In vitro release of bound (nonextractable) 14C residues from plants by corn leaves bomogenates

Corn leaves homogenates were found to release bound (nonextractable) ¹⁴C residues from the aerial portion of matured corn plants. The 22,000 g pellet and 10,000 g supernatant fractions were the most active in releasing the bound ¹⁴C residues. The released ¹⁴C residues comprised mainly 2-OH analogues of the $\text{N}$-monodealkylated analogues of atrazine. It is suggested that the enzymatic system in plants may cause metabolic conversion of bound residues.     

Release of soil bound (nonextractable) residues by various physiological groups of microorganisms∗

Abstract

Soil bound ¹⁴C‐labeled residues were released by four different physiological groups of microorganisms from an organic soil treated with ¹⁴C‐ring‐labeled prometryn [2‐(methylthio) ‐4,6‐bis(isopropylamino)‐s‐triazine]. The extent to which the different microbial populations released bound ¹⁴C residues (25–30% of the total bound ¹⁴C) from the Y‐irradiated soil after 28 days incubation did not differ considerably. Analysis of the extractable material from the incubated soil showed the presence of small amounts of the parent compound, and its hydroxy and mono‐N‐dealkylated analogues. Low level of ¹⁴CO₂ (1.5–3.0% of the total bound ¹⁴C) was evolved from the microbial systems indicating ring cleavage of the released material as being a very minor reaction.     

Influence of humic fractions on retention of isoproturon residues in two Moroccan soils

The influence of different fractions of soil organic matter on the retention of the herbicide isoproturon (IPU) has been evaluated. Water and methanol extractable residues of ¹⁴C labeled isoproturon have been determined in two Moroccan soils by β -counting–liquid chromatography. The quantification of bound residues in soil and in different fractions of soil humic substances has been performed using pyrolysis/scintillation-detected gas-chromatography. Microbial mineralization of the herbicide and soil organic matter has been also monitored. Retention of isoproturon residues after 30-days incubation ranged from 22% to 32% (non-extractable fraction). The radioactivity extracted in an aqueous environment was from 20% to 33% of the amount used for the treatment; meanwhile, methanol was able to extract another 48%. Both soils showed quantities of bound residues into the humin fraction higher than humic and fulvic acids. The total amount of residues retained into the organic matter of the soils was about 65 % of non-extractable fraction, and this percentage did not change with incubation time; on the contrary, the sorption rate of the retention reaction is mostly influenced by the clay fraction and organic content of the soil. Only a little part of the herbicide was mineralized during the experimental time.     

Bioavailability of bound 14C residues in rats from bean plants treated with 14C-deltamethrin

Bean plants were treated with deltamethrin labeled with ¹⁴C at the methyl or benzylic position. The aerial portion of the plants was exhaustively extracted with solvents and the extracted material containing bound ¹⁴C residues was fed to rats. After 4 days 60% and 53% of the dose was excreted in feces and 31% and 20% in the urine from rats fed extracted bean plants treated with deltamethrin labeled at the methyl and benzylic position, respectively. The data demonstrated that bound residues in bean plants treated with deltamethrin may be bioavailable in rats.     

Distribution,fate and formation of non-extractable residues of a nonylphenol isomer in soil with special emphasis on soil derived organo-clay complexes

Anthropogenic contaminants like nonylphenols (NP) are added to soil, for instance if sewage-sludge is used as fertilizer in agriculture. A commercial mixture of NP consists of more than 20 isomers. For our study, we used one of the predominate isomers of NP mixtures, 4-(3,5-dimethylhept-3-yl)phenol, as a representative compound. The aim was to investigate the fate and distribution of the isomer within soil and soil derived organo-clay complexes. Therefore, ¹⁴C- and ¹³C-labeled NP was added to soil samples and incubated up to 180 days. Mineralization was measured and soil samples were fractionated into sand, silt and clay; the clay fraction was further separated in humic acids, fulvic acids and humin. The organo-clay complexes pre-incubated for 90 or 180 days were re-incubated with fresh soil for 180 days, to study the potential of re-mobilization of incorporated residues. The predominate incorporation sites of the nonylphenol isomer in soil were the organo-clay complexes. After 180 days of incubation, 22 % of the applied ¹⁴C was mineralized. The bioavailable, water extractable portion was low (9 % of applied ¹⁴C) and remained constant during the entire incubation period, which could be explained by an incorporation/release equilibrium. Separation of organo-clay complexes, after extraction with solvents to release weakly incorporated, bioaccessible portions, showed that non-extractable residues (NER) were preferentially located in the humic acid fraction, which was regarded as an effect of the chemical composition of this fraction. Generally, 27 % of applied ¹⁴C was incorporated into organo-clay complexes as NER, whereas 9 % of applied ¹⁴C was bioaccessible after 180 days of incubation. The re-mobilization experiments showed on the one hand, a decrease of the bioavailability of the nonylphenol residues due to stronger incorporation, when the pre-incubation period was increased from 90 to 180 days. On the other hand, a shift of these residues from the clay fraction to other soil fractions was observed, implying a dynamic behavior of incorporated residues, which may result in bioaccessibility of the NER of nonylphenol.     

Plant availability of bound anilazine residues in a degraded loess soil

Abstract

The relative biological availability of [benzene ring‐U‐¹⁴C] and Ctriazine‐U‐¹⁴C] anilazine for maize plants was studied in a degraded loess soil in a standardized microecosystem. The total uptake of radiocarbon in the course of the 4‐week experiment was 3.1 and 4 % respectively of the radioactivity applied if anilazine was uniformly mixed into the soil immediately before beginning the experiment. However, if anilazine was subjected to a degradation at 65 % of the maximum water holding capacity of the soil and temperatures varying daily between 16 and 27°C for 100 days before the plant experiment then the uptake was reduced to 0.4 or 0.7 % respectively. The uptake from soil with non‐extractable (bound) anilazine residues was similarly low. The mineralization rate of aged and bound anilazine residues was below 0.1 % of the radioactivity applied. Up to 2/3 of the radioactivity present in the soil after the plant experiment remained in the humic fraction.     

Chemical and biological release of 14C‐bound residues from soil treated with 14C‐p,p'‐DDT

Abstract

¹⁴C‐p,p'‐DDT‐bound residues in soil can be released by treatment with concentrated sulphuric acid at ambient temperatures. Within 6 days, about 70% of the bound residues was released. Bound residues released after 9 months incubation with ¹⁴C‐DDT showed the presence of DDT and DDE only while bound residues released after 18 months, contained in addition 13% DDD.

Release of bound ¹⁴C‐residues also occurs readily following inoculation of the soil‐bound residues with fresh soil or with individual microorganisms. Almost complete release of bound residues was observed after incubation for 45 days. The rate of release was rapid during the first two weeks and decreased thereafter. TLC and HPLC analysis showed that the released residues contained DDE (about 80%) and a smaller amount of DDD. The disappearance of DDT from the released residues may be attributed to its microbiological degradation to DDE and DDD, shortly after its release.     

Fate of the veterinary antibiotic 14C-difloxacin in soil including simultaneous amendment of pig manure with the focus on non-extractable residues

The fate of ¹⁴C-labeled difloxacin (¹⁴C-DIF) was studied in time course experiments after application on soil (Ap horizon of silt loam) and amendment of authentic DIF containing pig manure (146 mL kg^?1; 4.17 MBq kg^?1; 0.85 mg kg^?1) or water (124 mL kg^?1; 0.42 MBq kg^?1; 0.09 mg kg^?1) for 56 and 120 days of incubation, respectively.

Mineralization of ¹⁴C-DIF was below 0.2% in both experiments after 56 days or 120 days. In the course of the experiments, portions of extractable radioactivity (Accelerated Solvent Extraction (ASE); acetonitrile-water) decreased to 19–21% depending only little on manure amendment. Non-extractable residues of ¹⁴C-DIF increased to 70–74% after 56 days and 120 days, respectively, and therefore were the main route of ¹⁴C-DIF in soil. According to radioanalytical HPLC and LC-MS/MS, only the parent compound was found in all extracts over the whole time of the experiment. According to fractionation of the non-extractable residues (NER) into particle size fractions, ¹⁴C portions were associated to the water used for fractionation, the silt and clay fractions, whereas no radioactivity was detected in the sand fraction. The majority of ¹⁴C was found within the clay fractions.

Fractionation of humic components showed that radioactivity derived from ¹⁴C-DIF was associated with humic acids, fulvic acids, humins and minerals and very little with soluble, non-humic HCl fraction. The highest portions of radioactivity were found in the fulvic acid fraction. Results obtained by size exclusion chromatography (SEC) of the purified fulvic acids were similar for every sample analyzed. One large portion of ¹⁴C co-eluted with fulvic acids of a molecular weight below 910 g mol^?1. Both fractionation methods demonstrated that the parent compound DIF or initial metabolites were rapidly integrated into humic materials and, thus, were major components of NER.     

Fate in soil of 14C-sulfadiazine residues contained in the manure of young pigs treated with a veterinary antibiotic

The fate of ¹⁴C-labeled sulfadiazine (¹⁴C-SDZ) residues was studied in time-course experiments for 218 days of incubation using two soils (A_p horizon of loamy sand, orthic luvisol; A_p horizon of silt loam, cambisol) amended with fresh and aged (6 months) ¹⁴C-manure [40 g kg^?1 of soil; 6.36 mg of sulfadiazine (SDZ) equivalents per kg of soil], which was derived from two shoats treated with ¹⁴C-SDZ. Mineralization of ¹⁴C-SDZ residues was below 2% after 218 days depending little on soil type. Portions of extractable ¹⁴C (ethanol-water, 9:1, v/v) decreased with time to 4–13% after 218 days of incubation with fresh and aged ¹⁴C-manure and both soils. Non-extractable residues were the main route of the fate of the ¹⁴C-SDZ residues (above 90% of total recovered ¹⁴C after 218 days). These residues were high immediately after amendment depending on soil type and aging of the ¹⁴C-manure, and were stable and not remobilized throughout 218 days of incubation. Bioavailable portions (extraction using CaCl₂ solution) also decreased with increasing incubation period (5–7% after 218 days). Due to thin-layer chromatography (TLC), 500 μg of ¹⁴C-SDZ per kg soil were found in the ethanol-water extracts immediately after amendment with fresh ¹⁴C-manure, and about 50 μg kg^?1 after 218 days. Bioavailable ¹⁴C-SDZ portions present in the CaCl₂ extracts were about 350 μg kg^?1 with amendment. Higher concentrations were initially detected with aged ¹⁴C-manure (ethanol-water extracts: 1,920 μg kg^?1; CaCl₂ extracts: 1,020 μg kg^?1), probably due to release of ¹⁴C-SDZ from bound forms during storage. Consistent results were obtained by extraction of the ¹⁴C-manure-soil samples with ethyl acetate; portions of N-acetylated SDZ were additionally determined. All soluble ¹⁴C-SDZ residues contained in ¹⁴C-manure contributed to the formation of non-extractable residues; a tendency for persistence or accumulation was not observed. SDZ's non-extractable soil residues were associated with the soluble HCl, fulvic acids and humic acids fractions, and the insoluble humin fraction. The majority of the non-extractable residues appeared to be due to stable covalent binding to soil organic matter.     

Fate of hexachlorobenzene-14C in wheat plants and soil under outdoor conditions

Residues of hexachlorobenzene-¹⁴C were found in all parts of wheat plants grown from treated seeds or in contaminated soil. Besides the parent compound and bound residues in plants and soil, very small amounts of soluble acidic metabolites were present in plants, which were characterized and determined quantitatively.     

Effect of organic amendment on degradation and formation of bound residues of methabenzthiazuron in soil under constant climatic conditions

Abstract

The degradation of [phenyl‐U‐¹⁴C]methabenzthiazuron (MBT) and formation of bound residues in the surface soil of an orthic luvisol were studied under constant climatic conditions (20°C, 40 % of maximum water holding capacity). In two treatments (with and without preincubation in the soil) maize straw was amended at a rate of 1.5 g/100 g dry soil in addition to the application of MBT. The mineralization of uniformly labeled maize straw was studied simultaneously. In additional flasks, MBT was incubated at 0, 10 and 30°C with and without addition of maize straw.

The turnover of the amended maize straw led to an enhanced dissipation of MBT which was mainly due to the formation of bound residues. This corresponded to a higher microbial activity in the soil after straw amendment and the intensive mineralization of the radiolabeled maize straw. About 2–3 % of the applied radioactivity from the radiolabeled maize straw was measured in the soil microbial biomass 10 and 40 days after application whereas ¹⁴C from MBT was only incorporated into soil microbial biomass in the treatments with straw amendment.

Within the bound residue fractions relatively more radioactivity was measured in fulvic and humic acids after straw amendment. Increasing temperatures promoted the dissipation of MBT and the formation of bound residues in both treatments, but without amendment of maize straw these effects were far less pronounced. The laboratory scale degradation experiment led to similar results as were found in a corresponding lysimeter study. Differences that were observed could be explained by different temperature regimes of the experiments and time of aging in soil.     

Metabolism of 14C‐carbaryl and 14C‐1‐naphthol in moist and flooded soils

Abstract

The metabolism of ¹⁴C‐carbaryl and ¹⁴C‐1‐naphthol in moist and flooded soils was studied in a continuous flow‐through system over a period of 28 days permitting ¹⁴C‐mass balance. The percent distribution of radiocarbon in organic volatiles, carbon dioxide, extractable and non‐extractable (bound) fractions of soils were determined. Organic volatiles could not be detected in both carbaryl and 1‐naphthol treated soils. More of ¹⁴CO₂ (25.6%) was evolved from moist than flooded soil (15.1%) treated with carbaryl. However, the mineralization of ¹⁴C‐1‐naphthol was negligible. The extractable radiocarbon was more in flooded soil (28.9%) than moist soil (5.5%) from carbaryl treatment. Less than one percent was present as parent compound, whereas carbaryl was mainly metabolized to 5‐hydroxy carbaryl in moist soil and to 4‐ and 5‐hydroxy carbaryl in flooded soil. The extractable radiocarbon amounted to 18.2 and 24.3% in moist and flooded soils respectively and the parent compound was less than one percent with 1‐naphthol treatment. Most of the radiocarbon was found as soil bound residues; the formation being more with 1‐naphthol than carbaryl. Humin fraction of the soil organic matter contributed most to soil bound residues of both carbaryl and 1‐naphthol.     

Soil bound residues of carbaryl and 1‐naphthol: Release and mineralization in soil,and uptake by plants

Abstract

¹⁴C‐carbaryl and ¹⁴C‐1‐naphthol form soil bound residues which get partially released when barley was grown. ¹⁴C‐residues could be detected in both shoot and root in the case of carbaryl treatment while only roots showed ¹⁴C‐residues in the case of 1‐naphthol. Flooding enhanced release of the bound residues while soil amendment did not. There was greater mineralization of bound residues of carbaryl than that of 1‐naphthol. Rice straw amendment enhanced mineralization.     

Comparative studies of the fate of atrazine‐14C and pentachlorophenol‐14C in various laboratory and outdoor soil‐plant systems

Abstract

Mass balance and fate of atrazine‐ ¹⁴C and pentachlorophenol‐ ¹⁴C (PCP‐ ¹⁴C) were studied in short‐term tests in a closed aerated laboratory soil‐plant system, using two concentrations in soil and two plant species, as well as under outdoor conditions for one vegetation period. In the laboratory, for both pesticides bioaccu‐mulation factors of radiocarbon taken up by the roots into plants were low. They were higher for lower (1 ppm) than for higher soil concentrations (6 ppm for atra‐zine, 4 ppm for pentachlorophenol) and varied with the plant species. Mineralization to ¹⁴CO₂ in soil was negatively related to soil concentration only for PCP‐ ¹⁴C. Conversion rates in soil including the formation of soil‐bound residues were higher for the lower concentrations of both pesticides than for the higher ones; conversion rates in plants were species‐dependent. In 14 terms of CO₂ formation and of conversion rates, PCP was less persistent in soil than was atrazine. For both pesticides, laboratory data on conversion and mineralization gave a rough prediction of their persistence in soil under long‐term outdoor conditions, whereas bio‐accumulation factors in plants under long‐term outdoor conditions could not be predicted by short‐term laboratory experiments.     

Soil-catalyzed oxidation of aniline

Aniline partially degraded in sterile soil to azobenzene, azoxybenzene, phenazine, form=anilide, and acetanilide. Nitrobenzene, $\text{p}$-benzoquinone, and unidentified species were possible products; substantial bound residues may also have formed. Soil-catalyzed conversion of aniline or [$\text{d}$₅]aniline seems evidenced by 6-24X more product recovery in sterile soil than in sterile water alone, a process inhibited by Na₂S₂O₄. Freundlich adsorption constants showed: azobenzene > azoxybenzene > phenazine > aniline.     

Laboratory studies on volatilization and mineralization of 14c‐p,p'‐DDT in soil,release of bound residues and dissipation from solid surfaces

Abstract

In support of field data, laboratory studies were conducted on volatilization, mineralization and binding of ¹⁴C‐p,p'‐DDT in soils at Sao Paulo. Incubation of soil for 6 weeks did not result in volatilized organics or mineralization; with >95% extractable radiocarbon in the form of p,p'‐DDT. Small amounts of bound residues (1.8%) were detected in soil. These data confirm the very slow dissipation of DDT in the field which presumably relates to the acidic pH of soil (4.5–4.8).

Bound ¹⁴C‐residues in soils treated with ¹⁴C‐p,p'‐DDT at Praia Grande and Sao Paulo could be released (5–21%) by sulphuric acid treatment. The released residue had the composition: 69–90% DDT, 7–32% DDD and 0–3% DDE. Incubation of soil bound ¹⁴C‐residues with fresh inoculum for 3 months did not result in release of ¹⁴C.

Dissipation from wooden surfaces was fairly slow. After 20 weeks, 74% of the applied radioactivity could be recovered; 44% hexane‐non‐extractable.     

Characterization of non-extractable residues of the fungicide dithianon in soil using 13C/14C-labelling techniques

The fate of the (14)C-labelled fungicide dithianon in soil is characterized by the formation of non-extractable, "bound" residues of approximately 63% of applied amount in 64 d. Humic acids containing these "bound" residues were isolated after conducting degradation studies of the active ingredient in an orthic luvisol under standardized conditions. In the same way, (13)C-labelled dithianon was incubated in an artificial soil which was produced by humification of (13)C-depleted straw in an incinerated soil. The "bound" residues of the (13)C-labelled dithianon in the humic acid fraction of the artificial soil were analyzed using (13)C-NMR techniques. There was no evidence of a covalent bonding of the residues to the humic substances. Results of polarity gradient high performance thin layer chromatography (AMD-HPTLC) of "bound" residues of the (14)C-labelled dithianon in the humic acid fraction indicate a sequestration process of metabolites into the humic substance as a possible binding mechanism.     

Behaviour of [ring-C] hydroxy-simazine in a parabraun soil

[Ring-¹⁴C] hydroxy-simazine was incubated in a parabraun soil for 102 days at 22°C and 65 % of the maximum water-holding capacity. Soil samples were analyzed according to two different extraction methods on the day of application as well as after 13, 62 and 102 days. Furthermore, the influence of air-drying the soil specimens on the extraction yield was also studied. Hydroxy-simazine was converted to ¹⁴CO₂ only up to 0.2 % of the applied radioactivity. A rapid bonding of the radioactivity to the organic mass of the soil was observed, especially in the fulvic acid and humin fractions. 62 days after beginning conversion these two fractions already altogether contained up to 85 % of the labelled ring carbon applied. Whereas in the humic acids only a maximum of 4.5 % was discovered.     

Fate of 3,3′,4,4′-tetrachloroazobenzene in soybean plants grown in treated soils

Three soils of varying organic matter (OM) concentrations (0, 1.7 and 57%) were treated with 3,3′4,4′-tetrachlorazobenzene (TCAB) at the 25 ppm level. Germinated soybeans$\text{(Glycine}$$\text{max}$ (L.) Merr.) were planted in the treated soils, along with controls, and grown for 12 days. The shoots, roots and soil were air-dried and analyzed for TCAB and 3,3′,4,4′-tetrachloroazoxybenzene (TCAOB). TCAB appears to translocate from the treated soil into the plant shoots and roots. Residue levels varied with the percentage organic matter of each soil; levels as high as 58.4 ppm were identified in roots of soybeans grown in 1.7% OM soil and 0.620 ppm in the shoots from 0% OM soil. TCAOB was identified in soil and root extracts with the highest levels in soybean roots grown in 0% OM soil, 0.317 ppm. Residues of TCAB and TCAOB decreased in soil and root and shoot tissues as percentage OM increased. Bound residues of TCAB were released from roots grown in 0% OM soil by refluxing with boron trifluoride methanol (BF₃CH₃OH).     

Metabolic fate of the 14C-labeled herbicide clodinafop-propargyl in a sediment–water system

The metabolic fate of ¹⁴C-phenyl-labeled herbicide clodinafop-propargyl (¹⁴C-CfP) was studied for 28 days in lab assays using a sediment–water system derived from a German location. Mineralization was 5.21% of applied ¹⁴C after 28 days exhibiting a distinct lag phase until day 14 of incubation. Portions of radioactivity remaining in water phases decreased at moderate rate to 18.48% after 28 days; 62.46% were still detected in water after 14 days. Soxhlet extraction of the sediment using acetonitrile released 35.56% of applied ¹⁴C with day 28, while 33.99% remained as non-extractable residues. A remarkable increase of bound ¹⁴C was observed between 14 and 28 days correlating with the distinct increase of mineralization. No correlation was found throughout incubation with microbial activity of the sediment as determined by dimethyl sulfoxide reduction. Dissolved oxygen and pH value of water phases remained almost constant for 28 days. Analyses of Soxhlet extracts of the sediment and ethyl acetate extracts of water phases by radio-TLC and radio-HPLC revealed that CfP was rapidly cleaved to free acid clodinafop (Cf), which was further (bio-) transformed. DT₅₀ values (based on radio-HPLC) were below 1 day (CfP) and slightly above 28 days (Cf). Further metabolites were not detected. Fractionation of humic and non-humic components of the sediment demonstrated that CfP's non-extractable residues were predominantly associated with fulvic acids up to 14 days of incubation (3.36%), whereas after 28 days, the majority of radioactivity was found in the humin/mineral fraction (13.30% of applied ¹⁴C). Due to high-performance size-exclusion chromatography of the fulvic acids fraction derived from assays incubated for 28 days, this portion of ¹⁴C was firmly, possibly covalently bound to fulvic acids and did not consist of CfP or Cf. Using an isolation strategy comprising preincubation of sediment with CfP and mineralization of ¹⁴C-CfP as criterion, a microorganism was isolated from the sediment examined. It grew on ¹⁴C-CfP as sole carbon source with evolution of ¹⁴CO₂. The bacterium was characterized by growth on commonly used carbon sources and 16S rDNA sequence analysis. Its sequence exhibited high similarity with that of Nocardioides aromaticivorans strain H-1 (98.85%; DSM 15131, JCM 11674).