Pyridazinone Herbicides Research Articles

Aquatic Risk Assessment of Herbicides in Freshwater Ecosystems of South Florida

Widespread, high-volume use and subsequent off-site transport of herbicides, specifically photosystem II inhibitors (PSII), on agricultural and noncultivated lands in south Florida has resulted in frequent detections in freshwater systems. In light of the current restoration efforts as part of the Comprehensive Environmental Restoration Plan (CERP), increased water flows containing detectable herbicide levels into the Everglades ecosystem and adjacent areas may have adverse consequences to the unique plant communities present in the region. The potential impact of individual herbicides to aquatic plant and algae species was examined using a probabilistic risk assessment approach. Risk was characterized for nine PSII herbicides (four triazines: ametryn, atrazine, simazine, and prometryn; two triazinones: hexazinone and metribuzin; two substituted ureas: diuron and linuron; and a uracil: bromacil) and a pyridazinone herbicide (norflurazon) by comparing the overlap of the 90th percentile surface water concentration (exposure benchmark) from the exposure distributions to the 10th percentile of effects (effects benchmark) determined from species sensitivity distributions of acute laboratory toxicity data. Overall, the risk of the individual herbicides was generally low. A Multiple Substance Potentially Affected Fraction (msPAF) risk approach also was utilized to examine the joint toxicity of the herbicide mixtures based on a concentration addition model. The risk associated with the herbicide mixture (mainly bromacil, diuron, and norflurazon) was determined to be high for Lee (site S79 on the Caloosahatchee River), Martin (site S80 on St. Lucie Canal), and St. Lucie (site S99 on C25 emptying into Indian River Lagoon) counties in south Florida. This study highlights the need to consider joint action of chemical mixtures as part of an ecological risk assessment.

Effect of Norflurazon on Resorcinolic Lipid Metabolism in Rye Seedlings

Norflurazon is a selective pyridazinone herbicide excessively employed in the control of many annual grasses and broad-leaved weeds. This chemical causes plant bleaching due to the inhibition of the carotenoid pigment biogenesis as well as induces irreparable changes to chloroplasts, which are considered the organelles where the biosynthesis of resorcinolic lipids takes place. Resorcinolic lipids, a group of phenolic compounds, constitute not only an essential part of the plant antifungal defense system, but also are an important component of the human cereal diet. The aim of this study was to investigate the effect of norflurazon on the biosynthesis of resorcinolic lipids in 5-day-old rye plants (Secale cereale L.) that were grown at three different temperatures under light or dark conditions. At all tested temperatures, norflurazon decreased the fresh biomass of light-grown rye seedlings and increased the weight of plants grown in darkness. Compared with respective controls, this herbicide caused an increase in total content of alkylresorcinols in both green and etiolated plants with the exception of dark-grown norflurazon-treated rye at 29 degrees C. The general level of saturated homologues was markedly decreased by norflurazon in all etiolated plants and in light-grown seedlings at 15 degrees C. Independent of thermal and light conditions, in all norflurazon-treated samples two alkylresorcinol derivatives predominated: 1,3-dihydroxy-5-n-heptadecylbenzene and 1,3-dihydroxy-5-n-nonadecylbenzene. Thus, our results suggest that norflurazon affected the metabolism of alkylresorcinols in rye seedlings and its action was dependent on external stimuli.

M-置換ジフェニルエーテル系化合物TOPEとKC6361の生理的作用特性

This study was conducted to examine physiological effects of m-substituted diphenylethers, TOPE (3-methylphenyl-4-nitrophenylether) and KC6361 [3-(N, N′-diethylcarbamoyl)-phenyl-4-nitrophenylether]. Both compounds are p-nitrodiphenylethers, but their A rings are substituted at meta position with methyl and diethylcarbamoyl group, respectively. TOPE was found to exert its herbicidal effect by causing swelling of meristematic regions of treated monocots and wrinkled expansion of cotyledon tissues of dicots, with and subsequent necrosis of the treated tissues. The mechanism of action of TOPE did not appear to be associated with the inhibition of the photosynthetic and mitochondrial electron transport, the porphyrin pathway, or the carotenoid biosynthesis. Rather, TOPE abnormally stimulated nucleic acid biosynthesis. These effects might result in ultimate death of the treated plants. KC6361, applied at preemergence at rates ranging from 0.5 to 1kg/ha, effectively controlled the weeds of amaranth (Amaranthus viridis), flatsedge (Cyperus iria), large crabgrass (Digitaria sanguinalis), and barnyardgrass (Echinochloa crus-galli), following the appearance of white foliage. However, the crops of soybean (Glycine max), rice (Oryza sativa), and corn (Zea mays) were not injured by KC6361 even at a rate of 2kg/ha, showing that KC6361 has a relatively good selectivity between weed and crop species. KC6361, like pyridazinone herbicides such as norflurazon [4-chloro-5-(methylamino)-2-(α, α, α-trifluoro-m-tolyl)-3 (2H)-pyridazinone], enhanced the growth of dwarf rice (O. sativa cv. Tanginbozu) in a certain concentration range. KC6361 increased phytoene and phytofluene levels in treated leaves, but decreased β-carotene level. These results imply that KC6361, similarly to norflurazon, has an inhibitory effect on phytoene and/or phytofluene desaturase in the carotenoid biosynthetic pathway. The effects of both TOPE and KC6361 occurred under light as well as under dark condition. Taken altogether, it can be concluded that physiological activities of the p-nitrodiphenylether compounds in which the A rings are substituted at meta position rather than at ortho and para position are totally distinct from those of the commercialized light-dependent p-nitrodiphenylether herbicides. Furthermore, their physiological activities vary with the substituted group at meta position of A ring.

S-1 Physiological Characteristics of m-substituted Diphenylethers, TOPE and KC6361

This study was conducted to examine physiological effects of m-substituted diphenylethers, TOPE (3-methylphenyl-4-nitrophenylether) and KC6361 [3-(N, N′-diethylcarbamoyl)-phenyl-4-nitrophenylether]. Both compounds are p-nitrodiphenylethers, but their A rings are substituted at meta position with methyl and diethylcarbamoyl group, respectively. TOPE was found to exert its herbicidal effect by causing swelling of meristematic regions of treated monocots and wrinkled expansion of cotyledon tissues of dicots, with and subsequent necrosis of the treated tissues. The mechanism of action of TOPE did not appear to be associated with the inhibition of the photosynthetic and mitochondrial electron transport, the porphyrin pathway, or the carotenoid biosynthesis. Rather, TOPE abnormally stimulated nucleic acid biosynthesis. These effects might result in ultimate death of the treated plants. KC6361, applied at preemergence at rates ranging from 0.5 to 1kg/ha, effectively controlled the weeds of amaranth (Amaranthus viridis), flatsedge (Cyperus iria), large crabgrass (Digitaria sanguinalis), and barnyardgrass (Echinochloa crus-galli), following the appearance of white foliage. However, the crops of soybean (Glycine max), rice (Oryza sativa), and corn (Zea mays) were not injured by KC6361 even at a rate of 2kg/ha, showing that KC6361 has a relatively good selectivity between weed and crop species. KC6361, like pyridazinone herbicides such as norflurazon [4-chloro-5-(methylamino)-2-(α, α, α-trifluoro-m-tolyl)-3 (2H)-pyridazinone], enhanced the growth of dwarf rice (O. sativa cv. Tanginbozu) in a certain concentration range. KC6361 increased phytoene and phytofluene levels in treated leaves, but decreased β-carotene level. These results imply that KC6361, similarly to norflurazon, has an inhibitory effect on phytoene and/or phytofluene desaturase in the carotenoid biosynthetic pathway. The effects of both TOPE and KC6361 occurred under light as well as under dark condition. Taken altogether, it can be concluded that physiological activities of the p-nitrodiphenylether compounds in which the A rings are substituted at meta position rather than at ortho and para position are totally distinct from those of the commercialized light-dependent p-nitrodiphenylether herbicides. Furthermore, their physiological activities vary with the substituted group at meta position of A ring.

Octadecatetraenoate synthesis in the unicellular alga Isochrysisgalbana: Studies with intact and broken chloroplasts

(1) Monogalactosyldiacylglycerol (MGD) is the major lipid component of Isochrysis galbana. In cells incubated for 3 h with [1− 14C]]acetate or [1− 14C]oleate, MGD contained 35.9% and 52.8%, respectively, of the label incorporated into cellular lipids. (2) 18:4 amounted to 50–60% of the total FA of MGD. Separation of MGD species of cells grown with [1− 14C]oleate on AgNO 3 impregnated plates revealed 20 distinct spots. The slowest spot was identified as dioctadecatetraenoyl MGD. Fast moving species were enriched with 18:1. (3) In cells incubated for 3 h with [1− 14C]oleate, approx. 60% of the radioactivity was associated with 18:1. Subsequent chase resulted in a gradual shift of label and after 48 h [ 14 C]18:1 declined to 10% and [ 14 C]18:4 reac This shift was also reflected in the labeling pattern of the MGD-species. Dioctadecatetraenoyl-MGD became labelled only after 24 h. (4) Addition of the substituted pyridazinone herbicide (SAN 9785) during the chase period inhibited [ 14 C]18:4 formation; [ 14 C]18:2 and [ 14 C]18:3 accumulated instead. (5) Isolated chloroplast readily incorporated [ 14C]oleate into MGD and PA. Considerable amounts of [ 14 C]18:1 were desaturated to 18:2 and 18:3. Only very small amounts of 18:4 were formed. O 2 was required for desaturation. Cofactor requirement could not be shown. (6) Membranes isolated from broken chloroplasts retained the ability to incorporate [ 14C]oleate into MGD and PA and desaturate 18:1 to 18:2 and 18:3.

Inhibition of the cation-induced reversible changes in excitation energy distribution in thylakoids of BASF 13.338-grown plants

Measurements of relative quantum yields of Photosystem II and Photosystem I partial reactions and room-temperature chlorophyll a fluorescence of thylakoids in high- and low-salt media showed that the cation-induced changes in the excitation energy distribution were inhibited in the thylakoids isolated from pea plants grown in the presence of sublethal concentration of the pyridazinone herbicide BASF 13.338. Simultaneous measurement of Photosystem II and Photosystem I fluorescence emission kinetics at 77 K showed that the ability of cations to regulate excitation energy spillover from Photosystem II to Photosystem I was inhibited in thylakoids of the BASF-grown plants. Cation regulation of the absorption cross-section of the photosystems was not affected. Electron microscope data revealed that the proportion of stroma membranes relative to grana membranes was markedly less in the thylakoids of the BASF-grown plants. Furthermore, when the thylakoids were resuspended in low-salt medium, no unstacking of the grana was detected. The observed inhibition of cation-induced spillover change was presumably due to loss of ability of these photosynthetic membranes to undergo unstacking in low-salt medium.

Differential Effects of the Substituted Pyridazinone Herbicide Sandoz 9785 on Lipid Composition and Biosynthesis in Photosynthetic and Non-Photosynthetic Marine Microalgae

The effect of the substituted pyridazinone, 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (Sandoz 9785), on fatty acid synthesis in two photosynthetic species (Chroomonas salina and Nannochloropsis oculata) and one non-photosynthetic species (Crypthecodiniun cohnii) of marine microalgae were examined. Effects were more obvious in C. salina than in C. cohnii or N. oculata. In C. cohnii the relative distribution amongst polyunsaturated fatty acids (PUFA) of radioactivity incorporated from 14C-acetate was not influenced by the herbicide to any great extent and no major changes in the fatty acid composition of lipid fractions were observed. In C. salina, Sandoz 9785 reduced the proportions of radioactivity recovered in 20: 5 (n−3) and 22: 6 (n−3) of the phospholipid fraction. The distribution pattern of radioactivity in the fatty acids of monogalactosyldiacylglycerol (MGDG) in this species was not greatly affected by the herbicide whereas its presence significantly reduced the proportions recovered in 18: 4 and 20: 1 in digalactosyldiacylglycerol (DGDG). The level of 20: 5 (n−3) in DGDG of C. salina was increased from 4.0 to 19.8% by growing the algae in the presence of Sandoz 9785. The only notable effect of the herbicide on the synthesis of PUFA in N. oculata was a reduction from 18.3% to 11.3% of the proportion of radioactivity recovered in 20: 5 in phospholipids. The herbicide had no effect on the distribution of radioactivity in PUFA of galactolipids or on the fatty acid composition of lipid fractions. The results are discussed in relation to the potential role of galactolipids and phospholipids as substrates for desaturations involved in the formation of long chain PUFA in marine microalgae.

Differential Effects of the Substituted Pyridazinone Herbicide Sandoz 9785 on Lipid Composition and Biosynthesis in Photosynthetic and Non-Photosynthetic Marine Microalgae

A comparison was made of the effects of the substituted pyridazinone herbicide, 4-chloro-5-(dimethylamino)-2-phenyl-3(2H) pyridazinone (Sandoz 9785; BASF 13–338) on the growth rate and lipid class synthesis of two species of photosynthetic (Chroomanas salina and Nannochloropsis oculata), and one species of non-photosynthetic (Crypthecodinium cohnil), marine microalgae. The growth rate, but not the total lipid content, of all three species was reduced by the herbicide. The percentage distribution of radioactivity incorporated by C. cohnii into lipid classes generally resembled the relative abundance of the classes and the presence of Sandoz 9785 resulted in only small changes in lipid class proportions or in the distribution of radioactivity. In C. salina, the proportion of radioactivity located in monogalactosyldiacylglycerol (MGDG) increased from 9.2% to 20.3%, and that in digalactosyldiacylglycerol (DGDG) from 6.3% to 12.7%, in the presence of the herbicide. In N. oculata the proportion of radioactivity recovered in DGDG, but not MGDG, was increased significantly by the presence of Sandoz 9785. The results suggest that Sandoz 9785 alters galactolipid metabolism in photosynthetic microalgae but has no great effect on lipid synthesis in non-photosynthetic species.

The Development of Daffodil Chromoplasts in the Presence of Herbicides SAN 9789 and SAN 9785

The effects of two pyridazinone herbicides (SAN 9789 and SAN 9785) were studied on the fine structure and carotenoid composition during the transformation of chloroplasts into chromoplasts in daffodil (Narcissus poëticus L.) flowers. SAN 9789 caused the absence of big characteristic carotene crystals and the appearance of numerous nonosmiophilic plastoglobuli in chromoplasts. The accumulation of all carotenoids was drastically reduced and the content of carotenes was 40-fold lower than in the control. SAN 9785 caused no gross abnormalities in ultrastructure of chromoplasts. The synthesis of carotenes was partially reduced to about 75%. In spite of this the accumulation of xanthophylls is two times higher than in the control. The interaction between chromoplast ultrastructure and carotenoid composition is discussed.

Inhibition of nitrate and nitrite reductase induction in wheat by sandoz 9785

The effect of a non-bleaching pyridazinone herbicide [4-chloro-5(dimethylamino)-2-phenyl-3(2H) pyridazinone; SANDOZ 9785; BASF 13-338] on the induction of nitrate reductase (NR) and nitrite reductase (NIR) has been studied in leaf segments and seedlings of wheat. The induction of both NR and NIR was inhibited, albeit differentially, in detached green leaves as well as intact green seedlings grown in the presence of SAN 9785 and nitrate. The inhibition of NR and NIR induction due to SAN 9785 treatment occurred in the absence of photobleaching (detached leaves) or in the presence of marginal photobleaching (intact seedlings). Seedlings grown in the presence of SAN 9785 without nitrate showed a reduced capacity for induction of NR and NIR. The NADH-DCPIP reductase activity was not inhibited in SAN 9785-grown seedlings. The cause for inhibition of NR activity was neither inactivation of NR in vivo or in vitro, nor a reduction in the level of soluble protein. Both DCMU and SAN 9785 inhibited 14CO 2 fixation and induction of NR and NIR under similar conditions of treatment.

Differential Effects of a Substituted Pyridazinone, BASF 13-338, on Pathways of Monogalactosyldiacylglycerol Synthesis in Arabidopsis

We have examined the effects of the substituted pyridazinone herbicide, 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)pyridazinone (BASF 13-338, Sandoz 9785), on the desaturation of linoleic acid (18:2) on different molecular species of monogalactosyldiacylglycerol (MGDG) and phosphatidylcholine (PC) in leaf tissue of Arabidopsis thaliana (L.) Heynh. Specific changes in lipid composition allowed identification of different substrates for desaturation of 18:2 to linolenic acid (18:3). 18:2/16:2 MGDG was desaturated in the chloroplast to form 18:3/16:3 MGDG. Levels of 18:3/16:3 MGDG were reduced by treatment with BASF 13-338, suggesting that both the formation of 18:3 at the sn-1 position, and the formation of 16:3 at the sn-2 position of 18:2/16:2 MGDG were inhibited by this compound. Kinetic studies using exogenously incorporated [(14)C] 18:1 indicated that 18:2/18:3 MGDG originated from an 18:2/18:3 diglyceride precursor derived from PC. The formation of 18:3 at the sn-1 position of 18:2/18:3 MGDG was also inhibited by BASF 13-338. In contrast the desaturation of 18:2 proposed to occur at the sn-2 position of PC outside the chloroplast, was not affected.

Effect of pyridazinone herbicides on lipid metabolism in groundnut ( Arachis hypogaea) leaves

The effect of five substituted pyridazinones (pyrazon, San 133-410H, San 9774, norflurazon, and San 6706) on lipid metabolism in groundnut ( Arachis hypogaea) leaves was investigated under nonphotosynthetic conditions. In experiments with leaf disks, the uptake of [1- 14C]acetate, [ 32P]orthophosphate, and [ 35S]sulfate was significantly inhibited by these herbicides and the magnitude of inhibition varied, depending on the substituents. When the incorporation of these precursors into lipids was measured and expressed as percentage of total uptake, no effect was observed in the case of [1- 14C]acetate but there was significant inhibition in the incorporation of the other two precursors, suggesting that pyridazinones interfere with the metabolism of the phospholipids and the sulfolipid. None of these compounds affected the uptake of [ methyl- 14C]choline but all inhibited its incorporation into phosphatidylcholine indicating that phosphatidylcholine metabolism is vulnerable to pyridazinones. The fatty acid synthetase of isolated chloroplasts assayed in the absence of light was inhibited 20–50% by the pyridazinones at 0.1–0.5 m M concentrations. San 9774 showed the most potent inhibition. In addition, the pyridazinone herbicides significantly inhibited sn-glycerol-3-phosphate acyltransferase(s) in both chloroplast and microsomal fractions but showed no effect on phosphatidic acid phosphatase. The magnitude of inhibition of fatty acid synthetase and acyltransferase(s) is related to the nature of the substituent groups on the herbicide. Trifluorophenyl substitution at position 2 or amino substitution at position 5 of the pyridazinone molecule caused the maximum inhibitory effect.

Sites of action of pyridazinone herbicides (SANDOZ 9785 and 9789) onChlorella protothecoides

WhenChlorella protothecoides cells were grown in the presence of 30, 100 and 300 μM of SANDOZ 9785 and 9789, there was a significant decrease in cell number and packed cell volume as compared to control. SANDOZ 9789 seems to be a stronger inhibitor of growth than SANDOZ 9785. The immediate effect of pyridazinone herbicides on photosynthesis showed that SANDOZ 9789 is a weaker inhibitor than SANDOZ 9785. There was no direct correlation between the inhibition of growth and photosynthesis by the herbicide treatment suggesting that the inhibition of photosynthesis is not the primary cause of growth inhibition. Experiments on growth, carried out with non-photosynthetic and non green cells also support this suggestion. The observed increase in cell size when the cells grown with SANDOZ 9785 suggests that cell division was inhibited by SANDOZ 9785.

Immediate effects of pyridazinone herbicides on photosynthetic electron transport in algal systems

Pyridazinone herbicides, SANDOZ 9785 (4-chloro-5-dimethylamino2-phenyl-3-(2H) pyridazinone), SANDOZ 9789 (4-chloro-5 (methylamino)-2-(α, α, α-trifluoro-m-tolyl-3-(2H) pyridazinone) and SANDOZ 6706 (4-chloro-5-(methylamino)-2-(α, α, α-trifluoro-m-tolyl-3-(2H) pyridazinone) inhibited photosystem II electron transport inChlorella protothecoides, when the herbicides were added to the assay medium. The inhibitory eficiency varied with the algal species and the nature of substitution of pyridazinones. Using 3 algal systemsviz., Chlorella, Scenedesmus andAnacystis, the I50 value of for the inhibition of photosynthesis of 3 substituted pyridazinones (SANDOZ 9785, SANDOZ 6706 and SANDOZ 9789) were determined. SANDOZ 9789 was found to be the weakest inhibitor of photosystem II electron transport (H2O→ benzoquinone) as compared to SANDOZ 9785 and SANDOZ 6706. In general, the order of inhibition could be given as SANDOZ 6706 >- SANDOZ 9785 > SANDOZ 9789. The I50 value of photosynthetic particles obtained fromChlorella cells was similar to that of whole cells, suggesting that the cell wall ofChlorella did not act as a barrier for the herbicide action. Studies on the light intensity dependence of SANDOZ 9785 inhibition of electron transport (H2O→ benzoquinone) showed that the light-dependent portion of the curve was more sensitive than the light independent portion of the curve. It is suggested that the site of action was on the reducing side of photosystem II.

Effects of Pyridazinone Herbicides during Chloroplast Development in Detached Barley Leaves III. Effects of SAN 6706 on Photosynthetic Activity and Chlorophyll-Protein Complexes

The photosynthetic activities of barley leaves in vivo and of thylakoids, isolated from the leaves, in vitro were studied during greening in the presence or absence of 2 × 10-4 м SAN 6706. The degree of chlorophyll bleaching increased from 32% at 24 h of greening to 60% at the final stage. Treated leaves were unable to carry out 14CO2-fixation, their fluorescence induction properties showed very limited, if any, photosystem II-activity, and the thylakoids isolated from the leaves were also inactive in mediating ferricyanide Hill reaction. The leaves, however, exhibited lightinduced quenching of fluorescence revealed by slow fluorescence induction measurements; and the thylakoids were active in mediating photosystem I-specific in vitro Mehler reaction. Thylakoid membranes of the chloroplasts isolated from treated leaves contained CP 1 and LHCP3 bands as revealed by polyacrylamide gel electrophoresis. From these results it is concluded that i) greening in the presence of SAN 6706 leads to the formation of inactive photosystem -II units; that ii) photosystem-I per se is active in vitro, and can, possibly, mediate cyclic electron transport in vivo coupled to the formation of ATP; and that iii) the presence of xanthophyll pigments is required for the assembly of the light-harvesting complex. The experiments were repeated with the application of SAN 9789, another pyridazinone compound, and yielded practically the same results as those obtained with SAN 6706. For practical reasons, only the results obtained with SAN 6706 are demonstrated throughout this communication.

Photosynthetic Properties of Green Barley Leaves after Treatment with Pyridazinone Herbicides—Comparison with the Effects of Diuron

Photosynthetic characteristics of detached green barley leaves after 72 h of treatment with 0·2 mol m−3 of the pyridazinone herbicides SAN 6706, SAN 9785 and SAN 9789 were studied. For comparison, the effects of 0·01 mol m−3 diuron were also investigated. Pyridazinone herbicides caused only a slight reduction of the total carotene content of the barley leaves. The total chlorophyll content, as well as the linolenic to linoleic acid ratio of chloroplast glycerolipids, however, remained unchanged. Diuron treatment caused total inhibition of electron transport, as revealed by fast fluorescence induction of leaves and the Hill reaction activity of chloroplasts. The 14CO2-nxation by the leaves and the light-induced fluorescence quenching were also completely inhibited in vivo by diuron. Pyridazinone herbicides left 20–40% of the 14CO2-fixation found in the control, in spite of the fact that their fast fluorescence induction tracings showed inhibition in the electron transport. Chloroplasts isolated from the leaves treated with pyridazinones were found to be highly active in mediating the ferricyanide-dependent Hill reaction. In order to test the ability of pyridazinones to inhibit photosynthetic electron transport in vivo, their ‘prompt’ effect on fluorescence was also investigated. It is concluded that pyridazinone herbicides can readily and rapidly enter the chloroplasts and inhibit the photosynthetic electron transport in vivo. The differences between the long-term effects of pyridazinones and those of diuron suggest differences in the inhibitory effectiveness on the various photosynthetic parameters between the two herbicide groups. It is suggested that pyridazinones can leave the chloroplasts during isolation owing to the loose binding onto the thylakoid membranes.

Uptake and metabolism of the phenylpyridazinone herbicide metflurazon during the bleaching and regeneration process of the green alga, Chlorella fusca

The uptake and metabolism of the pyridazinone herbicide metflurazon by the green alga Chlorella fusca was studied using a new and sensitive technique which is based on reverse-phase high-performance liquid chromatography. During the metflurazon-induced bleaching process, the agent is rapidly taken up and metabolized to the monomethyl derivative, norflurazon, by N-demethylation. Norflurazon accumulates during the treatment in the culture medium because Chlorella cells demethylate norflurazon to the less-phytotoxic SAN 9774 only to a very minor extent. It is shown that only 10% of the initial 1 μ M metflurazon is sufficient to induce the bleaching process. This amount is much too small to directly inhibit the photosynthetic electron transport, so that a contribution of this site of action to the bleaching process is not likely. Treated Chlorella cells are able to regenerate to photosynthetically active cells. This phenomenon is based on the inability of the regenerating algae to further take up and demethylate metflurazon. Fully regenerated cells are resistant to metflurazon because a treatment with 1 μ M metflurazon neither affects growth nor inhibits photosynthetic activity. This resistance results from an altered permeability of the cell membrane with respect to phenylpyridazinones and an inhibited N-demethylation of metflurazon. These results clearly show that metflurazon itself is nonphytotoxic, and has to be demethylated to norflurazon to exert its bleaching activity.

Comparison of the Action of Bleaching Herbicides

Among chlorosis-inducing herbicides that interfere with carotenoid synthesis two groups of different potency can be discriminated (group 1: aminotriazole amd haloxidine; group 2 with more extensive photodestructions: pyridazinone herbicides and difunon). After application of herbicides of group 2 colored carotenoids were completely absent and preexisting chlorophyll was degraded by photochemical reactions requiring high light intensity and O2, that occurred also at 0°C. In treatments with group 1 herbicides direct photodegradation of chlorophyll was not sufficient to generate the chlorosis. Light-induced interference with constituents of the chloroplast protein synthesis apparatus being more sensitive to photooxidative damage than chlorophyll, appeared to indirectly mediate the chlorosis. In the absence of chloroplast protein synthesis further chlorophyll accumulation is prevented. Photodegradation of chlorophyll in the presence of group 2 herbicides involved the participation of O2 - radicals and was accompanied by lipid peroxidation. In all herbicide treatments the catalase activity of the leaves was very low. Only in the presence of group 2 herbicides chloroplast enzymes of cytoplasmic origin (e.g. NADP-glyceraldehyde-3-phosphate dehydrogenase) were also inactivated. Rapid inactivation of catalase as well as of NADP-glyceraldehyde-3-phosphate dehydrogenase was induced by exposure of dim-light-grown herbicide-treated leaves to bright light, also at 0°C. In treatments with herbicides of group 2 also other peroxisomal enzymes (e.g. glycolate oxidate, hydroxy-pyruvate reductase) were affected. The elimination of these peroxisomal enzymes also appeared to depend on photooxidative processes of the chloroplast.

Effects of Pyridazinone Herbicides during Chloroplast Development in Detached Barley Leaves I. Effects on Pigment Accumulation and Fluorescence Properties

The effects of three differently substituted pyridazinone herbicides on the accumulation of photosynthetic pigments and on the fluorescence properties of greening barley leaves were studied. The two trifluoro-methyl derivatives, SAN 6706 and 9789 caused inhibition in the accumulation of carotenes only after the first 24 h of greening. By the 48th hour of greening no detectable amounts of carotenes were present in the treated leaves. Considerable amounts of xanthophylls however remained present, and no bleaching of the leaves was observed. It is suggested that SAN 6706 and 9789 prevent the formation of the enzyme(s) catalyzing the desaturation of phytoene and phytofluene. Fluorescence spectra indicate that the organization of pigment forms altered seriously in the treated leaves. This experimental system may be useful to study the action of pyridazinone herbicides without causing complete photobleaching of the photosynthetic apparatus. SAN 9785 had only minor effects on pigment accumulation and also on the organization of pigment forms. The possible explanations of the effects observed are dis­cussed.

Effects of Pyridazinone Herbicides during Chloroplast Development in Detached Barley Leaves II. Effects on Lipid Content, Fatty Acid Composition and Ultrastructure of Chloroplasts

The effects of three differently substituted pyridazinone herbicides were studied on the lipid content, fatty acid composition of glycerolipids, fatty acid synthesizing capacity and ultrastruc­ture of chloroplasts after 72 h of greening of etiolated barley leaves. SAN 9789 caused a nearly threefold increase in the total fatty acid content of chloroplasts. and this increase was accom­panied by an increase in the DGDG- and phospholipid-content as well. SAN 6706 and 9785 caused reduction in the linolenic to linoleic acid ratio in nearly all lipid classes, only PC was unaffected. Both herbicides reduced the saturated to unsaturated ratio in the PG, SAN 9789 had no effect on the fatty acid composition of the PG. Fatty acid synthesis was inhibited in the SAN 6706- and 9789-treated leaves as monitored by incorporation of labelled acetate, but all of the three pyridazinones stimulated the incorporation of labelled galactose into chloroplast galactolipids. SAN 6706 and 9789 also caused abnormal organization of chloroplast lamellar system.