Weed Research, Japan Vol.35(1) 36-43 (1990) 雑 草 研 究

Characteristics of Herbicidal Injury by biphenyl Ether Herbicides Oxyfluorfen and Bifenox in Lemna pausicostata HEGELM.

HiroshiMATSUMOTO, Shuuichi KOJIMA and Kozo ISHIZUKA

Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

action of the herbicides. We used the Lemna Introduction plant in the study. The plant absorbs solutes Diphenyl ether herbicides with an ortho from a culture medium by its fronds directly, substituent on one of the benzene rings are therefore herbicides can be applied via the known to require light for their phytotoxic culture medium. It was expected that the action6,21,24) In the presence of 02, they induce effect of the herbicides could be determined photooxidative peroxidation of unsaturated rapidly and reproducibly. fatty acids which causes the disruption of cell The objective of the study was to charac- membranes2,12,22). The formation of thiobar- terize the herbicidal action of diphenyl ethers bituric acid reacting materials and short oxyfluorfen (2-chloro-l-(3-ethoxy-4-nitro- chain hydrocarbons, peroxidative products of phenoxy)-4-(trifluoromethyl) benzene) and lipids were detected in the herbicides treated bifenox (methyl-5-(2, 4-dichlorophenoxy) -2- higher plants and unicellular algae13, 14,16,23,25). nitrobenzoate) in Lemna plants. Recent studies suggest that the toxicity of Materials and Methods the p-nitro diphenyl ether herbicides is depen- dent on their ability to accumulate a tetrapyr- Plant Materials role compound, protoporphyrin IX17-19) The aquatic floating plant Lemna pausicos- Involvement of photodynamic action of the tata HEGELM. strain 441 was grown axenical- chemical is proposed for the action of di- ly in 1 1 Erlenmeyer flasks with 500ml of half phenyl ether herbicides; however, the mecha- strength Hutner's medium containing 1% (w/ nism of initiation of lipid peroxidation of v) sucrose. Flasks were inoculated with three- fatty acids in the membrane is not yet fully or four-frond colonies and incubated at 25t understood. Furthermore, contradictory under continuous illumination by 10uE m-2 results were obtained on the involvement of s-1 of fluorescent white light. The plants photosynthetic electron transport in the initi- grew well in the medium and its colony num- ation of the above mentioned oxidative ber doubled every 2 or 3 days. Approximately processes. Many findings indicated that one month after inoculation the colonies of photosynthesis is not involved in the mecha- three- or four-fronds were harvested, rinsed nism of action of diphenyl ether herbicides in with distilled water and supplied for the higher plants2,3,5, 21,22) There is, however, evi- experiments. dence of photosynthetic involvement in some Herbicide Treatment unicellular algae14-16). Thirty colonies were transferred to a 100 Development of a sensitive new bioassay ml beaker containing 50ml of double distilled system is valuable in studying modes of water. Herbicides were added as ethanol

This work was reported at the 28th Annual Meeting of the Weed Science Society of Japan, 1989. -36- H.MATSUMOTO et al.:Herbicidal Injury by biphenyl Ether Herbicides 37

solution. The final ethanol concentration was 0.1%. Beakers were then exposed to light in a p-0 oxyf luorfen growth chamber at 25t under a photon flux density of 200uE m-2s-1 provided by sodium bifenox halide lamps at the plant surface. A- A chlomethoxynil Detection of Electrolyte Leakage and nitrofen Thoobarbituric Acid-Reacting Materials a chlornitrofe (TBARM) Content. -control Periodically, conductivity of the treatment solution was assayed with a conductivity meter (Model AOC-10, DENKI KAGAKU Co., LTD). TBARM, product of lipid perox- ide decomposition, in the treatment solution was assayed by a colorimetric reaction by a modified method of DERRICK et al 1). One- milliliter of the treatment solution was mixed with 1ml of thiobarbituric acid and 1ml of 10% trichloroacetic acid ; then the mixture was heated for 25 min in a boiling water bath. After cooling with an ice bath, the mixture was centrifuged at 1,300Xg for 10min. The amount of TBARM was estimated as malon- dialdehyde (MDA) by using its an extinction coefficient of 156 mM-1cm-1 at 535nm. Fig. 1. Effect of diphenyl ether herbicides (1 Results were expressed in nanomoles of pM) on electrolyte leakage from Lemna MDA. plant. Vertical bars represent standard Effect of Nitrogen Gas Substitution. errors. To determine the necessity of oxygen on herbicidal action, Lemna plants were trans- red in the plant within that 4-hr. The first ferred to a 100ml Erlenmeyer flask and visible effect of the herbicides in Lemna nitrogen gas was introduced to the flask at a plants was separation of the colonies into flow rate of 167ml/min. Conductivity of the individual fronds and this was observed in treatment solution was determined as above. conjunction with increasing conductivity. Bleaching of the plant was observed later. Results and Discussion Relative activity determined by the electro- Electrolyte leakage from Lemna was lyte leakage differed among the herbicides observed in all herbicide treatments at a and oxyfluorfen and bifenox exhibited the concentration as low as 1u M (Figure 1). In greatest activity among those tested. Thus, the experiment the herbicides were applied to all subsequent experiments were conducted the external bathing solution at time 0 and with these two herbicides and with the con- the plants were immediately exposed to light. centration of 1p M. This experiment also The leakage is thought to be due to a loss of indicated that action of the herbicides could selective permeability of cell membrane. It be detected by determination of conductivity was found after a lag period of 4-hr and this increase similarly to other experiments using may indicate that some phytotoxic event (s) leaf discs24,25);differing from leaf discs, how- perturbs the integrity of the membrane occur- ever, electrolyte leakage from intact plants

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can be determined in Lemna. Thus, the intact intensities on the activity of oxyfluorfen. The Lemna plant is considered a suitable material increase of conductivity was observed in light with which to investigate the relationship quantities higher than 200u E m-2s-1. By between physiological events caused by these decreasing the quantity of light, the effect of herbicides and their visible phytotoxic action. the chemical was delayed; however, once the Light was required for herbicidal activity leakage was initiated, its magnitudes were of oxyfluorfen in Lemna (Figure 2). It has nearly equal. At light intensities less than 100 been demonstrated that p-nitro diphenyl uE m-2s-1, no significant increase was detect- ether herbicides require light for their ed during 12-hr irradiation. These results activity6,21,24) In our experiment, when the suggest that an accumulation of photon to the plant was returned to darkness after 4-hr threshold level is necessary to initiate mem- light irradiation by 200uE m-2s-1, no brane disruption and that, once initiated, the increase of ion leakage was observed during membrane damage is continuously propagat- the following 8-hr incubation. However, 8-hr ed. irradiation caused a rapid increase of ion When nitrogen atmosphere was substituted leakage and this was not prevented by a for air, the effect of oxyfluorfen on ion leak- following dark treatment. age disappeared (Figure 4). The data indicat- Figure 3 shows the effect of various light ed that molecular oxygen is also required for

Light Dark ○-○12→Ohr ○-○800μE・m-2・sec-1

●-●8→4hr ●-●400

△-△4一>8hr △-△200 ▲-▲ 0→12hr ▲-▲ 100

□-□control □-□ 50 ■-■Control

Fig. 2. Effect of various lengths of light irradia- Fig. 3. Effect of various light intensities on tion on electrolyte leakage from Lemna electrolyte leakage from Lemna treated treated with 1pM oxyfluorfen. Vertical with 1 pM oxyfluorfen. Vertical bars bars represent standard errors. represent standard errors.

-38- H. MATSUMOTO et al.: Herbicidal Injury by biphenyl Ether Herbicides 39

oxyfluorfen (ail oxyfluorfen oxyfluorfen (N2 control (air) control A -A control(N2)

Fig. 5. MDA release from Lemna treated with 1 pM oxyfluorfen to external solution. Vertical bars represent standard errors.

Fig. 4. Effect of nitrogen gas substitution on of exogenously applied antioxidant or quen- electrolyte leakage from Lemna treated chers of active oxygen species on ion leakage with 1 p M oxyfluorfen. Vertical bars and TBARM production in Lemna was deter- represent standard errors. mined. Figure 6 shows protection against membrane disruption by a -tocopherol: lipid- the action of the herbicide. soluble antioxidant which scavenges lipid- Peroxidation of lipids in diphenyl ether soluble free radicals8)10), D-mannitol: hydrox- herbicide treated plants has been yl radical scavenger and hydroquinone: sing- reported13,17,23) To verify the peroxidative let oxygen scavenger7). Since electrolyte leak- disruption of the membrane in Lemna, pro- age from the plant was observed after a 4-hr duction of TBARM was determined. As herbicide treatment (Figure 1), these chemi- shown in Figure 5, MDA content in the exter- cals were applied to the bathing solution at nal solution was continuously increased with 4-hr after herbicide administration. The light exposure time. It is considered that chemicals showed partial protection from the electrolyte leakage correlates with perox- herbicidal toxicity, however, they functioned idative damage of the cell membrane. Oxy- most effectively against oxyfluorfen. They gen molecules are thought to play a role in showed greater protection when they were the peroxidative reaction of unsaturated applied simultaneously with the herbicides fatty acids leading to destruction of the (data not shown). Reversal of oxyfluorfen membrane9)19)22). action by a -tocopherol was also demonstrat- To obtain further information on the ed by MDA production (Figure 8). It should nature of oxidative reaction involved, effect be noted that a -tocopherol showed remark-

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ing of N, N-dimethyl p-nitroaniline11). There- Lulloxyfluorfen fore, the role of singlet oxygen in the her- bicidal action must be investigated. bifenox Influence of a photosynthetic electron transport on the phytotoxic processes was also studied. When the herbicide diuron, a photosynthetic electron transport inhibitor, was applied at 4-hr after oxyfluorfen or bifenox treatment, ion leakage from Lemna was partially protected (Figure 7). Eight hours after 1uM diuron application, 5.5% and 52.7%reduction of cellular leakage was determined for oxyfluorfen and bifenox, respectively. MDA formation by oxyfluorfen was also reduced by diuron (Figure 8). The results suggest that photosynthetic electron transport is involved in the action of the herbicides in this case. Contradictory results have been obtained on the involvement of photosynthetic electron transport with the herbicidal action of diphenyl ethers. DUKEet Fig. 6. Effect of exogenously applied a-toco- al.2,3), ENSMINGER and HESS5), ORR and pherol (100uM), D-mannitol (10mM) or HESS22) and MATSUNAKA21) demonstrated hydroquinone (1pM) on electrolyte leak- that photosynthesis was not required for the age from Lemna treated with 1 pM oxy- toxicity of diphenyl ether herbicides. In con- fluorfen (left) or bifenox (right). The trast, KUNERT and BOGER14) and LAMBERT chemicals were applied 4-hr after the et al.15,16) reported that oxyfluorfen action on herbicide treatment and the conductivity unicellular algae Scenedesmus and Bumil- was determined 12-hr after the herbicide treatment. leriopuswas completely protected by diuron. MATRINGEet al. demonstrated that the able protective activity to the plant after the pyrimidine derivative LS82-556 ((S) 3-N- destruction of the membrane had taken place. (methylbenzyl) carbamoyl-5-propionil-2, 6- From the data, participation of the active lutidine) which showed peroxidation of cell oxygen species in lipid peroxidation was membrance similar to diphenyl ethers suggested. Partial reversal of the herbicidal required photosynthesis in the mechanism of action with a -tocopherol and hydroxyl radi- action20). Our results also indicate that cal scavenger mannitol may implicate gener- photosynthetic electron flow is necessary for ation of free radical reaction in the plant4). full activity of the diphenyl ether herbicides However, our results also suggest involve- in Lemna plant. The mechanism of the ment of singlet oxygen in the oxidative reac- involvement is still unclear, however, reduc- tion. Peroxidation of lipids by singlet oxygen tion energy derived from photosynthesis and/ is known as a non-radical reaction and a- or oxygen molecule produced by photosystem tocopherol acts as a scavenger of the singlet II may participate with the reaction causing oxygen molecule7). HAWORTH and HESS peroxidation of unsaturated fatty acids of demonstrated that oxyfluorfen was a potent cell membranes. generator of singlet oxygen by using bleach-

-40- H.MATSUMOTO et al.: Herbicidal Injury by biphenyl Ether Herbicides 41

○-○oxyfluorfen ○-●bifenox

●-●oxyfluorfen-1-diuron (1uM) ●-●bifenox+diuron (1μM)

▲-▲Lcontrol(diuron) ▲-▲ control (diuron)

Fig. 7. Effect of diuron on electrolyte leakage from Lemna treated with 1uM oxyfluorfen or bifenox. Diuron was applied 4-hr after the herbicide treatment. Vertical bars represent standard errors.

Summary

The herbicidal mode of action of diphenyl ethers oxyfluorfen (2-chloro-l-(3-ethoxy-4- nitrophenoxy)-4-(trifluoromethyl) benzene) and bifenox (methyl-5- (2,4-dichlorophenoxy)- 2-nitrobenzoate) in Lemna pausicostata HEGELM. was investigated by monitoring an electrolyte leakage and a lipid peroxide de- composition product. Among the diphenyl ethers tested, these two herbicides caused intensive leakage in the plant after a lag period of 4-hr. Thiobarbituric acid-reacting material, product of lipid peroxide decom- position, content in a bathing solution was also increased. Light and oxygen were Fig. 8. Effect of exogenously applied chemicals required for their action and by decreasing on MDA release from Lemna treated the light quantity, the effect of oxyfluorfen with 1 pM oxyfluorfen. Diuron (1pM) was delayed. Exogenously applied antiox- or a-tocopherol (100pM) was simultane- idant cr-tocopherol, and the scavengers of ously with the herbicide and MDA con- tent was determined 24-hr after the trea- active oxygen species D-mannitol and hydro- tment. Light was continuously irradiated. quinone partially protected from herbicidal Vertical bars represent standard errors. injury. Protection was also partially afforded

-41- 42 Weed Research, Japan Vol. 35(1990) from phytotoxicity by the photosynthetic in higher plants. J. Agric. Food Chem. 33, electron transport inhibitor, diuron. Although 574-577. the mechanism of involvement of photosyn- 9) GILLHAM, D. J. and A. D. DODGE (1987) thesis in the action is still unclear, active Studies into the action of diphenyl ether oxygen species were suggested to be involved herbicides acifluorfen and oxyfluorfen. Part I. Activation by light and oxygen in leaf in the initiation of lipid peroxidation. tissue. Pestic. Sci. 19, 19-24. 10) GRAMS,G. W. and K. ESKINS (1972): Dye- Acknowledgment : The authors wish to sensitized photooxidation of tocopherols. thank Professor A. TAKIMOTO, Kyoto Correlation between singlet oxygen University, for his generous gift of Lemna reactivity and vitamin E activity. Biochem. pausicostata used in the study. 11, 606-608. 11) HAWORTH,P. and F. D. HESS (1988): The References generation of singlet oxygen (102) by the 1) DERRICK,P. M., A. H. COBBand K. E. PAL- nitrodiphenyl ether herbicide is independent LETT (1988): Ultrastructural effects of the of photosynthesis. Plant Physiol. 86, 672-676. diphenyl ether herbicide acifluorfen and the 12) KENYON, W. H., S. 0. DUKE and K. C. experimental herbicide M&B 39279. Pest. VAUGHN (1985): Sequence of effect of acif- Biochem. Physiol. 32, 153-163. luorfen on physiological and ultrastructural 2) DUKE, S. 0. and W. H. KENYON (1986) parameters in cucumber cotyledon discs. Photosynthesis is not involved in the mecha- Pest. Biochem. Physiol. 24, 240-250. nism of action of acifluorfen in cucumber 13) KUNERT, K. J. (1984): The diphenyl-ether (Cucumis sativus L.). Plant Physiol. 81,882- herbicide oxyfluorfen: A potent inducer of 888. Lipid peroxidation in higher plants. Z. Natur- 3) DUKE, S. O., K. C. VAUGHN and R. L. forsch. 39(C),476-481. MEEUSEN (1984) : Mitochondorial involve- 14) KUNERT, K. J. and P. BOGER (1981): The ment in the mode of action of acifluorfen. bleaching effect of the diphenyl ether oxy- Pest. Biochem. Physiol. 21,368-376. fluorfen. Weed Sci. 29, 169-173. 4) DUMELIN, E. E. and A. C. TAPPEL (1977) 15) LAMBERT, R. and P. BOGER (1984): Perox- Hydrocarbon gases produced during in vitro idative activity of oxyfluorfen with regard to peroxidation of polyunsaturated fatty acids carotenoids in Scenedesmus. J. Agric. Food and decomposition of pref ormed hydroper- Chem. 32. 523-526. oxides. Lipids 12, 894-900. 16) LAMBERT, R., G. SANDMANN and P. 5) ENSMINGER,M. P. and F. D. HESS (1985) BOGER : Mode of action of nitrodiphenyl Phtotosynthesis involvement in the mecha- ethers affecting pigments and membrane nism of action of diphenyl ether herbicides. integrity. In Pesticide Chemistry: Human Plant Physiol. 78, 46-50. Welfare and the Environment vol. 3, ed. by S. 6) FADAYOMI,0. and G. F. WARREN (1976): MATSUNAKA, D. H. HUTSON and S. D. The light requirement for herbicidal activity MURPHY, Pergamon Press, 97-102(1983). of diphenyl ethers. Weed sci. 24, 598-600. 17) LYDON,J. and 5.0. DUKE (1988): Porphyrin 7) FAHRENHOLTZ, S. R., F. H. DOLEIDEN,M. synthesis is required for photobleaching TROZZOLOand A. A. LAMOLA (1974): On activity of the p-nitrosubstituted diphenyl the quenching of singlet oxygen by a- ether herbicides. Pest. Biochem. Physiol. 31. tocopherol. Photochem. Photobiol. 20, 505- 74-83. 509. 18) MATRINGE, M. and R. SCALLA (1988) 8) FINCKH, B. F. and K. J. KUNERT (1985) Effect of acifluorfen-methyl on cucumber Vitamins C and E : An antioxidative system cotyledons: Porphyrin accumulation. Pest. against herbicide-induced lipid peroxidation Biochem. Physiol. 32, 164-172.

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19) MATRINGE, M. and R. SCALLA (1988) acifluorfen-methyl in excised cucumber Studies on the mode of action of acifluorfen- (Cucumis sativus L.) cotyledons. Plant methyl in nonchlorophyllous soybean cells. Physiol. 69, 502-597. Plant Physiol. 86, 619-622. 23) ORR, G. L. and F. D. HESS (1981) 20) MATRINGE, M., J. L. DUFOUR, J. LHER- Characterization of herbicidal injury by MINIER and R. SCALLA (1986): Characteri- acifluorfen-methyl in excised cucumber zation of the mode of action of the exper- (Cucumis sativus L.) cotyledons. Pest. Bio- imental herbicide LS82-556 ((S (-3-N- (methyl- chem. Physiol. 16, 171-178. benzyl) carbamoyl-5-propionyl-2, 6-lutidine). 24) VANSTONE, D. E. and E. H. STOBBE (1979) Pest. Biochem. Physiol. 26, 150-159. Light requirement of the diphenyl ether her- 21) MATSUNAKA, S. (1969): Acceptor of light bicide oxyfluorfen. Weed Sci. 27, 88-91. energy in photoactivation of diphenyl ether 25) VANSTONE, D. E. and E. H. STOBBE (1977) herbicides. J. Agric. Food Chem. 17, 171-175. Electrolytic conductivity-a rapid measure of 22) ORR, G. L. and F. D. HESS (1982): Mecha- herbicide injury. Weed Sci. 25, 352-354. nism of action of the diphenyl ether herbicide (Received July 18, 1989)

ジ フ ェニ ル エ ー テ ル 系 除 草 剤 oxyfluorfen お よび bifenox の

ア オ ウ キ ク サ に対 す る作 用 の 特 徴

松本 宏 ・小 島修 一 ・石塚 皓造

筑波大学応用生物化学系

摘 要

ジフェニルエーテル系除草剤は植物の内膜系の過酸化 をひきおこす ことが知 られているが, その作用機構は 完全には明らかにされていない。これ らの作用機構 を明 らかにする端緒 として, 本研究ではアオウキ クサを用 いて剤の作用発現の特徴 を検討 した。作用の指標 としてアオウキクサから外液への電解質の漏出と過酸化脂質 の分解物の定量 を行 った。まずいくつかの ジフェニルエーテル系剤の影響 を比較 した ところ, oxyfluorfen と bifenox の作用が強かった (Fig. 1)。 また薬剤の処理後4時 間を経てから, 膜機能の変化 によると思われ る電 解質の漏 出が測定された (Fig. 1)。作用の発現には4時 間をこえる光照射が必要であ り (Fig. 2), 照射強度 の強いほど作用は短時間で現れた(Fig.3)。 窒素ガス下では作用 は消失 した(Fig.4)。 マ ロンジアルデセ ド量 として求めた過酸化脂質の分解物含量 も薬剤処理によって増加 した (Fig. 5)。 しか しこれらの作用 は抗酸化 剤, 活性酸素消去剤, および光合成電子伝達阻害剤の添加 によって一部防御 された(Fig.6, 7, 8)。 これらの結 果から, これ らのジフェニルエーテル系除草剤 による過酸化には, 活性酸素および光合成電子伝達系の関与が 示唆 された。

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