A comparative study of the biodegradation of the surfactant sodium dodecyltriethoxy sulphate by four detergent-degrading bacteria

The 35S-labelled metabolites produced during biodegradation of sodium dodecyltriethoxy [35S]sulphate (SDTES) by four bacterial isolates were identified and quantified. All four isolates used ether-cleavage as the predominant primary degradation pathway. In two of the organisms, the etherase system (responsible for approx. 60-70% of primary biodegradation) liberated mono-, di- and triethylene glycol monosulphates in substantial proportions, the last two esters undergoing some further oxidation to acetic acid 2-(ethoxy sulphate) and acetic acid 2-(diethoxy sulphate), respectively. For these isolates, liberation of SO4(2-) directly from SDTES was also significant (30-40%) and the organisms were shown to contain alkyl sulphatases active towards SDTES. For the remaining two isolates, etherase action was even more important (responsible for greater than 80% of primary biodegradation) and was restricted almost totally to the alkyl-ether bond to generate mainly triethylene glycol sulphate, some of which was further oxidized. Very small amounts of diethylene glycol monosulphate were also produced, but its mono-homologue, and the oxidation products of both these esters, were absent. Small amounts of inorganic sulphate (approx. 10%) were liberated by these isolates and one of them also produced compounds tentatively identified as intermediates of omega-/beta-oxidation.     

Metabolite production during the biodegradation of the surfactant sodium dodecyltriethoxy sulphate under mixed-culture die-away conditions

Sodium dodecyltriethoxy sulphate (SDTES), either pure or as a component of commercial surfactant mixtures, underwent rapid primary biodegradation by mixed bacterial cultures in OECD screen and river-water die-away tests. Inoculation of [35S]SDTES-containing solutions with OECD screen test media acclimatized to surfactants or their degradation products led to production of various 35S-labelled glycol sulphates and their oxidation products, all known to occur during degradation of [35S]SDTES by pure bacterial isolates. Triethylene glycol monosulphate was the major catabolite together with smaller amounts of di- and monoethylene glycol monosulphates implying, by analogy with pure cultures, that ether-cleavage was the major primary biodegradation step. The oxidation product (carboxylate derivative) of each glycol sulphate was also detected together with metabolites tentatively identified as omega-/beta-oxidation products of the dodecyl chain. Relatively little SO2-4 was liberated directly from SDTES but mixed cultures derived from sewage could metabolize the sulphated glycols to SO2-4. The environmental relevance of these degradation routes was established by following metabolite production from [35S]SDTES in full-scale river-water die-away tests. Triethylene glycol sulphate was formed first, then rapidly oxidized to acetic acid 2-(diethoxy sulphate) which persisted as the major metabolite for 2-3 weeks. Small amounts of sulphated derivatives of di- and monoethylene glycols were also detected during the same period. Very little SO2-4 was formed directly from SDTES but large amounts accompanied the eventual disappearance of glycol sulphate derivatives. None of the 35S-labelled organic metabolites was persistent and, whenever [35S]SDTES was a component of a commercial mixture, all ester sulphate was completely mineralized to 35SO4(2-) within 28 d.     

Central noradrenergic activity and the formation of glycol sulfate metabolites of brain norepinephrine

The present study utilized intraventricular injection of Na₂³⁵SO₄ to detect drug induced changes in the $\text{in vivo}$ formation of the two major metabolites of rat brain norepinephrine (NE) - the sulfate conjugates of 3-methoxy-4-hydroxyphenylglycol (MOPEG-SO₄) and 3,4-dihyd (DOPEG-SO₄). Assays involved the hypothalamus only. Rats pretreated with clonidine showed a reduced formation of both MOPEG-³⁵SO₄ and DOPEG-³⁵SO₄ after intraventricular Na₂³⁵SO₄ as well as reduced synthesis of ³H-NE from intraventricular ³H-tyrosine. Phenoxybenzamine (POB) produced increases in the synthesis of both ³⁵S-labeled conjugates and ³H-NE. Neither drug altered the loss of exogenous ³H-MOPEG-SO₄ but clonidine increased both the accumulation of labeled sulfate and the sulfation of exogenous MOPEG in pheniprazine treated rats. These results show that the rates of formation of the labeled glycol sulfates are sensitive indicators of changes in brain NE turnover but can also be influenced by factors involved in sulfation that are unrelated to NE turnover. Blockade of NE synthesis with alpha methyltyrosine did not affect resting or POB-elevated levels of the labeled conjugates until stores of NE were reduced by 40%. The latter findings suggest that central noradrenergic neurons can release and metabolize NE at a normal rate despite synthesis blockade so long as adequate stores of NE are available.     

Enzymatic sulfation of a large molecular weight glycoprotein associated with pig brain membranes

Pig brain membranes catalyze the transfer of [³⁵S]sulfate from 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate into two macromolecular endogenous acceptors. Several operational enzymatic properties of the sulfotransferase activity have been defined. An apparent K_m = 0.65 μm for 3′-phosphoadenosine 5′-phosphosulfate has been determined for the pig brain in vitro sulfotransferase system. Direct proof for the absolute requirement of the 3′-phosphate moiety of 3′-phosphoadenosine 5′-phosphosulfate is presented. The nucleotide end product, 3′,5′-ADP, is a potent competitive inhibitor of the pig brain sulfotransferase activity. One of the major products enzymatically labeled during incubation with 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate is a membrane-bound glycoprotein of high molecular weight. The sulfated glycoprotein appears to be an integral membrane glycoprotein, requiring 1% Triton X-100 for extraction. An ³⁵S-labeled oligosaccharide, released by mild base treatment, contains O-sulfate ester groups and at least one N-acetylneuraminic acid residue. The sulfated glycoprotein has an apparent molecular weight of 198,000. Under the same in vitro conditions [³⁵S]sulfate is also incorporated into a membrane-associated ³⁵S-labeled proteoglycan having the properties of heparan sulfate. The ³⁵S-labeled proteoglycan is electrostatically bound to the pig brain membranes, and can be readily extracted with 1 m NaCl.     

Xenoestrogenic short ethoxy chain nonylphenol is oxidized by a flavoprotein alcohol dehydrogenase from <Emphasis Type="Italic">Ensifer</Emphasis> sp. strain AS08

The ethoxy chains of short ethoxy chain nonylphenol (NPEO_av2.0, containing average 2.0 ethoxy units) were dehydrogenated by cell-free extracts from Ensifer sp. strain AS08 grown on a basal medium supplemented with NPEO_av2.0. The reaction was coupled with the reduction in 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and phenazine methosulfate. The enzyme (NPEO_av2.0 dehydrogenase; NPEO-DH) was purified to homogeneity with a yield of 20% and a 56-fold increase in specific activity. The molecular mass of the native enzyme was 120 kDa, consisting of two identical monomer units (60 kDa). The gene encoding NPEO-DH was cloned, which consisted of 1,659 bp, corresponding to a protein of 553 amino acid residues. The deduced amino acid sequence agreed with the N-terminal amino acid sequence of the purified NPEO-DH. The presence of a flavin adenine dinucleotide (FAD)-binding motif and glucose–methanol–choline (GMC) oxidoreductase signature motifs strongly suggested that the enzyme belongs to the GMC oxidoreductase family. The protein exhibited homology (40–45% identity) with several polyethylene glycol dehydrogenases (PEG-DHs) of this family, but the identity was lower than those (approximately 58%) among known PEG-DHs. The substrate-binding domain was more hydrophobic compared with those of glucose oxidase and PEG-DHs. The recombinant protein had the same molecular mass as the purified NPEO-DH and dehydrogenated PEG400-2000, NPEO_av2.0 and its components, and NPEOav10, but only slight or no activity was found using diethylene glycol, triethylene glycol, and PEG200. English edition: The paper was edited by a native speaker through American Journal Experts ().     

Release of O-sulfate groups under mild acid hydrolysis conditions used for estimation of N-sulfate content

The treatment of chondroitin sulfate isolated from cultured B16 mouse melanoma cells with 0.04 M HCl at 100°C for 90 min released up to 45% of O-sulfate residues as free inorganic sulfate. In addition to the release of inorganic sulfate, extensive degradation of this polysaccharide as well as of cartilage chondroitin sulfate, pig rib cartilage proteoglycan, heparin and hyaluronic acid was also evident under these conditions. The above hydrolysis conditions are used for characterizing ³⁵S-labeled heparan sulfates synthesized by cultured cells and to calculate ratio of N- and O-sulfates in these molecules. Our results suggest that caution in necessary in interpreting the results of mild acid hydrolysis of glycosaminoglycans.     

In vivo biosynthesis and turnover of 35S-labeled glomerular basement membrane

Renal glomerular basement membrane was labeled in vivo by the injection of tracer amounts of radioactive sulfate into normal adult rats. The biosynthesis and turnover of [³⁵S]glycosaminoglycans in purified basement membrane was determined from the specific activity of ³⁵S in pronase digests of basement membranes isolated 1–7 days after injection. Peak radioactive labeling occurred 24 h after injection following which the specific activity of basement membrane sulfate, expressed as cpm/μg uronic acid, progressively declined over the ensuing period of study. The biologic half-life of radioactive sulfate in basement membrane was estimated at about 7 days, which is within the range previously reported for [³⁵S]glycosaminoglycans in whole renal cortex. The findings indicate that ³⁵S-labeled components of glomerular basement membrane have a relatively rapid turnover.     

Effect of stress on sulfated glycol, metabolites of brain norepinephrine.

The present study examined the effect of footshock stress on the formation of the two major metabolites of rat brain norepinephrine (NE) - the sulfate conjugates of 3-methoxy-4-hydroxyphenylglycol (MOPEG-SO₄) and 3,4-dihydroxyphenylglycol (DOPEG-SO₄). Rats receiving intraventricular injections of either ³HNE or Na₂³⁵SO₄ prior to 0.5 hour of footshock showed significant and comparable increases in both sulfated glycols labeled with ³H or ³⁵SO₄. Elevations were greatest in the hypothalumus using Na₂³⁵SO₄. In pheniprazine pretreated rats footshock did not increase the production of MOPEG-³⁵SO₄ from intraventricular labeled sulfate given alone or in combination with various doses of exogenous MOPEG. The results indicate that neuronally released brain NE is metabolized to form both MOPEG-SO₄ and DOPEG-SO₄. The increase in these metabolites results from an increased glycol production and not from a stress-induced activation of brain sulfation mechanisms.     

CHEMICAL SYNTHESIS OF [3H]CEREBROSIDE [35S]SULFATE AND ITS IN VIVO METABOLISM IN RAT BRAIN

—Double-labeled sulfatide containing [3-³H]lignoceric acid and [³⁵S]sulfate was synthesized and injected intracerebrally into 28-day-old rats. The ³H-labeled sulfatide was synthesized by condensing (RS)-[3-³H]lignoceroyl chloride with lysosulfatide which had been obtained by saponification of sulfatide. The ³⁵S-labeled sulfatide was synthesized by using [³⁵S]sulfuric acid for sulfating 2′, 4′, 6′-tri-benzoyl-galactosyl N-fatty acyl, N-benzoyl-3-0-benzoyl-sphingosine, which had been obtained by per-benzoylation followed by solvolysis of calf brain nonhydroxycerebrosides. The perbenzoylated [³⁵S]sul-fatide was then subjected to mild alkaline saponification. Eight hours following the injection, the brain lipids contained various radioactive sphingolipids in addition to sulfatides. Fourteen per cent of the injected ³H was recovered in total lipids, and 26% of this was found in sulfatide. Nonhydroxy- and hydroxyceramides, nonhydroxy- and hydroxycerebrosides, and polar lipids contained 7, 1, 8, 3, and 22 per cent of the ³H found in total lipids, respectively. On the other hand, only 6% of the ³⁵S injected was recovered in total lipids; 63% of this was found in sulfatide, 5% in a mixture of seminolipid and cholesterol sulfate and 10% in a water-soluble material.     

Degradation of 35S-labeled metallothionein in the liver and the kidney of Brindled mice: Model for Menkes' disease

An amino acid analysis of the renal copper-binding protein of heterozygous Brindled mice indicated that the protein labeled with L-[³⁵S]cystine was metallothionein.The metabolism of ³⁵S-labeled hepatic and renal metallothionein of adult normal (Mo^+/+) and heterozygous (Mo^br/+) Brindled mice was investigated without prior induction with metals. After incorporation of L-[³⁵S] cysteine into hepatic and renal metallothionein, ³⁵S-labeled metallothionein is normally degraded with two half-lives (liver: 11.6 ± 1.3 hours and 3.1 ± 0.3 days; kidney: 8.22 ± 0.08 hours and 3.5 ± 1.2 days). However, ³⁵S-labeled renal metallothionein of the heterozygous Brindled mice is exclusively degraded with a half-life of 3.1 ± 0.2 days.The results imply that the mutation in Brindled mice causes an impaired renal reabsorption of copper (transport of copper from the tubular cells into the blood circulation).     

Formation of the fe-s cluster of ferredoxin in lysed spinach chloroplasts

In vitro formation of the ³⁵S-labeled Fe-S cluster of ferredoxin (Fd) has been achieved by incubating apo-Fd and [³⁵S]cysteine with osmotically lysed chloroplasts of spinach (Spinacia oleracea). Correct integration of the ³⁵S-labeled Fe-S cluster into Fd was verified on the basis of the following: (a) Under nondenaturing conditions, ³⁵S-labeled holo-Fd showed the same electrophoretic mobility as authentic holo-Fd; (b) ³⁵S-labeled holo-Fd showed an ability to bind Fd-NADP⁺ reductase; (c) the ³⁵S-labeled moiety was removed from the Fd polypeptide by TCA treatment but not by 2-mercaptoethanol treatment; (d) externally added pea II apo-Fd was converted to ³⁵S-labeled holo-Fd. This reconstitution was dependent on both ATP and light, and formation of the ³⁵S-labeled Fe-S cluster was observed upon addition of ATP or when an ATP generation-system was constructed in the light. In contrast, ATP-consuming systems abolished the Fe-S cluster formation. A non-hydrolyzable ATP analog was unable to serve as an ATP substitute, indicating the requirement of ATP hydrolysis for cluster formation. GTP was able to substitute for ATP, but CTP and UTP were less effective. Fe-S cluster formation in lysed chloroplasts was stimulated by light even in the presence of added ATP. Light stimulation was inhibited by DCMU or methyl viologen but not by NH₄⁺. NADPH was able to substitute for light, indicating that light energy is required for the production of reducing compounds such as NADPH in addition to the generation of ATP. These results confirm the requirement of light for the Fe-S cluster formation observed previously in intact chloroplasts.     

Application of isotope dilution analysis to the determination of sulfate in glycoproteins and oligosaccharides

Isotope dilution analysis, using a probe of ³⁵S-labeled BaSO₄, is proposed for the determination of sulfate in hydrolysates of glycoproteins and other glycoconjugates. A scaled-down version of the method of Klockow is presented. A modified radioassay in strongly acidic conditions, effective in the range of 0.5 to 10 nmol sulfate per sample, has been developed.     

Sulfate reduction and inorganic carbon assimilation in acidic thermal springs of the Kamchatka peninsula

Thermoacidophilic sulfate reduction, which remains a poorly studied process, was investigated in the present work. Radioisotope analysis with ³⁵S-labeled sulfate was used to determine the rates of dissimilatory sulfate reduction in acidic thermal springs of Kamchatka, Russia. Sulfate reduction rates were found to vary from 0.054 to 12.9 nmol SO₄/(cm³ day). The Oil Site spring (Uzon caldera, 60°C, pH 4.2) and Oreshek spring (Mutnovskii volcano, 91°C, pH 3.5) exhibited the highest activity of sulfate-reducing prokaryotes. Stable enrichment cultures reducing sulfate at pH and temperature values close to the environmental ones were obtained from these springs. Analysis of the 16S rRNA gene sequences revealed that a chemolithoautotrophic bacterium Thermodesulfobium sp. 3127-1 was responsible for sulfate reduction in the enrichment from the Oil Site spring. A chemoorganoheterotrophic archaeon Vulcanisaeta sp. 3102-1 (phylum Crenarchaeota) was identified in the enrichment from Oreshek spring. Thus, dissimilatory sulfate reduction under thermoacidophilic conditions was demonstrated and the agents responsible for this process were revealed.     

Characteristics of human chondrocyte cultures in completely defined medium

Summary Chondrocytes derived from normal human adult articular cartilage were established and maintained for over 5 months in a completely defined medium without the addition of serum or any other growth factors. At the end of 5 months, these cells were still metabolically active. The cells incorporated [³H]thymidine into DNA, incorporated [³⁵S]sulfate into proteoglycans, and exhibited lysosomal enzyme activities. The³⁵S-labeled proteoglycans isolated from the culture medium had elution profiles on high pressure liquid chromatography (HPCL) similar to those observed from proteoglycans from other mammalian sources. This self-contained growth competence may reflect a need produced by the unusual avascular and alymphatic character of articular cartilage. This research was supported, in part, by Grant AM22057 from the National Institutes of Health, Bethesda, MD.     

Sulfolipid metabolism in chlorella

When S-deficient cells of Chlorella cllipsoidea were incubated in radio-sulfate in light or in aerobic darkness for 1 hour, equal amounts of radioactivity were found in sulfolipid and glutathione but none was detected in sulfoquinovosyl glycerol which is one of the major S-compounds in this alga. No assimilation of radiosulfate was observed under anaerobic darkness.

To elucidate the function of sulfolipid in algal cells uniformly ³⁵S-labeled Chlorella cells were transferred to S-deficient culture medium or unlabeled normal culture medium and the changes of radioactivity in sulfolipid and the related compounds were followed. A) On incubating ³⁵S-labeled algal cells in S-deficient medium under photosynthetic conditions, the amounts of radioactivity in sulfate, sulfoquinovosyl glycerol and sulfolipid decreased rapidly. B) When ³⁵S-labeled cells were cultured photoautotrophically in unlabeled medium, no decrease of radioactivity was observed in sulfoquinovosyl glycerol and sulfolipid. C) A decrease of ³⁵S-sulfolipid and an increase of ³⁵S-sulfoquinovosyl glycerol were observed when the uniformly ³⁵S-labeled algal cells were illuminated in CO₂-free air.

When S-deficient Chlorella cells were incubated in ³⁵S-sulfolipid under photosynthetic conditions, significant radioactivity was found in the insoluble fraction of the cells. A similar result was observed when normal Chlorella cells were incubated in ¹⁴C-sulfolipid and CO₂-free air.

It is inferred from these observations that sulfolipid is a reservoir of sulfur and carbon compounds.

In order to ascertain if the sulfolipid is involved in the mechanism of photosynthetic oxygen evolution, the rate of photosynthesis was measured during the incubation of ³⁵S-labeled cells in a S-deficient medium. Parallelism was not observed between the rate of photosynthetic activity and the decrease of sulfolipid.

     

Defect in 3′-phosphoadenosine 5′-phosphosulfate synthesis in brachymorphic mice: I. Characterization of the defect

Biosynthesis of the undersulfated proteoglycan found in brachymorphic mouse (bm/ bm) cartilage has been investigated. Similar amounts of cartilage proteoglycan core protein, as measured by radioimmune inhibition assay, and comparable activity levels of four of the glycosyltransferases requisite for synthesis of chondroitin sulfate chains were found in cartilage homogenates from neonatal bm/bm and normal mice, suggesting normal production of glycosylated core protein acceptor for sulfation. When incubated with ³⁵S-labeled 3′-phosphoadenosine 5′-phosphosulfate (PAPS), bm/bm cartilage extracts showed a higher than control level of sulfotransferase activity. In contrast, when synthesis was initiated from ATP and ³⁵SO₄^2?, mutant cartilage extracts showed lower incorporation of ³⁵SO₄^2? into endogenous chondroitin sulfate proteoglycan (19% of control level) and greatly reduced formation of PAPS (10% of control level). Results from coincubations of normal and mutant cartilage extracts exhibited intermediate levels of sulfate incorporation into PAPS and endogenous acceptors, suggesting the absence of an inhibitor for sulfate-activating enzymes or sulfotransferases. Degradation rates of ³⁵S]PAPS and of ³⁵S-labeled adenosine 5′-phosphosulfate (APS) were comparable in bm/bm and normal cartilage extracts. Specific assays for both ATP sulfurylase (sulfate adenylyltransferase; ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (adenylylsulfate kinase; ATP:adenylylsulfate 3′-phosphotransferase, EC 2.7.1.25) showed decreases in the former (50% of control) and the latter (10–15% of control) enzyme activities in bm/bm cartilage extracts. Both enzyme activities were reduced to intermediate levels in extracts of cartilage from heterozygous brachymorphic mice (ATP-sulfurylase, 80% of control; APS kinase, 40–70% of control). Furthermore, the moderate reduction in ATP sulfurylase activity in bm/bm cartilage extracts was accompanied by increased lability to freezing and thawing of the residual activity of this enzyme. These results indicate that under-sulfation of chondroitin sulfate proteoglycan in bm/bm cartilage is due to a defect in synthesis of the sulfate donor (PAPS), resulting from diminished activities of both ATP sulfurylase and APS kinase, although the reduced activity of the latter enzyme seems to be primarily responsible for the defect in PAPS synthesis.     

Fate of 3-Allyloxy-1,2-benzisothiazole 1,1-Dioxide (Oryzemate®) in Rice Plants

Studies on absorption, translocation and metabolism of 3-allyloxy-1,2-benzisothiazole 1,1-dioxide (Oryzemate), a new rice blast controlling agent, in rice plants were undertaken using the ³⁵S-labeled preparation. Oryzemate was administrated to the plants by liquid application. Uptake of labeled compound into rice plants was demonstrated by autoradiography and quantitative ³⁵S-analyses. Preferential accumulation of radioactive compounds in the leaves was observed. The metabolites in the plants were identified as allyl o-sulfamoylbenzoate, saccharin and N-d-glucopyranosylsaccharin by cochromatography and cocrystallization with synthesized authentic compounds. Most of ³⁵S-labeled compounds accumulated in the plants were saccharin and N-d-glucopyranosylsaccharin. A small amount of allyl o-sulfamoylbenzoate was detected and Oryzemate was detectable in trace quantities at all harvest.     

Catabolism of 3-indole acetic acid in protoplasts from etiolated seedlings of scots pine (Pinus sylvestris L.)

Protoplasts preparated from dark grown seedlings of Pinus sylvestris L. were incubated with 3-indole (1-¹⁴C) acetic acid and 3-indole (2-¹⁴C) acetic acid (IAA). Three catabolites were consistently produced in the (2-¹⁴C) IAA feeds, one of which co-chromatographed with 3-indole methanol on reversed phase high performance liquid chromatography (HPLC). Protoplasts feed with (1-¹⁴C) IAA produced only one labelled catabolite. The non-decarboxylated compound formed was highly polar on reversed phase HPLC, both in the ion suppression and the ion pair mode. The substance was not hydrolysable at pH 11 and 100° indicating that it is not a conjugated form. Effects of time of incubation, pH and the cofactors hydrogen peroxide and 2,4-dichlorphenol on the catabolic rate of IAA are discussed.Abbreviations BSA bovine serum albumin - DCP 2,4-dichlorophenol - HPLC high performance liquid chromatography - IAA 3-indole acetic acid - IAld 3-indole carboxaldehyde - ICA 3-indole carboxylic acid - IM 3-indole methanol - MES 4-morpholineethane sulfonic acid - MO 3-methyl-oxindol - MnO 3-methylene-oxidol - OxIAA 3-oxindole acetic acid - PEG polyethylene glycol     

Non-collagen protein and proteoglycan in renal glomerular basement membrane

Extraction of rat glomerular basement membrane, purified by osmotic lysis and sequential detergent treatment, with 8 M urea containing protease inhibitors solubilizes protein that is devoid of hydroxyproline and hydroxylysine. This material represents 8–12% of total membrane protein, elutes mainly as two high molecular weight peaks on agarose gel filtration, and is associated with glycosaminoglycans. Isolated rat renal glomeruli incorporate [³⁵S]sulfate into basement membrane from which this non-collagenous ³⁵S-labeled fraction can be subsequently solubilized. The radioactivity incorporated into urea-soluble glomerular basement membrane eluted primarily with the higher molecular weight peak (M_r greater than 250 000). Cellulose acetate electrophoresis after pronase digestion of the urea-soluble fraction revealed glycosaminoglycan that was resistant to digestion with Streptomyces hyaluronidase and chondroitinase ABC, sensitive to nitrous acid treatment, and contained [³⁵S]-sulfate. The findings indicate that one of the non-collagenous components of glomerular basement membrane is a proteoglycan containing heparan sulfate.     

Metabolic Fate of O-Ethyl S,S-Diphenyl Phosphorodithiolate (Hinosan®) in Rice Plant

Metabolism and residual fate of O-ethyl S,S-diphenyl phosphorodithiolate (Hinosan®) applied on rice plant was examined by using ³⁵S-labeled or ³²P-labeled compound. Ion exchange chromatography, thin-layer chromatography and gas-liquid chromatography with flame thermionic detector or flame photometric detector were applied for identification of water soluble and toluene soluble metabolites of Hinosan. Degradation of Hinosan at the initial stage of metabolism was mainly the cleavage of P-S linkage, and a large portion of phenyl dihydrogen phosphorothiolate and a minor portion of O-ethyl S-phenyl hydrogen phosphorothiolate were found as water soluble metabolites. Phenylthio radical released on the production of the above mentioned metabolites was recovered as diphenyl disulfide, which was finally converted to sulfuric acid through benzenesulfonic acid. Triphenyl phosphorotrithiolate and O,O-diethyl S-phenyl phosphorothiolate were produced by transesterification between molecules of Hinosan at the initial stage of metabolism. Examination of metabolites in rice grains showed that sulfur and phosphorus atoms in Hinosan were incorporated into neutral or cationic substances probably after several steps of chemical transformation.