymnaeae sp. after 24 and 48 h exposure.Fig. 6. Effects of BTO1 on the number of fronds (=lines with squares) and the length of roots (=lines with cir-cles) in L. minor after 7 d exposure.Fig. 1. Survival rate (%) of D. magna after 24 h expo-sure on different BTO1 concentrations.Concentration (mg l -1)0102030405060708090100Fig. 2. Survival rate (%) of L. variegatus as a function of concentrations of BTO1 after 24 and 48..4900294017641058635381229137824930Concentration (mg l -1)Fig. 3. Survival rate (%) of D. rerio as a function of con-centrations of BTO1 after 24 and 96 h exposure.102030405060708090Concentration (mg l -1)Fig. 4. Survival (%) of A. aquaticus as a function of BOD1 after 24 (=lines with circles) and 48 (=lines withsquares) h exposure.102030405060708090Concentration (mg l -1)102030405060708090100Concentration (mg l -1)04900294017641058635381229137824930Concentration (mg l -1)Growth inhibition (%)Hagner, M. et al. Acute toxicity of birch tar oil2005). The higher tolerance of A. aquaticus, also a crustacean, can be explained by its larger body size (4-12 mm, van Hattum 1995), around twice as long as D. magna (Koivisto 1995). The sensitivity of L. variegatus can be a consequence of its thin surface epithelium, while the shell of Lymnaea sp. evidently provides these fauna with efficient protection against harmful substances, despite their disability to close the frontal aperture of the shell due to the lacking operculum (Olsen et al. 2005). According to von der Ohe and Liess (2004), most species of Oligochaeta worms and Isopoda crustaceans (A. aquaticus) are less sensitive to organic compounds and metals than D. magna, a Branchiopoda crustacean, while molluscs are commonly the least sensitive group to organic compounds. Our results corroborate these findings and further show that while the impact of moderate to high concentrations of BTO1 on aquatic organisms was low, a small additional burden may have disproportionately large impacts.Surprisingly, the duckweed Lemna minor ap-peared to react positively to BTO1 by increasing the number of fronds and the length of roots at low BTO1 concentrations. Other studies have shown that L. minor has reacted positively to certain or-ganic toxicants, which was speculated to be derived from hormone-like effects at low chemical expo-sure levels (Sherry et al. 1997, Song and Huang 2005).In our case, the plants may have benefited from growth promoting nutrients in the birch tar oils, but this needs to be investigated in more de-tail. Interestingly, a similar response pattern was missing in algae (S. gracilis) in the present study.According to the Categories of Ecotoxicity for pesticides (Kamrin 2000), the toxicity of a pesticide-active ingredient is qualitatively classi-fied to be very highly toxic to aquatic organisms if its LC50value is less than 100 µg l-1. The sub-stance is considered nontoxic if the LC50value is over 100 000 µg l-1. We found that the majority of acute toxicity values of BTO1 were above this limit with the exception of V. fisheri, whereby the exact IC50value could not be obtained. In comparison to other pesticides such as Malathion, a widely used organophosphorus insecticide, the LC50for D. magna is 1–2.35 µg l-1 (LC5024 and 48 h; Keller and Ruessler 1997, Cano et al. 1999), and for D. rerio is 19.8 mg l-1 (LC5096 h; Lange et al. 1995). As with BTO, Glyphosate (herbicide) can stimu-late the growth of L. minor in (EC50= 2 µg l-1; 14 d) (Hartman and Martin 1984) but is toxic at low concentrations to D. magna (EC50= 95.96 µg l-1;48 h) (Alberdi et al. 1996). In the current study, the EC50values of BTO1 for the organisms testedTable 1. The acute toxicity (EC50-values with 95% confidence limits) of BTO1 for the tested aquatic organisms.Test species Duration of test E/L/IC50(mg l-1)95% L.C.I(mg l-1)a95% U.C.I(mg l-1)bDaphnia magna EC50Asellus aquaticus LC5048 h 397314492Lumbriculus variegatus LC5048 h 176134 232Vibrio fisheri IC5030 min <30--Lemna minor (root) IC507 d 231157 305Lemna minor (frond) IC507 d2*******Lymnaea sp. LC5048 h 866659 1132Scenedesmus gracilis IC5072 h---Danio rerio LC5096 h320 297 337a 95% lower confidence interval (LCI)b 95% upper confidence interval (UCI)EC = effect, LC = lethal and IC = inhibition concentrationVol. 19(2010): 24–33.ranged from between <30 and 866 mg BTO1 l-1 and is therefore much less toxic to most aquatic organ-isms than, for example, Malathion or Glyphosate.The very steep dose response curve and high effective concentration indicates that BTO is only baseline toxic (Könemann 1981). Therefore it is unlikely that the effective compound/compounds represent just a fraction of the compounds in BTO1, or that the proportion of such biologically effective substances can vary between production runs.Furthermore, it is possible that, while a par-ticular effective compound(s) in BTO can elicit a response by a target organism, it can be practically non-toxic for many aquatic organisms when ex-isting in a mixture. Interestingly, aquatic animals appear to be more sensitive to birch tar oils than soil animals (Hagner et al. 2010), which effect is likely to stem from the difference in the amounts of bioavailable fractions of BTO between aquatic and terrestrial environments. Because BTO is a suspension with a complex mixture composition it was not possible for us to determine real dis-solved/bioavailable BTO1 concentrations and its changes in test medium over time. More investiga-tions are required to determine the risks of BTO1 to the aquatic environment. The tests of S. gracilis and V. fisheri should be repeated with higher/lower concentrations to get a comprehensive assessment of BTO1.All risk related topics of BTO belongs to the next step of the project and in future envi-ronmental Quality Standards will be derived using data available from this and other studies (Hagner et al. 2010, Lindqvist et al. 2010).In contrast to some other toxic mixtures, such as creosote or municipal wastewaters, BTO has to be treated as a single chemical compound given the lack of comprehensive information on the active compounds present in the distillate. Furthermore, to better understand the mechanism behind the sen-sitivity of aquatic or terrestrial organisms to birch tar oils, a thorough investigation of the chemical composition of BTO is required. Knowledge of the chemical composition of BTO will also be essen-tial for EU registration and future permitted use as a biological plant protection product. In conclu-sion, our toxicity tests showed that a wide range of aquatic organisms are, to some extent, sensitive to BTO, but further suggest that BTO does not pose a severe hazard to aquatic biota. We deduce that, unless BTO is not applied in the immediate vicinity of water bodies, no special precaution is required.ReferencesAccinelli, C., Vicari, A., Pisa, P.R. & Catizone, P. 2002. Losses of atrazine, metolachlor, prosulfuron and triasul-furon in subsurface drain water. I. Field results. Agron-omie 22: 399−411.Alberdi, J.L. Sáenz, M.E., Di Marzio, W.D. & Tortorelli, M.C. 1996. Comparative acute toxicity of two herbicides, para-quat and glyphosate to Daphnia magna and D. spinu-lata. Bulletin of Environmental Contamination and Toxi-cology 57: 229−235.American Chemical Society 2007: Homepage. Updated(29. April 200). Cited 13 June 2007. Available on the internet: /expertise/cascontent/registry/reg-syst.html?WT.mc_id=casrn708&WT.srch=1.Cano, E., Jimenez, A., Cabral, J.A. & Ocete, M.E. 1999. Acute toxicity of Malathion and new surfactant “Genapol OXD 080” on species of rice basins. Bulletin of Envi-ronmental Contamination and Toxicology 63: 133-138. Connell, D., Lam, D., Richardson, B. & Wu, R. 1999. In-troduction to ecotoxicology. Blackwell Publishing. Great Britain. 170 p.EC 1996. Technical guidance documents in support of the Commission Directive 93/67/EEC on risk assessment for new notified substances and the Commission Regulation (EC) 1488/94 on risk assessment for existing substanc-es. European Comission, Brussels, Belgium. Haanstra, L., Doelman, P., & Oude Voshaar, J.H. 1985. The use of sigmoidal dose response curves in soil ecotoxico-logical research. Plant and Soil 84: 293−297. Hagner, M. 2005. Koivutisle torjunta-aineena: vaikutuk-set lehtokotiloon (Arianta arbustorum) ja maaperään (in Finnish). 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第42卷第2期2021年3月水生态学杂志Journal of HydroecologyVol.42,No.2Mar.20211)01：10.15928/j.16743075.201905120118微囊藻毒素急性暴露对斑马鱼卵巢的损伤效应张同舟，张学振，刘婉婧，展春华(华中农业大学水产学院教育部长江经济带大宗水生生物产业绿色发展工程研究中心，湖北武汉430070)摘要：为了探究蓝藻(Cyanobacteria)大量爆发产生的微囊藻毒素(microcystins,MCs)对鱼类的生殖毒性，以斑马鱼(Danio rerio)为实验对象，采取腹腔注射毒性最强的microcystin-LR(MC-LR)方式，研究MC-LR对斑马鱼卵巢的损伤效应及其作用机制.对性成熟雌性斑马鱼腹腔注射50”g/kg和200”g/kg MC-LR,在注射3、9、24、48h后取卵巢分析其生理活性指标的变化.结果显示，染毒24h后,200”g/kg剂量组斑马鱼性腺指数(gonad somatic index,GSI)14.14与对照组性腺指数16.98相比显著降低(P<0.05),其他组别无显著变化；卵巢发生卵母细胞空泡化、卵母细胞膜与滤泡细胞层连接组织缺失等病理现象;MC-LR显著抑制了斑马鱼卵巢蛋白磷酸酶(protein phosphatase2A,PP2A)活性，并激活促成熟因子(maturation promoting factor,MPF)活性;MC-LR处理后，斑马鱼卵巢内丝裂原活化蛋白激酶(mitogcn-activatcd protein kinase,M APK)家族中的p38M A PK、E：RK1/2的转录水平显著上调JNK未发生显著变化.研究表明,MC-LR抑制斑马鱼卵巢PP2A活性，并激活MPF活性与MAPK信号通路中ERK1/2与p38MAPK的转录水平，进而干扰其卵母细胞的发育进程并产生生殖毒性.关键词：微囊藻毒素；生殖毒性；腹腔注射；斑马鱼；蛋白激酶中图分类号：X174文献标志码:A文章编号：1674-3075(2021)02-0116-08蓝藻(Cyanobacteria)大量爆发会产生天然毒素，给水生生物造成严重危害，其中最常见的是微囊藻毒素(microcystins,MCs)(谢平,2009)。

第37卷㊀第12期2019年12月环㊀境㊀工㊀程Environmental EngineeringVol.37㊀No.12Dec.㊀2019不同水体硬度条件下Cu 2+对不同生长阶段斑马鱼的毒性∗廖㊀伟1,2,3㊀刘大庆1㊀冯承莲1,2㊀金小伟4㊀刘㊀娜1㊀白英臣1,2㊀吴丰昌1(1.中国环境科学研究院环境基准与风险评估国家重点实验室,北京100012;2.南昌大学资源环境与化工学院鄱阳湖环境与资源利用教育部重点实验室,南昌330031;3.江西省灌溉试验中心站,南昌330201;4.中国环境监测总站,北京100012)摘要:以斑马鱼为研究对象,开展了不同硬度水质条件下Cu 2+对不同生长阶段斑马鱼的毒性研究㊂结果表明:同等水质条件下,Cu 2+对不同阶段斑马鱼的毒性顺序为成鱼>幼鱼>仔鱼;同一阶段的受试斑马鱼,水质硬度与Cu 2+对斑马鱼毒性呈负相关(R 2>0.90),硬度为50,125,250mg /L 时,96h-LC 50几何平均值分别为0.20,0.24,0.35mg /L ㊂通过Cu 2+对斑马鱼仔鱼㊁幼鱼㊁成鱼和全生命周期平均急性毒性与水体硬度对数拟合计算得到的硬度斜率分别为0.301㊁0.471㊁0.279和0.359㊂研究结果可为重金属的生物有效性和生态风险评估提供理论依据㊂关键词:不同生长阶段;斑马鱼;硬度斜率;急性毒性;水质基准DOI:10.13205/j.hjgc.201912013TOXICITY OF COPPER (Cu 2+)TO ZEBRAFISH AT DIFFERENT LIFE STAGESUNDER DIFFERENT WATER HARDNESSLIAO Wei 1,2,3,LIU Da-qing 1,FENG Cheng-lian 1,2,JIN Xiao-wei 4,LIU Na 1,BAI Ying-chen 1,2,WU Feng-chang 1(1.State Key Laboratory of Environmental Criteria and Risk Assessment,Chinese Research Academy of Environmental Sciences,Beijing 100012,China;2.Key Laboratory of Poyang Lake Environment and Resource Utilization,Ministry of Education,School ofEnvironmental and Chemical Engineering,Nanchang University,Nanchang 330031,China;3.Jiangxi Irrigation ExperimentCentral Station,Nanchang 330201,China;4.China National Environmental Monitoring Center,Beijing 100012,China)Abstract :Acute toxicity of copper (Cu 2+)at different life stages of zebrafish under different hardness (as CaCO 3)wasstudied in this work.The results showed that the acute toxicity of copper to zebrafish at different life stages was different,andthe 96h-LC 50value of copper were in the sequence of larvae-stage-zebrafish>juvenile-stage-zebrafish >adult-stage-zebrafish;as the hardness increased,the acute toxicity of copper to Zebrafish was decreased.There was a significant linear correlationbetween the acute toxicity of copper to zebrafish and its hardness (R 2>0.90).The geometric mean values of 96h-LC 50for copper at different life stages of zebrafish were 0.20,0.24,0.35mg /L in a series of hardness of 50,125,250mg /Lrespectively;the hardness slope of toxicity of copper to the larvae-stage,juvenile-stage,adult-stage and the whole-life-stage of zebrafish was 0.301,0.471,0.279and 0.359,namely.In conclusion,the results could provide theoretical basis for the bioavailability and ecological risk assessment on heavy metals.Keywords :different life stage;zebrafish;hardness slope;acute toxicity;water quality criteria㊀㊀㊀㊀㊀㊀㊀㊀∗水体污染控制与治理科技重大专项(2017ZX07301005-001)㊂收稿日期:2019-03-100㊀引㊀言铜是参与生物机体细胞代谢的重要元素[1],过量的铜对水生生物产生毒性[2-4]㊂我国已颁布的关于铜的GB 3838 2002‘地表水环境质量标准“中,Ⅰ类水质铜的标准为0.01mg /L,Ⅱ~Ⅵ类水质铜的标准为1mg /L [5],GB 11607 1989‘渔业水质标准“为0.01mg /L [6],这些标准对我国水生态环境保护意义重大㊂但是随着我国经济社会的进一步发展,特别是为控制藻类水华㊁鱼类养殖而使用的CuSO 4及含铜工业废水等大量排放[7],使得铜成为水生态环境中环㊀境㊀工㊀程第37卷对水生生物潜在风险最大的重金属元素之一[3,8-10]㊂本研究对我国铜的水质标准修订㊁保护水生态系统的稳定性具有重要意义㊂硬度是影响铜对水生生物毒性的重要水质参数之一[11-14]㊂我国幅员辽阔,各地区水体硬度相差巨大,全国总硬度<150mg/L的软水和极软水面积占总面积的42%,150~300mg/L的适度硬水占34%[15]㊂另外,同地区时间不同,硬度也不同,如北京密云库区1991 2011年硬度为30.3~446mg/L[16]㊂考虑我国本土区域性水环境特征,研究硬度对生物急性毒性的影响,有利于推动我国水环境质量标准修订㊂斑马鱼原产于印度㊁孟加拉等热带地区溪流,在我国各实验室广泛用于毒性测试[17-19],是研究水生生物水质基准良好的模式生物之一[20]㊂已有研究将斑马鱼分为幼鱼㊁成鱼阶段进行毒性实验[21],本研究在该基础上将斑马鱼不同阶段进一步细分,详细了解其变化规律;旨在探索不同生长阶段斑马鱼对污染物铜敏感性差异的变化规律及硬度的影响,以期为铜的生态风险评估及水质基准提供理论依据㊂1㊀实验部分1.1㊀实验材料五水硫酸铜(CuSO4㊃5H2O)购于上海国药试剂厂,优级纯;铜标准溶液购于国家标准物质网;二水氯化钙(CaCl2㊃2H2O)㊁七水硫酸镁(MgSO4㊃7H2O)㊁碳酸氢钠(NaHCO3)㊁氯化钾(KCl)等试剂购于国药集团,分析纯㊂AB系斑马鱼(Danio rerio)购自中科院水生生物研究所,并在实验室自行繁殖㊂1.2㊀仪器设备电感耦合等离子发射质谱(ICP-MS,Agilent7500a,美国);YSI多参数水质分析仪(YSI;YSI Professional Plus,美国);人工智能气候箱(MGC-350HP-2,上海一恒科学仪器有限公司);流水养殖系统(CASA,无锡中科水质环境技术有限公司)㊂1.3㊀实验设计不同生长阶段包括斑马鱼仔鱼(孵化后1,10, 17d)㊁幼鱼(孵化后24,31,60d)和成鱼(孵化后90, 120d)等阶段㊂硬度范围包括标准毒性试验推荐的标准水(250mg/L),代表性选取的软水(50mg/L)和中硬水(125mg/L)㊂1.4㊀实验方法斑马鱼培养参照斑马鱼培养手册和GB21807 ㊀㊀表1㊀不同水体硬度条件下浓度设置Table1㊀Configuration of Cu2+concentration underdifferent water hardness硬度/(mg㊃L-1)鱼龄/dρ(Cu2+)/(mg㊃L-1) 50(软水)1,10,17,24,31,60,90,1200,0.05,0.09,0.16,0.29,0.52,0.70,0.94125(中硬水)1,10,17,24,31,60,90,1200,0.05,0.09,0.16,0.29,0.52,0.70,0.94250(标准水)1,10,17,24,31,60,90,1200,0.09,0.16,0.29,0.52,0.70,0.94,1.26 2008‘化学品鱼类胚胎和卵黄囊仔鱼阶段的短期毒性试验“[22,23],温度为(25ʃ1)ħ,光强为1000lux,光照条件为光照ʒ黑暗=16hʒ8h㊂急性毒性实验参照GB/T13267 91‘水质物质对淡水鱼(斑马鱼)急性毒性测定方法“[24]和OECD203Guideline for Testing of Chemicals Fish Acute Toxicity Test[25],暴露实验采用超纯水配制的重组水,不同硬度的重组水通过改变水中Ca2+和Mg2+的浓度来实现㊂重组水配好后曝气24h,使用时需ρ(DO)>6mg/L,pH值稳定在7.8ʃ0.2㊂每个暴露液浓度设置3个平行,每个平行放入10尾斑马鱼,每24h更换1次暴露液,并对暴露液中铜浓度取水样实测㊂观察记录24,48,72,96h 斑马鱼死亡的数目(用玻璃棒轻触鱼的尾部,没有反应即认为已死亡),及时清出死鱼㊂试验过程中,空白组死亡率低于10%㊂1.5㊀统计分析实验所得数据采用SPSS22.0软件处理,计算24,48,72,96h的半致死浓度及其95%置信区间,利用一元线性回归分析半致死浓度与硬度的相关性㊂2㊀结果与分析2.1㊀不同硬度条件下Cu2+对斑马鱼仔鱼毒性情况实验选取了孵化后1,10,17d斑马鱼小鱼作为仔鱼阶段实验用鱼,分别研究了硬度为50,125, 250mg/L的水质条件下,Cu2+对斑马鱼孵化后1,10, 17d仔鱼阶段急性毒性,采用更能表示毒性数据集中趋势的几何平均方法[26]计算了不同硬度条件下Cu2+对斑马鱼仔鱼(24,48,72,96h)-LC50平均值,如图1所示㊂可知:Cu2+对仔鱼阶段斑马鱼的半数致死浓度随着时间的延长而降低,说明溶液中Cu2+对斑马鱼仔鱼毒性随着时间增加而增大;不同硬度水质条件下,Cu2+对斑马鱼仔鱼毒性随着硬度增加而降低㊂2.2㊀不同硬度条件下Cu2+对斑马鱼幼鱼毒性情况实验选取了孵化后24,31,60d的斑马鱼作为幼鱼阶段试验用鱼,分别研究了硬度为50,125,27第12期廖㊀伟,等:不同水体硬度条件下Cu 2+对不同生长阶段斑马鱼的毒性硬度: Ѳ 50mg /L;--ʻ--125mg /L; ә 250mg /L㊂图1㊀不同硬度条件下Cu 2+对斑马鱼仔鱼(24,48,72,96h)-LC 50变化Fig.1㊀Acute toxicity of copper on the larvae stage of zebrafish:the average and geometric mean values of (24,48,72,96h)-LC 50250mg /L 水质条件下,Cu 2+对斑马鱼孵化后24,31,60d 幼鱼阶段急性毒性,采用几何平均方法计算不同硬度条件下Cu 2+对斑马鱼仔鱼(24,48,72,96h )-LC 50平均值,如图2所示㊂硬度: Ѳ 50mg /L;--ʻ--125mg /L; ә 250mg /L㊂图2㊀不同硬度条件下Cu 2+对斑马鱼幼鱼(24,48,72,96h)-LC 50变化Fig.2㊀Acute toxicity of copper on the juvenile stage of zebrafish:the average and geometric mean values of (24,48,72,96h)-LC 50由图2可知:Cu 2+对幼鱼阶段斑马鱼的半数致死浓度随着时间的延长而降低,说明溶液中Cu 2+对斑马鱼幼鱼毒性随着时间增加而增大;不同硬度水质条件下,Cu 2+对斑马鱼幼鱼毒性随着硬度增加而降低㊂2.3㊀不同硬度条件下Cu 2+对斑马鱼成鱼毒性情况实验选取了孵化后90,120d 斑马鱼作为成鱼阶段实验用鱼,分别研究了硬度为50,125,250mg /L 水质条件下,Cu 2+对斑马鱼孵化后90,120d 成鱼阶段急性毒性,采用几何平均方法计算不同硬度条件下Cu 2+对斑马鱼成鱼(24,48,72,96h)-LC 50平均值,如图3所示㊂由图3可知:Cu 2+对成鱼阶段斑马鱼的半数致死硬度: Ѳ 50mg /L;--ʻ--125mg /L; ә 250mg /L㊂图3㊀不同硬度条件下Cu 2+对斑马鱼成鱼(24,48,72,96h)-LC 50变化Fig.3㊀Acute toxicity of copper on the adult stage of zebrafish:the average and geometric mean values of (24,48,72,96h)-LC 50浓度随着时间的延长而降低,说明溶液中Cu 2+对斑马鱼成鱼毒性随着时间增加而增大;不同硬度水质条件下Cu 2+对斑马鱼成鱼毒性随着硬度增加而降低㊂2.4㊀不同水体硬度与Cu 2+对斑马鱼急性毒性的关系硬度是影响水体重金属对水生生物毒性的重要因素之一,多数重金属对水生生物毒性与硬度有函数关系[27]㊂根据实验结果,参考USEPA 发布的铜㊁镉对水生生物急性毒性硬度斜率的计算方法[27,28],将硬度及各阶段斑马鱼96h-LC 50值对数转换后,利用一元线性回归分析方法,拟合了Cu 2+对斑马鱼仔鱼㊁幼鱼㊁成鱼及斑马鱼全生命周期急性毒性与水体硬度的关系,结果如表2所示㊂拟合计算得出:Cu 2+对斑马鱼仔鱼㊁幼鱼㊁成鱼和斑马鱼全生命周期硬度斜率分别为0.301㊁0.471㊁0.279和0.359;相关系数R 2分别为0.891㊁0.949㊁0.963和0.936,随着斑马鱼生长,急性毒性与水质硬度相关性逐渐增强㊂铜的主要致毒机理为Cu 2+进入细胞质膜,增加质膜的渗透性,造成K +以及其他离子流失[29]及高浓度的Cu 2+造成腮损伤[30],从而导致死亡㊂目前大部分学者认为构成硬度的Ca 2+㊁Mg 2+和Cu 2+在细胞膜上存在竞争吸附关系,并提出生物配体模型[31],所以随着斑马鱼生长发㊀㊀表2㊀不同水体硬度条件下Cu 2+对不同阶段斑马鱼急性毒性效应影响线性拟合Table 2㊀Linear fitting results for acute toxicity of copper ofzebrafish in different hardness重金属受试生物阶段拟合方程R 2铜斑马鱼仔鱼ln(96h-LC 50)=0.3006ln H -2.28840.8909幼鱼ln(96h-LC 50)=0.4713ln H -3.79040.9490成鱼ln(96h-LC 50)=0.2788ln H -3.15950.9631全周期ln(96h-LC 50)=0.3592ln H -3.06940.9358㊀㊀注:H 为水体硬度(以CaCO 3计),mg /L㊂37环㊀境㊀工㊀程第37卷育和调节机制的完善,Cu2对斑马鱼急性毒性与水质硬度相关性逐渐增强㊂3㊀讨㊀论已有研究表明,水体硬度能降低重金属对水生生物毒性[32,33]㊂USEPA将水体硬度作为研究重金属水质基准必须考虑的因素,再分析不同硬度条件下生物毒性数据,利用污染物对水生生物毒性硬度斜率公式,将水质条件转换为同一硬度水平进行比较,科学地比较污染物毒性效应大小并更严谨地推导水质基准阈值㊂本研究通过求出Cu2+对不同阶段斑马鱼毒性几何平均值,将毒性几何平均值与溶液硬度拟合得到相关线性方程,表明硬度与各阶段毒性几何平均值显著相关(R2>0.90),在推导其他硬度下Cu2+对各阶段斑马鱼毒性大小时得出的数值可靠㊂研究得出: Cu2+对斑马鱼全生命周期硬度斜率为0.359,研究数值低于周永欣等[34]研究铜对草鱼㊁鲢鱼㊁大鳞副泥鳅等毒性得出的全生命周期硬度斜率0.751~1.021,熊小琴等[14]研究铜对稀有鮈鲫的全生命周期硬度斜率为0.727,以及USEPA[27]制定的铜基准硬度斜率为0.942,与王伟莉等[35]研究得出的0.51更接近㊂实验结果表明,生物种类以及不同生命阶段均影响污染物基准硬度斜率,研究水体硬度与重金属生物毒性相关关系,有利于掌握重金属对生物体生物有效性的计算,丰富不同硬度重金属对生物体生物有效性的数据库㊂4㊀结㊀论1)同等水质条件下,Cu2+对斑马鱼不同生长阶段毒性顺序为:成鱼>幼鱼>仔鱼㊂2)通过改变水体硬度,影响Cu2+对斑马鱼各生长阶段急性毒性,Cu2+对斑马鱼仔鱼㊁幼鱼㊁成鱼和全生命周期96h-LC50与水质硬度线性呈负相关,相关系数分别为0.891㊁0.949㊁0.963和0.936㊂硬度为50,125,250mg/L条件下,96h-LC50几何平均值分别为0.20,0.24,0.35mg/L㊂3)通过实验计算得出,Cu2+对斑马鱼仔鱼㊁幼鱼㊁成鱼和全生命周期平均急性毒性与水体硬度对数拟合计算得到硬度斜率分别为0.301㊁0.471㊁0.279和0.359,丰富了硬度对重金属生物体生物有效性的数据库㊂参考文献[1]㊀Festa R A,Thiele D J.Copper:an essential metal in biology[J].Current Biology,2011,21(21):877-883.[2]㊀吴丰昌,冯承莲,曹宇静,等.我国铜的淡水生物水质基准研究[J].生态毒理学报,2011,6(6):617-628.[3]㊀Donnachie R L,Johnson A C,Moeckel C,et ing risk-ranking of metals to identify which poses the greatest threat tofreshwater organisms in the UK[J].Environmental Pollution,2014,194(7):17-23.[4]㊀张旭,付卫强,冯承莲,等.我国淡水中铜的水质基准及生态风险评估研究[J].环境工程,2016,34(5):156-160. 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溢油污染物对紫贻贝的急性毒性效应高祥;杨阳;林雨霏【摘要】选择紫贻贝(Mytilus edulis)作为受试生物,测定燃料油、原油分散液(WAF)以及添加消油剂后的乳化液(DWAF)对紫贻贝的急性毒性效应参数.结果表明,随着溢油污染物浓度的升高,紫贻贝的死亡率逐渐增大;当浓度为5 mg/L时,各实验组中紫贻贝的死亡率达60%以上.加入消油剂后,燃料油对紫贻贝的急性毒性效应降低,而原油升高.%The Mytilus edulis was taken as subjects to test the toxicity of crude and fuel oil water accommodated fractions (WAF)and their dispersed water accommodated fractions(DWAF).According to the result,with the increase of the concentra-tion of spilled oil pollutants,its death rate is increasing;the death rate is more than 60%with the concentration of 5 mg/L.After adding the oil dispersant,the acute toxicity effect of the fuel oil decreased,and the crude oil increased.【期刊名称】《船海工程》【年(卷),期】2018(047)002【总页数】4页(P109-111,115)【关键词】紫贻贝;WAF与DWAF;急性毒性;96h-LC50【作者】高祥;杨阳;林雨霏【作者单位】中国海洋大学化学化工学院,山东青岛266100;中国海洋大学化学化工学院,山东青岛266100;国家海洋局海洋减灾中心,北京100194【正文语种】中文【中图分类】U698.7紫贻贝(Mytilus edulis)俗称海虹，为软体动物门，双壳纲，贻贝目，贻贝科，贻贝属，广泛分布于世界各海区[1]。

戊唑醇对斑马鱼的急性毒性及生物富集效应吴迟;刘新刚;何明远;董丰收;徐军;吴小虎;郑永权【期刊名称】《生态毒理学报》【年(卷),期】2017(012)004【摘要】为评价杀菌剂戊唑醇对水生生态系统的影响,以斑马鱼(Brachydanio rerio)为试验生物,采用半静态法分别研究了戊唑醇原药和其6种不同剂型对斑马鱼的急性毒性以及戊唑醇原药对斑马鱼的生物富集效应.结果表明:97.4%戊唑醇原药、25%戊唑醇水乳剂、430 g·L-1戊唑醇悬浮剂、25%戊唑醇可湿性粉剂、80 g·L-1戊唑醇悬浮种衣剂、250 g·L-1戊唑醇乳油、80%戊唑醇水分散粒剂对斑马鱼96h-LC50分别为8.73、3.35、6.44、9.68、4.60、0.892和6.81 mg·L-1;选择97.4%戊唑醇原药进行斑马鱼生物富集试验,在9.00×10-2和0.900 mg·L-12个处理浓度下连续暴露8 d,相应的富集系数(BCF8 d)分别为27.7和25.4.根据?化学农药环境安全评价试验准则?毒性划分标准,除250 g·L-1戊唑醇乳油对斑马鱼急性毒性属于高毒,其他剂型均为中毒,且戊唑醇属于中等富集性农药.该研究为戊唑醇田间安全使用提供理论依据.【总页数】8页(P302-309)【作者】吴迟;刘新刚;何明远;董丰收;徐军;吴小虎;郑永权【作者单位】沈阳农业大学植物保护学院,沈阳110161;中国农业科学院植物保护研究所,北京100193;北京依科世福科技有限公司,北京100096;中国农业科学院植物保护研究所,北京100193;北京依科世福科技有限公司,北京100096;中国农业科学院植物保护研究所,北京100193;中国农业科学院植物保护研究所,北京100193;中国农业科学院植物保护研究所,北京100193;中国农业科学院植物保护研究所,北京100193【正文语种】中文【中图分类】X171.5【相关文献】1.3种戊唑醇剂型对斑马鱼的急性毒性评价 [J], 王雅东;林庆胜;李祥英;夏晓明2.3种戊唑醇剂型对斑马鱼的急性毒性评价 [J], 王雅东;林庆胜;李祥英;夏晓明;3.戊唑醇对斑马鱼的急性毒性与安全评价 [J], 李俊;陈迎丽;段亚玲;杨鸿波4.叶菌唑对斑马鱼的急性毒性及生物富集效应 [J], 李如美; 戴争; 王维; 张霞; 郇恒尚; 李丹丹; 高宗军5.氰氟草酯对斑马鱼的急性毒性和生物富集效应 [J], 涂杰;沈艳;刘晓敏;武健;董旭;朱伟华;张勇因版权原因，仅展示原文概要，查看原文内容请购买。