Characterisation of bound residues of [3H]triforine in barley grain grown in the field

Barley was grown in an experimental field and at growth stage J (during the stem extension stage when the second node of the stem was formed and the next-to-last leaf was just visible), the aerial part of the plants was sprayed with an aqueous emulsion of a mixture of triforine and [³H]triforine (uniformly labelled in the piperazine ring) at the normal rate of 250 g triforine ha^?1. The barley was harvested when ripe, and the grain was analysed separately. Extraction of the grain with methanol left methanol-insoluble solids containing an amount of radioactivity (the bound residue) which represented 75% of the total radioactivity incorporated into the grain. Methanol acidified with hydrochloric acid extracted a further 7% of the triforine-derived bound residues in the form of radioactive iminodiacetic acid (1.1%), glycine (3.3 %), serine (0.9%), ethanolamine (0.2%) and unidentified compounds (1.5 %); in the grain, these compounds or their precursors had thus been complexed to grain constituents. Aqueous 0.03M sodium hydroxide extracted a further 27% of the total tritium which had been incorporated by means of α chemical bonds into the protein fraction; acid hydrolysis of the proteins yielded radioactive glycine (9.2%), serine (3.9%) and unidentified compounds (13.9%) which could have been a mixture of a large number of other amino-acids. The plant solids (which contained 41% of the total tritium) left after the alkaline aqueous extractions, were processed and separated into tritiated cellulose (4%) and starch (37%) fractions. The starch was hydrolysed and the resulting glucose was converted into the osazone (34%). After being recrystallised several times, the osazone contained a constant specific radioactivity, indicating that [³H]glucose was present. This glucose may be considered as having a carbon skeleton mainly originating biochemically from some metabolites of [³H]triforine; this tritiated glucose may also be considered as indirect evidence of the biochemical oxidation, by transamination, of the metabolites of [³H]triforine; in barley grain, the tritiated glucose (or at least a part of it) was anabolised into proteins. No piperazine was observed in the bound residues in the grain.     

Characterisation of bound residues of [3H]triforine in the straw of barley grown in the field

Barley was grown in an experimental field and, at the growth stage J (during the stem extension stage when the second node of the stem was formed and the next-to-last leaf just visible), the aerial part of the plants was sprayed with an aqueous emulsion of a mixture of triforine and [³H]triforine (uniformly labelled in the piperazine ring) at the recommended rate of 250 g triforine ha^?1. Barley was harvested when ripe, and the straw was analysed separately. Extraction of the straw with methanol left methanol-insoluble solids containing an amount of radioactivity (the bound residue) which represented 88 % of the tritium (as with all the subsequently quoted percentages, relative to the total tritium incorporated into the straw). Methanol acidified with hydrochloric acid extracted a further 8% of the triforine-derived bound residues as radioactive iminodiacetic acid (0.4%). glycine (3.2%), serine (2.0%), ethanolamine (0.2%) and unidentified compounds (2.2%); a neutral detergent solution extracted a further 3% of the radioactive triforine-derived bound residues as unidentified compounds; these radioactive compounds, which were dissolved by the acidified methanol and by the neutral detergent solution, were thus complexed in the straw to straw constituents. An acid detergent solution extracted a further 58% of the total tritium, which was incorporated by means of σ chemical bonds into the hemicelluloses fraction. Acid hydrolysis of the hemicelluloses yielded a mixture of mono-saccharides; these were converted into a mixture of osazones (50%), recrystallised several times, and had a constant specific radioactivity, indicating that the mono-saccharides were tritiated. The plant solids (which contained 19% of the total tritium), left by the acid-detergent extraction, were processed and separated into the tritium-containing cellulose (13%) and lignin (6%) fractions. No piperazine was observed in the bound residue of straw. Biochemical pathways are suggested for the radioactive metabolites of [³H]triforine.     

Metabolism of [2,5-14c]piperazine in barley plants

The shoots of barley plants root-treated with [2,5-¹⁴C]piperazine were analysed 30 days after treatment. Methanol extraction left a solid residue which contained 31.9% of ¹⁴C (all percentages refer to the total of ¹⁴C incorporated into the shoots); further extraction with acidified methanol and dimethyl sulphoxide dissolved respectively 3.2% and 5.8% of ¹⁴C. The initial methanol extract contained radioactive piperazine (16.8%), iminodiacetic acid (8.6%), glycine (15.4%), oxalic acid (7.2%), and un-identified compounds (20.1%). In barley, piperazine is the product of the most advanced metabolism so far identified of the fungicide triforine, 1,4-bis(2,2,2-trichloro-1-formamidoethyl)piperazine; the results obtained here show that piperazine is certainly not the end-product of the metabolism of triforine in barley.     

The nature of bound residues derived from triforine in barley plants

The shoots of barley plants root-treated with [³H]-triforine fungicide (uniformly labelled in the piperazine ring) were analysed 30 days after treatment. Methanol extraction left a solid plant residue which contained 33% of the total ³H which had been incorporated into the shoots. Methanol acidified with hydrochloric acid extracted a further 18% of the triforine-derived bound residues as N-[2,2,2-trichloro-1-(piperazin-1-yl)ethyl]formamide (8%), and piperazine (10%). In the plants, these compounds had thus been complexed to plant constituents. Hot dimethyl sulphoxide (DMSO) extracted a further 13% of the total ³H, leaving a solid residue (mainly cellulose) which contained 2% of ³H, perhaps incorporated into the cellulose. Evaporation of the solvent from the DMSO extract gave a solid, the radioactivity of which (13%) could not be extracted by methanol. A part (7%) of this radioactivity could be released by successive hydrolysis with amyloglucosidase and β-glucosidase, which generated a complex mixture of polar and water soluble unknown radioactive compounds not including piperazine. These latter compounds would be the products of extensive metabolism of triforine (and its metabolite piperazine) bound to, or incorporated into starch. Most (11%) of the radioactivity of this solid could be released by hydrochloric acid hydrolysis, which also generated a complex mixture of polar and water soluble unknown radioactive compounds not including piperazine; a part (4%) of them could have been associated with lignin in the plant.     

Transport of the systemic fungicide cela W 524 (triforine) in barley plants. II. Uptake and metabolism

After soil drench application of triforine (5 mg) to 35 barley plants in pots, a maximum of 7.5 μg a.i./g shoots (fresh weight) was found on the second day. The biological (effective) half-life was 9 to 10 days. Doses of 50 mg resulted in impaired absorption rather than increased foliar concentration. On day 8 piperazine comprised 20% of the radioactivity of the four metabolites found in the shoots. An additional metabolite appears solely in the soil.     

Metabolism of the fungicide triforine in barley plants

The metabolic fate of [³H]-triforine in barley plants has been studied. 15 and 30 days after treatment, unchanged triforine amounted to 57.5 and 43.2% respectively, and of three soluble metabolites detected, piperazine (0.3 and 4.0%) and N-[2, 2,2-trichloro-1-(piperazin-1-yl)ethyl] formamide (12.9 and 8.4%), the latter for the first time as a metabolite in plants, were identified using t.1.c. with four solvent systems. The solid residue contained 23.1 and 38.2% of bound label after 15 and 30 days respectively.     

Zum Metabolismus von Phenylharnstoff-Herbiziden VII. Metabolismusaufklärung und Bilanzierung des Verbleibs von Buturon-14C nach Anwendung bei Winterweizen.

Metabolism of Phenylurea Herbicides. VII. Metabolism Studies and Balance of the Fate of Buturon-¹⁴C after Application to Wheat. Radioactivity counts at harvest showed that 89.1% of the label was recoverable. Of this 50.1% was detected in the soil, 12.6% in the straw, 3.7% in the roots and 1.3% in the grain, while 16.2% was converted to radioactive CO₂. Only about 50% of the radioactivity in the plant material was extractable. This part of the activity consisted mainly of strongly polar metabolites, while the four less polar buturon metabolites accounted for only up to 12% each.     

Uptake,distribution and metabolic fate of 3H-triforine in plants .I. Short-term experiments

Most of the label, present in the roots of bean, tomato and barley seedlings after short-term root-treatment with ³H-triforine, appeared only weakly adsorbed on to the root tissue and was desorbed after transplanting in fungicide-free soil. Label taken up accumulated almost exclusively in the leaves present at the time of treatment; all leaves expanding after termination of the treatment remained virtually devoid of radioactivity. In “adult” plants label was usually present in a concentration gradient from roots to youngest leaves; in tomato plants, however, distribution of label was rather irregular. Time-course studies with bean and barley plants revealed that the aerial parts were gradually supplied with label, from old to youngest leaves, the maximum relative accumulation of radioactivity slowly moving acropetally. Under the experimental conditions chosen, triforine was converted nearly quantitatively to one metabolite, which almost certainly is different from any known non-enzymic breakdown product of the fungicide.     

The analysis of crops and soils for the triazine herbicide cyanazine and some of its degradation products. I. Development of method

Radiochemical techniques have been used to develop efficient procedures for the extraction of residues of cyanazine herbicide [‘BLADEX’,

1 BLADEX and FORTROL are Shell registered Trade Marks.

^a ‘FORTROL’,^a 2-chloro-4-(1-cyano-1-methylethylamino)-6-ethylamino-1,3,5-triazine] and its metabolites 2-chloro-4-(1-carbamoyl-1-methylethylamino)-6-ethylamino-1,3,5-triazine ( II ), 2-chloro-4-(1-cyano-1-methylethylamino)-6-amino-1,3,5-triazine ( V ) and 2-chloro-4-(1-carbamoyl-1-methylethylamino)-6-amino-1,3,5-triazine ( VI ) from crops and soils. Partition and column chromatographic techniques have been established for the purification of the extracts. The full analytical procedure is described and the final determination of all four compounds is by g.l.c. with electron capture detection with blank values for field samples generally 0.02 part/million and with good recoveries.     

Metribuzin metabolism as the basis for tolerance in barley (Hordeum vulgare L.)*

Metabolism of ¹⁴C-5 (ring)-metribuzin was studied in Steptoe (tolerant) and Morex (susceptible) barley (Hordeum vulgare L.) cultivars, 1, 4, and 8 days following a single application to roots. Both cultivars contained similar ether-soluble metribuzin metabolites and five water-soluble metabolites. Water-soluble compounds increased from 12 to 53% of the total ¹⁴C recovered for Steptoe and 5–17% for Morex between 1 day and 8 days, respectively, whereas the percentage of ether-soluble metabolites decreased. Ninhydrin reacting compounds were the major water-soluble metabolites in the leaf blades 8 days after treatment. On a d.p.m. mg^?1 dry weight basis, Steptoe leaves had five times more water-soluble material than Morex leaves and half the quantity of ether-soluble compounds. Metribuzin comprised 83 and 89% of the ether-soluble compounds in leaves of Morex and Steptoe, respectively, at 8 days. Terminal radioactivity comprised between 19% and 26% of total radioactivity for both cultivars as early as 1 day after application, with little change over 8 days. Rapid metabolism of metribuzin to non-phytotoxic water-soluble conjugates and terminal residues was the major mechanism responsible for the differential tolerance between these two barley cultivars.     

The role of translocation in selectivity of herbicides with reference to MCPA and MCPB

The distribution of 2.5 mM-[¹⁴C]MCPA

1 MCPA = (2-methyl-4-chlorophenoxyacetic acid).

and [¹⁴C]MCPB

2 MCPB = (4-(2-methyI-4-chlorophenoxy) butyric acid).

formulated as sodium salts with 0.05% tergitol, have been investigated when applied to an isolated leaf/agar block sink system of Vicia faba L. Results are presented which show the effect on absorption and translocation of method of application, age of treatment leaf, treatment period, pretreatment with 5 μM ATP and removal of cuticle wax by chloroform wipe treatment. Significant negative correlations between cuticle wax fixation/translocation and leaf tissue content/translocation were recorded for both compounds, particularly MCPB, suggesting the involvement of both physicochemical and metabolic components in the process of absorption and translocation. The nature of the mechanisms involved was further investigated using isolated cuticle/epidermal systems and tightly-coupled mitochondria isolated from roots of V. faba L. The results suggest that non-movement of MCPB in this species is largely due to its enhanced retention within the cuticle and to the fact that it is a more effective uncoupler of oxidative phosphorylation than MCPA. The importance of these findings in terms of the relative efficiency of translocation of MCPA and MCPB when applied in vivo is discussed.     

The behaviour of residues of flamprop-isopropyl and its breakdown products in barley

Residue data are reported for flamprop-isopropyl ( I ) in barley grain and straw samples following applications of the herbicide to crops grown in eight countries. The samples were analysed for I and its hydrolysis product N-benzoyl-N-(3-chloro-4-fluorophenyl)-DL -alanine ( II ). Following recommended applications (normally 1 kg ha^?1 at Feekes scale G-I/J), residues of I and II in the grain were low (90% were <0.02 mg kg^?1 for I , 86% were <0.06 mg kg^?1 for II , levels which were essentially the limits of determination). Residues in straw were higher and more variable, but again 63 and 77% of samples were below 1 mg kg^?1 for I and II , respectively.     

Hydrolysis of the triazine herbicide,cyanazine

The hydrolysis of cyanazine

1 Cyanazine is the proposed common name for the herbicide sold under the Shell registered trade name BLADEX.

(2-chloro-4-cyanoisopropylamino-6-ethylamino-1,3,5-triazine) has been studied using ¹⁴C-ring labelled compound over a temperature range of 25° to 75 °C and over a range of pH values from 1.5 to 12. The activation energies and the activation entropy changes during hydrolysis showed there was a different mechanism under acid and alkaline conditions. The only product identified after hydrolysis in acid solutions was 2-hydroxy-4-carboxyisopropylamino-6-ethylamino-1,3,5-triazine. In alkaline solution the same hydroxy-acid was the end-product, but 2-chloro-4-amidoisopropylamino-6-ethylamino-1,3,5-triazine was isolated as an intermediate. The variation of the specific rate constants with temperature for hydrolytic catalysis by H⁺, OH^? was determined, thus enabling the hydrolytic half-life of cyanazine to be calculated at any pH and temperature.     

The metabolic behavior of chlorotoluron in wheat and soil

The degradation of chlorotoluron, 1-(3-chloro-4-methylphenyl)-3,3-dimethylurea, was investigated in laboratory and field-grown wheat and soil. Thin-layer cochromatography and, partially, derivatization and mass spectroscopy were used to elucidate the structures of the metabolites. Wheat treated with 4-methyl[¹⁴C]-phenyl-labeled chlorotoluron rapidly metabolized the herbicide using two independent mechanisms: (I) oxidation of the 4-methylphenyl group to yield 4-hydroxy-methylphenyl and 4-carboxyphenyl derivatives; and (II) N-demethylation. Mechanism (I) clearly predominated over mechanism (II). Young wheat degraded the herbicide mainly to 4-hydroxy-methylphenyl derivatives with only a small fraction being additionally N-monodemethylated. Most of both metabolites was conjugated, most probably, with glucose. In straw and grains of mature field-grown summer wheat treated postemergence with labeled chlorotoluron at a rate corresponding to 2 kg active ingredient/hectare 2.8 ppm and 0.12 ppm radioactivity equivalent to chlorotoluron were found, respectively. About 50% of this terminal radioactivity was nonextractable by organic solvents. No chlorotoluron or its N-demethylated derivatives were present in either plant part. About 40% of the radioactivity in straw consisted of 4-carboxyphenyl derivatives half of which were N-mono- or didemethylated. The rest of the terminal radioactivity was mainly in form of the 4-hydroxymethylphenyl derivative of chlorotoluron. Less than 20% of the soluble metabolites was present as conjugates. In soil mechanism (II) exceeded mechanism (I). At harvest of the wheat the 0.4 ppm radioactivity of the 0- to 30-cm soil layer was composed of 43% chlorotoluron, 36% N-mono- and 3% N-didemethylated chlorotoluron, as well as 13% 4-carboxyphenyl derivatives partly N-demethylated.     

Metabolism of dibutyl-14C-labeled dibutylaminosulfenyl derivative of carbofuran in the cotton plant

The fate of the di-n-butylaminosulfenyl moiety in 2,3-dihydro-2,2-dimethyl-7-benzofuranyl (di-n-butylaminosulfenyl)(methyl)carbamate (DBSC or Marshal) was studied in the cotton plant at 1, 3, 6, and 10 days following foliage treatment with [di-n-butylamino-¹⁴C]DBSC. Dibutylamine and two major radioactive metabolites were obtained following extraction of the plant tissue with a methanol-buffer containing N-ethylmaleimide (NEM), a sulfhydryl scavenger which was added to prevent the cleavage of the NS bond during the workup procedure. The most adundant radioactive material recovered from plants was identified as a product arising from the reaction between NEM and dibutylamine. Extraction of plant tissue with straight methanol-buffer solution or with methol-buffer containing other sulfhydryl scavengers resulted in 57–86% of the applied radioactivity being recovered as dibutylamine in the organosoluble fraction. When [¹⁴C]dibutylamine was applied to cotton leaves, most of the radioactivity, i.e., 96% of the total recovered radioactivity, was found in the organosoluble fraction as dibutylamine. Dibutylamine is the major metabolite of [di-n-butylamino-¹⁴C]DBSC in the cotton plant.     

Radiochemical distribution and decline studies with bromoxynil octanoate in wheat

Bromoxynil octanoate labelled with ¹⁴C in the ring or in the cyano-group was applied to wheat seedlings at the two-leaf or fully-tillered stage and at rates equivalent to up to 16 oz a.i./acre. The plants were grown either in environmental chambers under controlled conditions for up to 28 days, or outdoors under field conditions for various periods up to harvest. Initially, elimination of radioactivity occurred more rapidly with bromoxynil-cyano-[¹⁴C]-octanoate than with bromoxynil-ring-[¹⁴C]-octanoate, indicating metabolic attack on the cyano group. Under outdoor conditions with ring-[¹⁴C]-herbicide applied at the two-leaf stage, only 12% of the radioactivity was retained after 28 days, principally in the treated leaves. When application was made at fully-tillered stage, about 33% of the ¹⁴C was retained after 56 days, almost entirely in the treated senescent leaves at the base of the plant. There was very little translocation of the herbicide or of any major metabolite. The level of radioactivity in harvested grain and in straw more than 7.5 cm above the ground was very low, even after very late application of ring-[¹⁴C]-labelled herbicide. The amount of bromoxynil octanoate, together with any metabolite retaining part of the aromatic ring, did not collectively exceed the equivalent of approx. 0.01 parts/million bromoxynil octanoate.     

Metabolism of [14C]dieldrin in bluegill fish

The exposure of bluegill fish to 50 parts per billion [¹⁴C]dieldrin in a static system resulted in the absorption of 73.00% of the radioactivity in 48 hr. Following transfer of the fish to clean water, only 16.20% of the absorbed radiolabel was eliminated in 23 days. Out of the 93.65% of the absorbed radioactivity recovered, 9 radioactive spots were isolated which included unchanged dieldrin (74.39%), pentachloroketone (8.17%), and aldrin-trans-diol (8.04%) as major metabolites.     

Behavior of 14C oryzalin in soil and plants

Radiochemical studies of field soil treated with ¹⁴C oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfanilamide) indicated that the compound was readily degradable. One year after soil treatment with oryzalin, 45% of the original radioactivity had dissipated, 25% was extractable, and 30% was “soil bound”. The extractable fraction contained oryzalin and several degradation products, some of which were isolated and identified. No single degradation product accounted for more than 3% of the applied oryzalin. The “soil-bound” radioactivity was extractable with hot alkali. No significant radioactive residues were detectable in either seed or forage of soybean and wheat plants. No specific metabolites of oryzalin were identified in soybean plants. Trace amounts of radioactivity found in plant tissue appeared to be associated with the various plant constituents.     

Hydrolysis and photolysis of the fungicide triforine

The main decomposition products formed from triforine by hydrolysis in solution and by photolysis were isolated and identified. In aqueous solution at 21°, hydrolysis proceeds rapidly by formation of chloride ions and, through several intermediates, 1-(dihydroxyacetyl)piperazine and piperazine. Photolysis by ultraviolet light in the absence of water leads preferentially to the removal of one side chain, the second side chain being attacked more slowly. In aqueous solution, triforine is rapidly destroyed by ultraviolet light. N-(2,2-Dichlorovinyl)formamide was isolated as an intermediate photodecomposition product.     

The behaviour of residues of benzoylprop-ethyl and its breakdown products in wheat

Samples of wheat grain and straw have been analysed from trials with the wild oat herbicide benzoylprop-ethyl ( I ) in several countries. Following recommended commercial treatments (application of 1.0–1.6 kg ha^?1 at Feekes growth stage G-J), total residues of I and its hydrolysis product N-benzoyl-N-(3,4-dichlorophenyl)-DL - alanine (free and conjugated) were low and in the majority of instances they were < 0.01 mg kg^?1 in samples of grain from the UK, although rather higher residues were detected in some grain samples from other countries. Residues in straw were higher, but normally did not exceed 2 mg kg^?1, and were rather variable, possibly as a result of differences in agricultural practice.