Antioxidative enzymes in seedlings of Nelumbo nucifera germinated under water.

Pesticide Biochemistry and Physiology69,112–117(2001)doi:10.1006/pest.2000.2518,available online at onEffects of the Fungicide Folpet on the Activities of Antioxidative Enzymesin Duckweed(Lemna minor)H.Teisseire1and G.VernetLaboratoire d’Eco-Toxicologie,Unite´de Recherche Vigne et Vins de Champagne E.A.2069,Faculte´des Sciences, Universite´de Reims Champagne-Ardenne,BP1039,F-51687REIMS-02,FranceReceived March2,2000,accepted October2,2000In a previous paper we reported the phytotoxicity of the fungicide folpet[N-(trichloromethylthio)phtalimide]in the aquatic floating macrophyte Lemna minor.The objectives of this study were to investigate themodifications of several antioxidative enzymatic activities in fronds of L.minor during a96-h exposure toa sublethal folpet concentration(33␮M).The specific activities of enzymes of the Halliwell–Asada pathway,namely,ascorbate peroxidase and glutathione reductase,increased after24h of exposure,reaching155and273%of the control level at96h,respectively.A fast induction of glutathione S-transferase activity wasobserved after6h of folpet exposure.From data presented it cannot be concluded whether glutathione S-transferase was involved in folpet metabolization or in peroxide scavenging.The fungicide was also foundto stimulate the activities of two H2O2-scavenging enzymes,catalase and pyrogallol peroxidase.Stimulationof catalase was very rapid(as soon as12h after exposure)and very strong since the activity was252%ofthe control after48h of exposure.Induction of pyrogallol peroxidase was less important;nevertheless,itreached66%at96h.No effect of the fungicide on guaiacol peroxidase was observed.As suggested by thesimultaneous and important induction(55to173%)of antioxidative enzymatic defenses of L.minor,thegeneration of reactive oxygen species by the fungicide was proposed and the involvement of a possibleoxidative stress in the phytotoxicity of folpet was discussed.᭧2001Academic PressKey Words:antioxidant enzymes;folpet;fungicide;Lemna minor;N-(trichloromethylthio)phtalimide;oxidative stress.INTRODUCTION cells,folpet(or the intermediary thiophosgene)preferentially reacts with SH groups and is Fungicidal activity of folpet[N-(trichloro-thereby able to inhibit various enzymes,suchmethylthio)phtalimide]was first reported in as the monooxygenases of rat liver(4).These 1952and it was introduced by the Standard Oil inhibitions were evoked to explain the fungicidal Company(1).It is still one of the most widely effects of folpet(3)and its hepatotoxicity(4). used fungicides in vineyards to control mildew In a previous study,the toxicity of the fungicide and other fungal diseases in grapes.Physiologi-for a nontarget species,the floating macrophytecally,folpet toxicity is not yet fully elucidatedLemna minor,was reported(5).That phytotoxic-and it has been proposed that the fungicide(likeity of folpet was still observed with some pear other N-trichloromethylthio fungicides,such asand apple varieties(1).Except for the two above captan or captafol)is a multisite inhibitor(2).cited studies,no more literature dealing with the Siegel and Sisler(3)suggested that the N-trichlo-mechanisms of folpet phytotoxicity is available. romethylthio moiety(SCCl3)of this moleculeIn order to determine whether enzymatic inhibi-was primarily responsible for its fungicidaltions are involved in that phytotoxicity,the in effects through binding to proteins and cellularvivo effects of the fungicide on the activities of components which led to their inactivation.Inseveral enzymes of L.minor are investigated inthe present study.Because of their importance 1To whom correspondence and reprint requests shouldin stress responses and during oxidative proc-be addressed.Fax:ϩ33.326.91.33.42.E-mail:guy.vernet@univ-reims.fr.esses,we mainly focused on the changes in the1120048-3575/01$35.00Copyright᭧2001by Academic PressAll rights of reproduction in any form reserved.EFFECTS OF FOLPET ON DUCKWEED ANTIOXIDATIVE ENZYMES113activities of enzymatic antioxidative defenses of(50ml)in crystallizing dishes before the plants L.minor during a96-h exposure to folpet.Thatwere added.Blank formulation served as a con-included activities of glutathione reductase trol and controls without formulation were alsorun.Because of the fast hydrolysis of folpet,the (GR,2EC1.6.4.2),ascorbate peroxidase(APO,EC1.11.1.7),catalase(CAT,EC1.11.1.6),guai-contamination media were daily renewed.The acol peroxidase(G-POD,EC1.11.1.7),pyrogal-crystallizing dishes were placed in the controlled lol peroxidase(P-POD,EC 1.11.1.7),and environment room as above.Then at different glutathione S-transferase(GST,EC2.5.1.18).times of exposure to folpet(0,6,12,24,36,48,72,and96h)samples were collected for MATERIALS AND METHODS enzyme extraction.Plant Material,Growth Conditions,and ChemicalsTreatment ProceduresFolpet(formulated as Folpan500,99% L.minor L.(duckweed)was collected from purity)and blank formulations were obtained an artificial pond of the University campus.The from Makhteshim Agan France.All other chemi-plants were disinfected by immersing the fronds cals were from Sigma(Saint Quentin,France). in NaOCl(0.01M)for20s and rinsing withdistilled water.The stock cultures were main-Enzyme Extraction and Proteintained in2-liter plastic(PVC)aquariums con-Determinationtaining500ml of inorganic growth medium(5)To obtain the enzyme extract,200–250mg that consisted of KNO3,202mg LϪ1;KH2PO4,of fronds was homogenized in2ml of cold 50.3mg LϪ1;K2HPO4,27.8mg LϪ1;K2SO4,potassium phosphate buffer(0.1M,pH7.0).17.4mg LϪ1;MgSO4,7H2O,49.6mg LϪ1;The homogenate was centrifuged for6min at CaCl2,11.1mg LϪ1;Na2-EDTA,10mg LϪ1;12,000g and the supernatant centrifuged again FeSO4,7H2O,6mg LϪ1;H3BO3,5.72mg LϪ1;for16min at27,000g.The latter supernatant MnCl2,4H2O,2.82mg LϪ1;ZnSO4,0.6mgwas used as the enzyme extract.Protein content LϪ1;(NH4)Mo7O24,4H2O,43␮g LϪ1;CuCl2,was determined according to Bradford(6)using 2H2O,8␮g LϪ1;CoCl2,6H2O,54␮g LϪ1.bovine albumin for calibration.Before the medium was autoclaved its pH wasadjusted to6.5.The aquariums were placed inEnzyme Assaysa controlled environment room at25Ϯ1ЊCunder continuous illumination provided by cool APO activity was monitored as the decrease white fluorescent lamps(Sylvania Grolux F36in absorbance of ascorbate at290nm(7).The W)with a light intensity of2500Ϯ100lux reaction mixture contained50mM potassiumphosphate(pH6.5),1mM Na-ascorbate,20mM (ca.40␮mol PAR mϪ2sϪ1)and plants weresubcultured twice a week.H2O2,and enzyme extract(60␮g protein)in a For experiments,approximately30double-final volume of1ml.To determine the part of fronded colonies of L.minor(200–250mg)were a possible nonenzymatic oxidation of ascorbate taken from the stock cultures and exposed to33in the⌬A290measured,controls were systemati-␮M folpet according to the procedure described cally realized in a reaction mixture withoutH2O2.Correction was also done for the low, in Teisseire et al.(5).Briefly,folpet,formulatedas Folpan500,was dissolved in growth medium nonenzymatic oxidation of ascorbate by H2O2.GR was determined from the increase in 2Abbreviations used:APO,ascorbate peroxidase;AsA,absorbance at412nm according to Smith et al. ascorbic acid;CAT,catalase;CDNB,1-chloro-2,4-dinitro-(8).The reaction mixture contained K-phosphate benzene;DTNB,5,5Ј-dithiobis(2-nitrobenzoic acid);G-(0.2M,pH7.5),EDTA(1mM),GSSG(1mM), POD,guaiacol peroxidase;GR,glutathione reductase;GSH,NADPH(0.1mM),DTNB(0.75mM),and reduced glutathione;GSSG,oxidized glutathione;GST,glu-enzyme extract(40␮g protein)in a final volume tathione S-transferase;P-POD,pyrogallol peroxidase;PSI,photosystem I;ROS,reactive oxygen species.of1ml at25ЊC.114TEISSEIRE AND VERNETFor GST,the reaction mixture consisted of induced a significant 38%increase of GST activ-ity (Fig.1).Then,it linearly rose to 11nmol 0.1M K-phosphate (pH 6.5),0.4mM CDNB (as a 40mM stock solution in 99.9%EtOH),1mg protein Ϫ1min Ϫ1(180%of the control)at 96h.Fronds of L.minor exposed to a blank mM GSH,and enzyme extract (100␮g protein)in a final volume of 1ml at 25ЊC.Enzyme activ-formulation displayed a regular increase in their GST activity from 6to 36h,when a steady state ity was measured by monitoring the increase in absorbance at 340nm during the conjugation of was reached.Folpet did not significantly modify G-POD GSH to CDNB (9).CAT activity was assayed by following the activity,while the P-POD continuously increased during the treatment of L.minor with consumption of H 2O 2at 240nm,according to Aebi (10),in 50mM K-phosphate (pH 6.5),the fungicide (Fig.1).The maximum was obtained after 96h of exposure,when the activ-containing 15mM H 2O 2and enzyme extract (50␮g protein),in a final volume of 1ml.ity was 166%of the control.Figure 1also showed that a slight increase of P-POD activity P-POD was determined directly by following the formation of purpurogallin at 430nm (2.5occurred in response to the exposure to a blank formulation.mM Ϫ1cm Ϫ1)as described by Kno ¨rzer et al.(11).The assay mixture contained 50mM K-phos-Treatments of L.minor with folpet also resulted in a strong stimulation of CAT activity phate (pH 6.5),20mM pyrogallol,5mM H 2O 2,and enzyme extract (15␮g protein)in a final from 12to 48h of exposure (Fig.1),when a maximum was reached (252%of the control).volume of 1ml.For G-POD,the reaction mixture consisted of Then,although it steadily decreased,the activity of CAT remained very high since it was again 50mM K-phosphate (pH 6.5),1mM H 2O 2,0.25%(v/v)guaiacol,and enzyme extract (5057␮mol mg protein Ϫ1min Ϫ1at 96h (177%of the control).Likewise,despite a transient ␮g protein),in a final volume of 1ml.The enzyme activity was measured by monitoring decrease in the first hours of the experiment,a blank formulation linearly stimulated CAT activ-the increase in absorbance at 470nm (26.6mM Ϫ1cm Ϫ1)during the polymerization of guai-ity from 24to 72h,when it reached a maximum level (221%of the control).Then,although it acol into tetraguaiacol (12).slightly decreased,the activity of CAT still Statisticsremained very important (205%of the control after 96h of exposure to the fungicide).In all experiments three replicates were per-APO activity regularly increased during the formed for each time of exposure to folpet and 96h of treatment with folpet (Fig.1).A maxi-all experiments were repeated three times.Data mum stimulation of 55%was observed at 96h.presented here are the means ϮSD of three inde-A blank formulation also caused the APO activ-pendent experiments.Significance of differ-ity to increase up to 36h of exposure and then ences between samples was determined by a a steady state was reached (125%of the control).Student’s t test and P values Յ0.05were consid-DISCUSSIONered significant.In this work,the effects of folpet on the enzy-RESULTSmatic defenses against oxidative stress in L.minor fronds were examined.First,we investi-Folpet induced a strong increase of the GR activity of L.minor (Fig.1).It was significant gated the activity of two main enzymes of the Halliwell–Asada pathway,APO and GR.APO (P Ͻ0.05)from 24to 96h,where a maximum activity of 54␮mol mg protein Ϫ1min Ϫ1(273%catalyzes the reduction of H 2O 2into water using AsA as a donor of electrons (7).By regenerating of the control)was reached.Blank formulation had no effect on GR activity.GSH from GSSG,and thus sustaining its role of a redox buffer,GR also plays a key role in theAs little as 6h of exposure to the fungicideEFFECTS OF FOLPET ON DUCKWEED ANTIOXIDATIVE ENZYMES115 FIG.1.Enzymatic activities in control(ⅷ)and blank formulation(⅜)and folpet-treated(᭢)fronds of L.minor during a96-h exposure(concentration of folpet,33␮M).Data are mean valuesϮSD of three independent experiments with triplicates.The symbols*andϩ,respectively,indicate a significant difference(PϽ0.05) between blank formulation-treated controls and folpet-treated plants and between the two controls(with or without blank formulation).Activities of GR,GST,P-POD,and G-POD were expressed as nmol or␮mol of product formedиmg proteinϪ1иminϪ1,while APO and CAT activities were expressed as␮mol of substrate consumedиmg proteinϪ1иminϪ1.H2O2-scavenging pathway.Our results showed and mainly H2O2,in the cells.Likewise,stimula-tion of GR is a common response to oxidative that folpet led the APO activity to progressivelyincrease,while the strongest stimulation of GR stress generated by a wide range of factors, occurred.Induction of APO was previouslyincluding physical ones such as heat and drought reported as a consequence of exposure to oxida-(14)and pollutants such as pesticides or ozone tive stress-generating compounds(e.g.,13)and(11,15).A very strong and rapid stimulation of joined with the increase in production of ROS CAT activity occurred during the treatment of116TEISSEIRE AND VERNETL.minor with folpet.CAT is an heme-containing formation of ROS induced by folpet we sug-gested above might explain the stimulation of enzyme which catalyzes the reduction of H2O2GST after a treatment with the fungicide. (16).Like APO and GR,induction of CAT wasThe present study was done to determine often reported in literature on stress adaptationwhether enzymatic inhibitions were involved in mechanisms and various sources of stress(phys-the phytotoxicity of folpet,as suggested to ical injuries,pollutants,pathogen agents)wereexplain the fungicidal effects of this molecule known to stimulate CAT(13,17).P-POD activ-(3)and its hepatotoxicity(4).Our results clearly ity was also stimulated by folpet and likewiseshowed that none of the six tested enzymes were the induction of peroxidases is a commoninhibited by the fungicide and on the contrary response to oxidative stress,caused by biotic ormost of them were stimulated.If it could not abiotic agents(18,19).A possible explanationbe concluded from the low number of enzymes for these stimulations evoked the capacity ofmeasured whether folpet was an enzymatic peroxidases to reduce H2O2using phenolic com-inhibitor in L.minor,our results suggested pounds or flavonoids as donors of electronsanother possible effect of the fungicide.Indeed, (20,21).inductions of antioxidative enzymes were The fast and strong mobilization of CATreported during numerous oxidative events and joined with the induction of APO and P-POD,the similarity between these responses and that which were a part of the defenses against oxida-induced by folpet suggested that the fungicide tive stress,suggested that folpet might,directlymight also generate the production of ROS. or indirectly,generate overproduction of ROSAccording to this hypothesis,the phytotoxicity in L.minor cells.According to this hypothesis,of folpet might,directly or indirectly,result from the stimulation of GR might be a cellular adapta-an oxidative stress.In order to confirm that tion process to cope with a possible perturbationhypothesis,further research is now required and of the redox state of the cell generated bythe first stage will consist in the following of these ROS.the H2O2levels in cells of L.minor during fol-Folpet also caused an important induction ofpet treatments.GST.GST is implied in the detoxification ofvarious herbicides(2,4-D,atrazine,metolachlorACKNOWLEDGMENTS...)by conjugating GSH onto them and it wascommonly reported to be stimulated by these This work was financed in part by Europol’Agro through molecules(22).However,in the present case the program“Toxicologie–e´cotoxicologie des pesticides et and despite the observed induction,it could notdes me´taux lourds.”Due thanks are given to Dr.J.Roederer,Makhteshim-Agan,France,for the gift of Folpan500and be concluded from our data whether conjugationfor blank formulation.with GSH was implied in folpet detoxificationand further studies were required to provide anyREFERENCESadditional arguments about a possible metaboli-zation of the fungicide by GST.In addition, 1. 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樱桃叶单宁的分布及累积特征张超越; 张茹涵; 梁栋; 李海刚; 王守箐; 刘林【期刊名称】《《中国果菜》》【年(卷),期】2019(039)010【总页数】5页(P64-67,70)【关键词】樱桃叶; 单宁; 积累; 组织化学; 超微技术【作者】张超越; 张茹涵; 梁栋; 李海刚; 王守箐; 刘林【作者单位】临沂大学药学院山东临沂276005【正文语种】中文【中图分类】Q945单宁是由植物细胞合成的一类天然酚类化合物，产量仅次于纤维素、半纤维素和木质素[1]。

单宁可分为水解性单宁和非水解性单宁，后者又称凝缩性单宁或原花青素[1-5]。

水解性单宁是没食子酸与多元醇酯化产物及其衍生物，最常见的多元醇是β-D-葡萄糖[2-3]。

非水解性单宁是黄酮类化合物的聚合物，最常见的单体是儿茶素和表儿茶素[4-5]。

单宁溶于水，能吸收紫外线，与蛋白质反应，引起蛋白质结构和性质发生变化，从而破坏其正常功能，这一性质赋予单宁杀菌功能[6]。

单宁与蛋白质作用的性质使草食性动物不愿取食那些积累大量单宁的植物，从而使植物免受这些动物伤害[7]。

紫外线破坏蛋白质和DNA 等生物大分子的结构和功能，细胞通过积累单宁保护蛋白质和DNA 免受紫外辐射伤害[8-9]。

因此，积累单宁是植物的一种自我保护机制[6-9]。

研究表明，单宁具有抗氧化及抑制肿瘤等生物活性[10]。

由于上述特性，单宁在食品、化妆品、医药、制革、造纸、冶金、水处理等许多领域得到应用。

了解植物叶器官单宁分布的特征，有助于分析其抵御病原菌侵染和紫外辐射伤害机制。

樱桃是栽培面积较大的果树，但樱桃叶单宁积累的特征研究还鲜有报道。

本研究采用组织化学和超微技术对樱桃叶单宁分布进行了显微和超微分析，了解了单宁积累及其分布特点，从而揭示樱桃单宁的紫外辐射和病原菌防御机制，同时拓宽对樱桃生物学的认识，为单宁资源研究增添信息，也为樱桃叶的开发利用提供理论参考。

1 材料与方法1.1 植物材料供试樱桃（Cerasus pseudocerasus）品种为金红樱桃，栽种于临沂大学校园内，树龄6 年，常规管理，长势良好。

作物学报ACTA AGRONOMICA SINICA 2013, 39(11): 2046−2054 / ISSN 0496-3490; CODEN TSHPA9E-mail: xbzw@DOI: 10.3724/SP.J.1006.2013.02046转codA基因提高番茄植株的耐热性李枝梅1窦海鸥1卫丹丹1孟庆伟1 Tony Huihuang CHEN 2杨兴洪1,*1山东农业大学生命科学学院 / 作物生物学国家重点实验室, 山东泰安 271018; 2 Department of Horticulture, Oregon State University,Corvallis, OR 97331, USA摘要: 以野生型番茄(cv. Moneymaker)和转codA番茄为材料, 用不同温度(25、30、35、40、45和50℃)分别处理2 h, 测定叶片净光合速率(P n)、PSII最大光化学效率(F v/F m)、过氧化氢(H2O2)含量、丙二醛(MDA)含量、相对电导率(REC)和抗氧化酶活性等生理指标; 42℃高温处理0、3和6 h后, 检测热响应基因的表达量以及D1蛋白的含量, 研究高温胁迫对上述参数的影响, 探讨转codA基因提高番茄叶片耐热性的机制。

结果表明, 高温胁迫下, 转codA基因番茄叶片P n和F v/F m的抑制程度明显低于野生型, H2O2、MDA的积累量以及REC均低于野生型, 而且明显增强了过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)的活性。

此外, 转codA基因番茄叶片中抗氧化酶基因和热胁迫基因的表达水平均高于野生型, 而D1蛋白的降解水平低于野生型。

转codA基因番茄体内合成的甜菜碱提高了转基因番茄的耐热性, 这与提高和维持较高的抗氧化酶活性、促进热激响应基因的表达及减缓D1蛋白的降解等有关。

关键词:codA基因; 番茄; 高温胁迫; 耐热性; 甜菜碱codA Transgenic Tomato Plants Enhance Tolerance to High Temperature StressLI Zhi-Mei1, DOU Hai-Ou1, WEI Dan-Dan1, MENG Qing-Wei1, Tony Huihuang CHEN2, and YANGXing-Hong1,*1 State Key Laboratory of Crop Biology / College of Life Sciences, Shandong Agricultural University, Tai’an 271018, China;2 Department of Horti-culture, Oregon State University, Corvallis, OR 97331, USAAbstract: The effects of codA gene on photosynthesis, activities of antioxidative enzymes, the expression of the heat responsegenes and the accumulation of D1 protein in tomato leaf under different temperature stresses were investigated to reveal themechanism of thermotolerance in codA-transgenic tomato plants. The wild type (cv. Moneymaker) and codA transgenic tomatoplants were treated with 25, 30, 35, 40, 45, and 50℃ respectively for two hours, then net photosynthetic rate (P n), the maximalefficiency of PSII photochemistry (F v/F m), hydrogen peroxide (H2O2) content, malondialdehyde (MDA) content, relative electricconductivity (REC) and activities of antioxidative enzymes were detected. After 42℃ heat stress for 0, 3, 6 hours, the expressionsof antioxidant enzyme genes and heat stress genes and the accumulation of the D1 protein were also determined. The results demonstrated that under high temperature stress, the inhibition degree of P n and F v/F m in codA transgenic tomato plants was lowerthan that in wild type plants. The accumulation of H2O2, MDA and REC in codA transgenic tomato plants was less than that inwild type plants. And codA transgenic tomato plants also greatly enhanced the activities of catalase (CAT), superoxide dismutase(SOD), peroxidase (POD), and ascorbate peroxidase (APX). Moreover, the expression levels of antioxidant genes and heat re-sponse genes in codA transgenic tomato plants was higher than those in wild type plants and the degradation degree of D1 proteinin codA transgenic tomato plants were lower than that in wild type plants. It indicated that codA transgenic tomato plants enhance thermotolerance via maintaining higher activities of antioxidant enzymes, accelerating the expression of heat response genes andreducing the degradation of D1 protein.Keywords:codA gene; Tomato; High temperature stress; Thermotolerance; Glycinebetaine本研究由国家重点基础研究发展计划(973计划)项目(2009CB118500), 国家高技术研究发展计划(863计划)项目(2012AA10A309), 国家自然科学基金项目(30970229)和教育部高校博士点基金(20103702110007)资助。

西北植物学报,2006,26(11):2297-2301Acta Bot.Boreal.-Occident.Sin. 文章编号:1000-4025(2006)11-2297-055-氨基乙酰丙酸对秋冬季大棚西瓜叶片光合作用及抗氧化酶活性的影响康　琅,程　云,汪良驹*(南京农业大学园艺学院,南京210095)摘　要:以秋冬季塑料大棚中盆栽西瓜幼苗为材料,研究了50～200mg/L5-氨基乙酰丙酸(A LA)处理对低温弱光条件下西瓜植株光合特性、碳水化合物积累、抗氧化酶活性和丙二醛(M DA)含量的影响。

结果表明,外源A L A处理显著提高了西瓜幼苗叶片净光合速率(Pn)、气孔导度(G s)和蒸腾速率(T r)。

A L A处理还显著提高了叶片SO D 和PO D等抗氧化酶活性,但对CA T及AP X活性影响不大,暗示A LA处理可能主要通过提高植株SOD和PO D 活性来促进西瓜幼苗活性氧代谢,从而保护细胞光合性能。

处理后30d分析表明,A L A处理植株干物质重量比对照高出42%～54%,淀粉含量增加62.2%～207.0%,可溶性糖含量也增加32.0%～87.1%。

以上结果说明,A LA 处理可以增强低温弱光条件下西瓜幼苗抗氧化系统活性,促进光合作用与光合产物积累,并促进植株生长。

关键词:5-氨基乙酰丙酸;抗氧化酶;碳水化合物;光合特性;西瓜中图分类号:Q945.78文献标识码:AEffects of5-Aminolevulinic Acid(ALA)on the Photosynthesis andAnti-oxidative Enzymes Activities of the Leaves of G reenhouseWatermelon in Summer and WinterKANG Lang,CH ENG Yun,WANG Liang-ju*(College of H orticultu re,Nanjing Agricultu ral University,Nanjing210095,China)A bstract:The study treated the seedling s of w atermelon po t planted in greenhouse w ith5-aminolevulinic acid(ALA)at50～200mg/L and studied their photosy nthetic characteristics,carbo hy drate accumulatio ns, anti-o xidative enzy me activitie s and M DA contents at low temperature under w eak lig ht.It w as show n that the treatments with exog enous A LA could significantly raise the ne t pho to synthetic rate,stom atal conduct-ance and transpiratio n rate of the leaves of w atermelon seedlings.A LA treatments raised the activities of such anti-oxidative enzy mes as SOD and POD,but exer ted little influence on the CA T and APX activities in the leaves of w atermelon seedlings,w hich im plied that the A LA treatm ents enhanced the m etabolism of ac-tive o xyg en species by raising the SOD and POD activities in w atermelon plants,thus maintaining the cellu-lar pho to synthetic capacity.30day s after the A LA treatments,the dry w eig hts,starch and solvable sug ar contents of treated w atermelo n plants increased separately by42%～54%,62.2%～207.0%and32.0%～87.1%co mpared w ith tho se of the control plants.The above results indicated that ALA treatm ents co uld improve the activities o f anti-oxidative enzy mes in w atermelo n seedling at lo w temperature under weak light,and promo ted the photosy nthesis and photosy nthate accumulatio ns as well as the plant grow th.收稿日期:2006-02-24;修改稿收到日期:2006-08-14基金项目:国家自然科学基金(30471181)作者简介:康　琅(1980-),女,硕士。

一氧化氮供体SNP对水稻根中由硒引起的脂质过氧化的调节作用摘要：研究以0.2和20 μmol/L Na2SeO3及一氧化氮供体硝普钠（SNP）处理对水稻根中根系活力和硫代巴比妥酸反应产物含量，愈创木酚过氧化酶（GPX）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）以及抗坏血酸过氧化物酶（APX）活性等生理生化指标的影响。

结果表明，1 μmol/L SNP处理显著提高0.2 μmol/L Na2SeO3处理下水稻根系活力，SNP通过促进CAT酶活性，缓解了膜脂过氧化；在20 μmol/L Na2SeO3处理下，1 μmol/L SNP明显促进SOD酶活性，但显著降低APX酶活性，显著降低根系活力。

NO对水稻根中Se引起的抗氧化活性变化具有调节作用，根系活力可以作为评价抗氧化活性的重要参考。

关键词：水稻；硒；一氧化氮；抗氧化作用Effects of Exogenous Nitric Oxide Donor SNP on Lipid Peroxidation Caused by Selenium in Roots of Rice SeedlingsAbstract：In this study，we reported some antagonist effects of exogenous nitric oxide on oxidative stress of rice induced by selenium. The root activity，the contents of TBARS and the activities of GPX，SOD，CAT and APX in roots of rice seedlings treated with varied concentr ations of selenium and 1 μmol/L SNP were investigated. The results showed that the root activity increased by treatment of SNP in 0.2 μmol/L Na2SeO3 group. SNP alleviated significantly the lipid peroxidation in rice seedlings via increasing CAT activities in rice root. In 20 μmol/L Na2SeO3 treated rice seedlings，SNP aggravated significantly the root activity loss via promoting SOD activities and repressing APX activity. Taken together，results suggested that NO regulates antioxidative activity caused by selenium in roots of rice seedlings. Root activity can be used as reference index of evaluating antioxidative activity.Key words：rice；selenium；nitric oxide；antioxidation一氧化氮（Nitric oxide，NO）是植物中一种重要的信号分子，在调节植物生长发育，促进植物细胞衰亡等方面发挥着重要作用[1]，进一步研究表明NO 在植物中的某些功能与它对活性氧（Reactive oxygen species，ROS）代谢水平的调节密切相关，如NO可作用于烟草中含血红素铁的过氧化氢酶（Catalase，CAT）和含非血红素铁的抗坏血酸过氧化物酶（Ascorbate peroxidase，APX），参与对活性氧代谢的调节[2]。

Hans Journal of Food and Nutrition Science 食品与营养科学, 2017, 6(2), 59-64Published Online May 2017 in Hans. /journal/hjfnshttps:///10.12677/hjfns.2017.62006Antioxidant Effect of Resveratrol and ItsControl Effect on Related DiseasesXiaochun Zhang, Qing Yang*College of Veterinary Medicine, Hunan Agricultural University, Changsha HunanReceived: May 4th, 2017; accepted: May 16th, 2017; published: May 24th, 2017AbstractResveratrol is a stilbene compounds, which is widely found in grapes, peanuts, Polygonum cuspi-datum, cassia, resveratrol and other plants and acts as a natural antioxidant. Resveratrol plays its antioxidant effect mainly by scavenging free radicals, inhibiting production of free radicals and li-pid peroxidation, regulating activity and gene expression of antioxidant enzymes. Oxidative dam-age is related to basic processes of many diseases. Therefore, it is very important to explore the preventive and therapeutic effects of resveratrol in the prevention and treatment of related dis-eases.KeywordsResveratrol, Antioxidation, Disease Control白藜芦醇抗氧化作用及其在相关疾病中的防治效果张晓春，杨青*湖南农业大学动物医学院，湖南长沙收稿日期：2017年5月4日；录用日期：2017年5月16日；发布日期：2017年5月24日摘要白藜芦醇(Resveratrol)属芪类化合物，广泛存在于葡萄、花生、虎杖、决明、藜芦等植物中的天然抗氧*通讯作者。

盐胁迫对小麦幼苗生理生化特性的影响张军;王新军;于浩世【摘要】押为了明确盐胁迫对小麦幼苗生理生化特性的影响，采用水培方法，以兰黑粒小麦和小偃15小麦为材料，对其在不同浓度的NaCl处理下叶片中超氧化物歧化酶（SOD）活性、过氧化物酶（POD）活性、丙二醛（MDA）含量、相对电导率、脯氨酸含量和可溶性蛋白质含量等生理指标进行测定。

结果表明：随着盐浓度的增加，兰黑粒小麦的SOD和POD活性先增后减，而小偃15小麦的SOD 和POD活性呈下降趋势；两种小麦的MDA含量、相对电导率和脯氨酸含量均随盐浓度的增加显著增大；可溶性蛋白质在2种小麦的变化趋势不尽一致。

2个供试品种均有一定的抗盐能力，兰黑粒小麦抗盐性相对较强。

%To study the effects of NaCl stress of wheat on its physiological and biochemical characteristics, Lanhei wheat and Xiaoyan 15 were taken as tested materials. The superoxide dismutase (SOD) activity, peroxidase (POD) activity, MDA content, relative conductivity, the proline content and the soluble protein content were investigated after different treatments. The results showed that under NaCl stress, compared with the each control, the activities of SOD and POD of Lanhei showed the trend of from increasing to decreasing, while the Xiaoyan15 showed the decreased; MDA content, relative conductivity, the proline content increased significantly as the stress decreased; whereas the trend of soluble protein was different between the 2 cultivars. Both of them had resistance to salt, and Lanhei wheat was a relatively higher salt-tolerant wheat.【期刊名称】《商洛学院学报》【年(卷),期】2015(000)004【总页数】4页(P59-62)【关键词】盐胁迫;兰黑粒小麦;小偃15小麦【作者】张军;王新军;于浩世【作者单位】商洛学院生物医药与食品工程学院/秦岭植物良种繁育中心，陕西商洛 726000;商洛学院生物医药与食品工程学院/秦岭植物良种繁育中心，陕西商洛726000;商洛学院生物医药与食品工程学院/秦岭植物良种繁育中心，陕西商洛726000【正文语种】中文【中图分类】S512作物产量的形成受多种因素的影响，其中干旱和盐碱造成的减产高达40%以上［1］。

The effects of grape seed extract fortification on the antioxidant activity and quality attributes of bread葡萄籽提取物对面包的抗氧化活性和质量属性的影响Abstract: The antioxidant activity change of breads added with grape seed extract (GSE) was investigated. The results showed that bread with the addition of GSE had stronger antioxidant activity than that of blank bread, and increasing the level of GSE addition further enhanced the antioxidant capacity of the bread. However, thermal processing caused antioxidant activity of GSE added to bread to decrease by around 30–40%. We also studied the effect of GSE on the formation of detrimental Ne-(carboxymethyl) lysine (CML), a famous advanced glycation endproduct in bread. According to the results, GSE could reduce CML in bread and acted in a dose-dependent manner. Meanwhile, except for an acceptable colour change, adding GSE to bread had only little effect on the quality attributes of the bread. Altogether, our findings indicate that GSE-fortified bread is promising to be developed as a functional food with relatively lower CML-related health risks, yet a high antioxidant activity.Keywords: Antioxidant activity Grape seed extract Bread Advanced glycation endproducts摘要：对加入葡萄籽提取物（GSE）对面包的抗氧化活性的变化进行了研究。

Adopted:19 July 2006OECD GUIDELINES FOR THE TESTING OF CHEMICALS Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test INTRODUCTION1. OECD Guidelines for the Testing of Chemicals are periodically reviewed in the light of scientific progress and applicability to regulatory use. This updated Guideline is designed to assess potential effects of substances on seedling emergence and growth. As such it does not cover chronic effects or effects on reproduction (i.e. seed set, flower formation, fruit maturation). Conditions of exposure and properties of the substance to be tested must be considered to ensure that appropriate test methods are used (e.g. when testing metals/metal compounds the effects of pH and associated counter ions should be considered) (1). This Guideline does not address plants exposed to vapours of chemicals. The Guideline is applicable to the testing of general chemicals, biocides and crop protection products (also known as plant protection products or pesticides). It has been developed on the basis of existing methods (2) (3) (4) (5) (6) (7). Other references pertinent to plant testing were also considered (8) (9) (10). Definitions used are given in Annex 1.PRINCIPLE OF THE TEST2. The test assesses effects on seedling emergence and early growth of higher plants following exposure to the test substance in the soil (or other suitable soil matrix). Seeds are placed in contact with soil treated with the test substance and evaluated for effects following usually 14 to 21 days after 50 % emergence of the seedlings in the control group. Endpoints measured are visual assessment of seedling emergence, dry shoot weight (alternatively fresh shoot weight) and in certain cases shoot height, as well as an assessment of visible detrimental effects on different parts of the plant. These measurements and observations are compared to those of untreated control plants.3. Depending on the expected route of exposure, the test substance is either incorporated into the soil (or possibly into artificial soil matrix) or applied to the soil surface, which properly represents the potential route of exposure to the chemical. Soil incorporation is done by treating bulk soil. After the application the soil is transferred into pots, and then seeds of the given plant species are planted in the soil. Surface applications are made to potted soil in which the seeds have already been planted. The test units (controls and treated soils plus seeds) are then placed under appropriate conditions to support germination/growth of plants.4. The test can be conducted in order to determine the dose-response curve, or at a single concentration/rate as a limit test according to the aim of the study. If results from the single concentration/rate test exceed a certain toxicity level (e.g. whether effects greater than x% are observed), a range-finding test is carried out to determine upper and lower limits for toxicity followed by a multiple concentration/rate test to generate a dose-response curve. An appropriate statistical analysis is used to obtain effective concentration EC x or effective application rate ER x (e.g. EC25, ER25, EC50,ER50) for the most sensitive parameter(s) of interest. Also, the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) can be calculated in this test.1/21INFORMATION ON THE TEST SUBSTANCE5. The following information is useful for the identification of the expected route of exposure to the substance and in designing the test: structural formula, purity, water solubility, solubility in organic solvents, 1-octanol/water partition coefficient, soil sorption behaviour, vapour pressure, chemical stability in water and light, and biodegradability.VALIDITY OF THE TEST6. In order for the test to be considered valid, the following performance criteria must be met in the controls:•the seedling emergence is at least 70%;•the seedlings do not exhibit visible phytotoxic effects (e.g. chlorosis, necrosis, wilting, leaf and stem deformations) and the plants exhibit only normal variation in growth and morphology for that particular species;•the mean survival of emerged control seedlings is at least 90% for the duration of the study;•environmental conditions for a particular species are identical and growing media contain the same amount of soil matrix, support media, or substrate from the same source.REFERENCE SUBSTANCE7. A reference substance may be tested at regular intervals, to verify that performance of the test and the response of the particular test plants and the test conditions have not changed significantly over time. Alternatively, historical biomass or growth measurement of controls could be used to evaluate the performance of the test system in particular laboratories, and can serve as an intra-laboratory quality control measure.DESCRIPTION OF THE METHODNatural soil - Artificial substrate8. Plants may be grown in pots using a sandy loam, loamy sand, or sandy clay loam that contains up to 1.5 percent organic carbon (approx. 3 percent organic matter). Commercial potting soil or synthetic soil mix that contains up to 1.5 percent organic carbon may also be used. Clay soils should not be used if the test substance is known to have a high affinity for clays. Field soil should be sieved to 2 mm particle size in order to homogenize it and remove coarse particles. The type and texture, % organic carbon, pH and salt content as electronic conductivity of the final prepared soil should be reported. The soil should be classified according to a standard classification scheme (11). The soil could be pasteurized or heat treated in order to reduce the effect of soil pathogens.9. Natural soil may complicate interpretation of results and increase variability due to varying physical/chemical properties and microbial populations. These variables in turn alter moisture-holding capacity, chemical-binding capacity, aeration, and nutrient and trace element content. In addition to the variations in these physical factors, there will also be variation in chemical properties such as pH and redox potential, which may affect the bioavailability of the test substance (12) (13) (14).10. Artificial substrates are typically not used for testing of crop protection products, but they may be of use for the testing of general chemicals or where it is desired to minimize the variability of the natural2/21soils and increase the comparability of the test results. Substrates used should be composed of inert materials that minimize interaction with the test substance, the solvent carrier, or both. Acid washed quartz sand, mineral wool and glass beads (e.g. 0.35 to 0.85 mm in diameter) have been found to be suitable inert materials that minimally absorb the test substance (15), ensuring that the substance will be maximally available to the seedling via root uptake. Unsuitable substrates would include vermiculite, perlite or other highly absorptive materials. Nutrients for plant growth should be provided to ensure that plants are not stressed through nutrient deficiencies, and where possible this should be assessed via chemical analysis or by visual assessment of control plants.Criteria for selection of test species11. The species selected should be reasonably broad, e.g., considering their taxonomic diversity in the plant kingdom, their distribution, abundance, species specific life-cycle characteristics and region of natural occurrence, to develop a range of responses (8) (10) (16) (17) (18) (19) (20). The following characteristics of the possible test species should be considered in the selection:•the species have uniform seeds that are readily available from reliable standard seed source(s) and that produce consistent, reliable and even germination, as well as uniform seedling growth;•plant is amenable to testing in the laboratory, and can give reliable and reproducible results within and across testing facilities;•the sensitivity of the species tested should be consistent with the responses of plants found in the environment exposed to the substance;•they have been used to some extent in previous toxicity tests and their use in, for example, herbicide bioassays, heavy metal screening, salinity or mineral stress tests or allelopathy studies indicates sensitivity to a wide variety of stressors;•they are compatible with the growth conditions of the test method;•they meet the validity criteria of the test.Some of the historically most used test species are listed in Annex 2 and potential non-crop species in Annex 3.12. The number of species to be tested is dependent on relevant regulatory requirements, therefore it is not specified in this Guideline.Application of the test substance13. The substance should be applied in an appropriate carrier (e.g. water, acetone, ethanol, polyethylene glycol, gum Arabic, sand). Formulated products or formulations containing active ingredients and various adjuvants can be tested as well.Incorporation into soil/artificial substrate14. Substances which are water soluble or suspended in water can be added to water, and then the solution is mixed with soil with an appropriate mixing device. This type of test may be appropriate if exposure to the chemical is through soil or soil pore-water and that there is concern for root uptake. The water-holding capacity of the soil should not be exceeded by the addition of the test substance. The volume of water added should be the same for each test concentration, but should be limited to prevent soil agglomerate clumping.15. Substances with low water solubility should be dissolved in a suitable volatile solvent (e.g. acetone, ethanol) and mixed with sand. The solvent can then be removed from the sand using a stream of3/21air while continuously mixing the sand. The treated sand is mixed with the experimental soil. A second control is established which receives only sand and solvent. Equal amounts of sand, with solvent mixed and removed, are added to all treatment levels and the second control. For solid, insoluble test substances, dry soil and the chemical are mixed in a suitable mixing device. Hereafter, the soil is added to the pots and seeds are sown immediately.16. When an artificial substrate is used instead of soil, chemicals that are soluble in water can be dissolved in the nutrient solution just prior to the beginning of the test. Chemicals that are insoluble in water, but which can be suspended in water by using a solvent carrier, should be added with the carrier, to the nutrient solution. Water-insoluble chemicals, for which there is no non-toxic water-soluble carrier available, should be dissolved in an appropriate volatile solvent. The solution is mixed with sand or glass beads, placed in a rotary vacuum apparatus, and evaporated, leaving a uniform coating of chemical on sand or beads. A weighed portion of beads should be extracted with the same organic solvent and the chemical assayed before the potting containers are filled.Surface application17. For crop protection products, spraying the soil surface with the test solution is often used for application of the test substance. All equipment used in conducting the tests, including equipment used to prepare and administer the test substance, should be of such design and capacity that the tests involving this equipment can be conducted in an accurate way and it will give a reproducible coverage. The coverage should be uniform across the soil surfaces. Care should be taken to avoid the possibilities of chemicals being adsorbed to or reacting with the equipment (e.g. plastic tubing and lipophilic chemicals or steel parts and elements). The test substance is sprayed onto the soil surface simulating typical spray tank applications. Generally, spray volumes should be in the range of normal agricultural practice and the volumes (amount of water etc. should be reported). Nozzle type should be selected to provide uniform coverage of the soil surface. If solvents and carriers are applied, a second group of control plants should be established receiving only the solvent/carrier. This is not necessary for crop protection products tested as formulations.Verification of test substance concentration/rate18. The concentrations/rates of application must be confirmed by an appropriate analytical verification. For soluble substances, verification of all test concentrations/rates can be confirmed by analysis of the highest concentration test solution used for the test with documentation on subsequent dilution and use of calibrated application equipment (e.g., calibrated analytical glassware, calibration of sprayer application equipment). For insoluble substances, verification of compound material must be provided with weights of the test substance added to the soil. If demonstration of homogeneity is required, analysis of the soil may be necessary.PROCEDURETest design19. Seeds of the same species are planted in pots. The number of seeds planted per pot will depend upon the species, pot size and test duration. The number of plants per pot should provide adequate growth conditions and avoid overcrowding for the duration of the test. The maximum plant density would be around 3 - 10 seeds per 100 cm² depending to the size of the seeds. As an example, one to two corn, soybean, tomato, cucumber, or sugar beet plants per 15cm container; three rape or pea plants per 15 cm container; and 5 to 10 onion, wheat, or other small seeds per 15 cm container are recommended. The number of seeds and replicate pots (the replicate is defined as a pot, therefore plants within the same pot do4/21not constitute a replicate) should be adequate for optimal statistical analysis (21). It should be noted that variability will be greater for test species using fewer large seeds per pot (replicate), when compared to test species where it is possible to use greater numbers of small seeds per pot. By planting equal seed numbers in each pot this variability may be minimized.20. Control groups are used to assure that effects observed are associated with or attributed only to the test substance exposure. The appropriate control group should be identical in every respect to the test group except for exposure to the test substance. Within a given test, all test plants including the controls should be from the same source. To prevent bias, random assignment of test and control pots is required.21. Seeds coated with an insecticide or fungicide (i.e. “dressed” seeds) should be avoided. However, the use of certain non-systemic contact fungicides (e.g. captan, thiram) is permitted by some regulatory authorities (22). If seed-borne pathogens are a concern, the seeds may be soaked briefly in a weak 5 % hypochlorite solution, then rinsed extensively in running water and dried. No remedial treatment with other crop protection product is allowed.Test conditions22. The test conditions should approximate those conditions necessary for normal growth for the species and varieties tested (Annex 4 provides examples of test condition). The emerging plants should be maintained under good horticultural practices in controlled environment chambers, phytotrons, or greenhouses. When using growth facilities these practices usually include control and adequately frequent (e.g. daily) recording of temperature, humidity, carbon dioxide concentration, light (intensity, wave length, photosynthetically active radiation) and light period, means of watering, etc., to assure good plant growth as judged by the control plants of the selected species. Greenhouse temperatures should be controlled through venting, heating and/or cooling systems. The following conditions are generally recommended for greenhouse testing:•temperature: 22 o C ± 10 o C;•humidity: 70 % ± 25 %;•photoperiod: minimum 16 hour light;•light intensity: 350 ± 50 µE/m2/s. Additional lighting may be necessary if intensity decreases below 200 µE/m2/s, wavelength 400 - 700 nm except for certain species whose light requirementsare less.Environmental conditions should be monitored and reported during the course of the study. The plants should be grown in non-porous plastic or glazed pots with a tray or saucer under the pot. The pots may be repositioned periodically to minimize variability in growth of the plants (due to differences in test conditions within the growth facilities). The pots must be large enough to allow normal growth.23. Soil nutrients may be supplemented as needed to maintain good plant vigour. The need and timing of additional nutrients can be judged by observation of the control plants. Bottom watering of test containers (e.g. by using glass fiber wicks) is recommended. However, initial top watering can be used to stimulate seed germination and, for soil surface application it facilitates movement of the chemical into the soil.24. The specific growing conditions should be appropriate for the species tested and the test substance under investigation. Control and treated plants must be kept under the same environmental5/21conditions, however, adequate measures should be taken to prevent cross exposure (e.g. of volatile substances) among different treatments and of the controls to the test substance.Testing at a single concentration/rate25. In order to determine the appropriate concentration/rate of a substance for conducting a single-concentration or rate (challenge/limit) test, a number of factors must be considered. For general chemicals, these include the physical/chemical properties of the substance. For crop protection products, the physical/chemical properties and use pattern of the test substance, its maximum concentration or application rate, the number of applications per season and/or the persistence of the test substance need to be considered. To determine whether a general chemical possesses phytotoxic properties, it may be appropriate to test at a maximum level of 1000 mg/kg dry soil.Range-finding test26. When necessary a range-finding test could be performed to provide guidance on concentrations/rates to be tested in definitive dose-response study. For the range-finding test, the test concentrations/rates should be widely spaced (e.g. 0.1, 1.0, 10, 100 and 1000 mg/kg dry soil). For crop protection products concentrations/rates could be based on the recommended or maximum concentration or application rate, e.g. 1/100, 1/10, 1/1 of the recommended/maximum concentration or application rate. Testing at multiple concentrations/rates27. The purpose of the multiple concentration/rate test is to establish a dose-response relationship and to determine an EC x or ER x value for emergence, biomass and/or visual effects compared to un-exposed controls, as required by regulatory authorities.28. The number and spacing of the concentrations or rates should be sufficient to generate a reliable dose-response relationship and regression equation and give an estimate of the EC x. or ER x.. The selected concentrations/rates should encompass the EC x or ER x values that are to be determined. For example, if an EC50 value is required it would be desirable to test at rates that produce a 20 to 80 % effect. The recommended number of test concentrations/rates to achieve this is at least five in a geometric series plus untreated control, and spaced by a factor not exceeding three. For each treatment and control group, the number of replicates should be at least four and the total number of seeds should be at least 20. More replicates of certain plants with low a germination rate or variable growth habits may be needed to increase the statistical power of the test. If a larger number of test concentrations/rates are used, the number of replicates may be reduced. If the NOEC is to be estimated, more replicates may be needed to obtain the desired statistical power (23).Observations29. During the observation period, i.e. 14 to 21 days after 50 % of the control plants (also solvent controls if applicable) have emerged, the plants are observed frequently (at least weekly and if possible daily) for emergence and visual phytotoxicity and mortality. At the end of the test, measurement of percent emergence and biomass of surviving plants should be recorded, as well as visible detrimental effects on different parts of the plant. The latter include abnormalities in appearance of the emerged seedlings, stunted growth, chloris, discoloration, mortality, and effects on plant development. The final biomass can be measured using final average dry shoot weight of surviving plants, by harvesting the shoot at the soil surface and drying them to constant weight at 60o C. Alternatively, the final biomass can be measured using fresh shoot weight. The height of the shoot may be another endpoint, if required by regulatory authorities. A uniform scoring system for visual injury should be used to evaluate the6/21observable toxic responses. Examples for performing qualitative and quantitative visual ratings are provided in references (23) (24).DATA AND REPORTINGStatistical analysisSingle concentration/rate test30. Data for each plant species should be analyzed using an appropriate statistical method (21). The level of effect at the test concentration/rate should be reported, or the lack of reaching a given effect at the test concentration/rate (e.g., <x % effect observed at y concentration or rate).Multiple concentration/rate test31. A dose-response relationship is established in terms of a regression equation. Different models can be used: for example, for estimating EC x or ER x (e.g. EC25, ER25, EC50, ER50) and its confidence limits for emergence as quantal data, logit, probit, Weibull, Spearman-Karber, trimmed Spearman-Karber methods, etc. could be appropriate. For the growth of the seedlings (weight and height) as continuous endpoints EC x or ER x and its confidence limits can be estimated by using appropriate regression analysis (e.g. Bruce-Versteeg non-linear regression analysis (25)). Wherever possible, the R2 should be 0.7 or higher for the most sensitive species and the test concentrations/rates used encompass 20% to 80% effects. If the NOEC is to be estimated, application of powerful statistical tests should be preferred and these should be selected on the basis of data distribution (21) (26).Test report32. The test report should present results of the studies as well as a detailed description of test conditions, a thorough discussion of results, analysis of the data, and the conclusions drawn from the analysis. A tabular summary and abstract of results should be provided. The report must include the following:substance:Test- chemical identification data, relevant properties of the substance tested (e.g. log P ow, water solubility, vapour pressure and information on environmental fate and behaviour, ifavailable);- details on preparation of the test solution and verification of test concentrations as specified in paragraph 18.Testspecies:- details of the test organism: species/variety, plant families, scientific and common names, source and history of the seed as detailed as possible (i.e. name of the supplier, percentagegermination, seed size class, batch or lot number, seed year or growing season collected,date of germination rating), viability, etc.;- number of mono- and di-cotyledon species tested;- rationale for selecting the species;- description of seed storage, treatment and maintenance.7/21Testconditions:- testing facility (e.g. growth chamber, phytotron and greenhouse);- description of test system (e.g., pot dimensions, pot material and amounts of soil);- soil characteristics (texture or type of soil: soil particle distribution and classification, physical and chemical properties including % organic matter, % organic carbon and pH);- soil/substrate (e.g. soil, artificial soil, sand and others) preparation prior to test;- description of nutrient medium if used;- application of the test substance: description of method of application, description of equipment, exposure rates and volumes including chemical verification, description ofcalibration method and description of environmental conditions during application;- growth conditions: light intensity (e.g. PAR, photosynthetically active radiation), photoperiod, max/min temperatures, watering schedule and method, fertilization;- number of seeds per pot, number of plants per dose, number of replicates (pots) per exposure rate;- type and number of controls (negative and/or positive controls, solvent control if used);- duration of the test.Results:- table of all endpoints for each replicate, test concentration/rate and species;- the number and percent emergence as compared to controls;- biomass measurements (shoot dry weight or fresh weight) of the plants as percentage of the controls;- shoot height of the plants as percentage of the controls, if measured;- percent visual injury and qualitative and quantitative description of visual injury (chlorosis, necrosis, wilting, leaf and stem deformation, as well as, any lack of effects) by the testsubstance as compared to control plants;- description of the rating scale used to judge visual injury, if visual rating is provided;- for single rate studies, the percent injury should be reported;EC x or ER x (e.g. EC50, ER50, EC25, ER25) values and related confidence limits. Where -regression analysis is performed, provide the standard error for the regression equation, andthe standard error for individual parameter estimate (e.g. slope, intercept);- NOEC (and LOEC) values if calculated;- description of the statistical procedures and assumptions used;- graphical display of these data and dose-response relationship of the species tested.Deviations from the procedures described in this guideline and any unusual occurrences during test.the8/21LITERATURE(1) Schrader G., Metge K., and Bahadir M. (1998). Importance of salt ions in ecotoxicological testswith soil arthropods. Applied Soil Ecology, 7, 189-193.(2) International Organisation of Standards. (1993). ISO 11269-1. Soil Quality -- Determination ofthe Effects of Pollutants on Soil Flora – Part 1: Method for the Measurement of Inhibition of Root Growth.(3) International Organisation of Standards. (1995). ISO 11269-2. Soil Quality -- Determination ofthe Effects of Pollutants on Soil Flora – Part 2: Effects of Chemicals on the Emergence and Growth of Higher Plants.(4) American Standard for Testing Material (ASTM). (2002). E 1963-98. Standard Guide forConducting Terrestrial Plant Toxicity Tests.(5) U.S. EPA. (1982). FIFRA, 40CFR, Part 158.540. Subdivision J, Parts 122-1 and 123-1.(6) US EPA. (1996). OPPTS Harmonized Test Guidelines, Series 850. Ecological Effects TestGuidelines:- 850.4000: Background - Non-target Plant Testing;- 850.4025: Target Area Phytotoxicity;- 850.4100: Terrestrial Plant Toxicity, Tier I (Seedling Emergence);- 850.4200: Seed Germination/Root Elongation Toxicity Test;- 850.4225: Seedling Emergence, Tier II;- 850.4230: Early Seedling Growth Toxicity Test.(7) AFNOR, X31-201. (1982). Essai d’inhibition de la germination de semences par une substance.AFNOR X31-203/ISO 11269-1. (1993) Determination des effets des polluants sur la flore du sol: Méthode de mesurage de l’inhibition de la croissance des racines.(8) Boutin, C., Freemark, K.E. and Keddy, C.J. (1993). Proposed guidelines for registration ofchemical pesticides: Non-target plant testing and evaluation. Technical Report Series No.145.Canadian Wildlife Service (Headquarters), Environment Canada, Hull, Québec, Canada.(9) Forster, R., Heimbach, U., Kula, C., and Zwerger, P. (1997). Effects of Plant Protection Productson Non-Target Organisms - A contribution to the Discussion of Risk Assessment and Risk Mitigation for Terrestrial Non-Target Organisms (Flora and Fauna). Nachrichtenbl. Deut.Pflanzenschutzd. No 48.(10) Hale, B., Hall, J.C., Solomon, K., and Stephenson, G. (1994). A Critical Review of the ProposedGuidelines for Registration of Chemical Pesticides; Non-Target Plant Testing and Evaluation, Centre for Toxicology, University of Guelph, Ontario Canada.(11) Soil Texture Classification (US and FAO systems): Weed Science, 33, Suppl. 1 (1985) and SoilSc. Soc. Amer. Proc. 26:305 (1962).(12) Audus, L.J. (1964). Herbicide behaviour in the soil. In: Audus, L.J. ed. The Physiology andbiochemistry of Herbicides, London, New York, Academic Press, NY, Chapter 5, pp. 163-206.9/21。