The anaerobic degradation of detergent range fatty alcohol ethoxylates. Studies with 14C-labelled model surfactants

The anaerobic degradation of fatty alcohol polyglycol ethers was studied in a model sludge digester employing stearyl alcohol ethoxylate which was ¹⁴C-labelled either in the alkyl or in the heptaglycol chain. After 4-weeks' incubation of the ¹⁴C-compounds at 35°C in the presence of raw sludge as additional digestible substrate more than 80% of the initial radioactivity was found as methane and carbon dioxide. In addition, the major part of radioactivity in the digested sludge, corresponding to nearly 10% of added ¹⁴C, was attributable to biomass so that ultimate degradation of the two model surfactants amounted to more than 90%. Analysis of the small fraction of radiolabelled metabolites in the sludge supernatant allowed conclusions with regard to the anaerobic degradation route of linear alcohol ethoxylates. After primary biodegradation of the surfactant molecule by scission into the alkyl and poly(ethylene glycol) moieties the further biodegradation of the latter seems to proceed as under aerobic conditions, i.e. via oxidative or hydrolytic depolymerization steps. Eventually, ultimate biodegradation of the obtained monomers (C₂-units) leads to the formation of the gaseous end products.     

Biodegradability and toxicity of sulphonate-based surfactants in aerobic and anaerobic aquatic environments

Four types of commonly used sulphonate-based surfactants (alkane sulphonates, alpha-olefin sulphonates, sulphosuccinates and methyl ester sulphonates) were tested for their aerobic and anaerobic biodegradability as well as for their toxicity to Daphnia magna and Photobacterium phosphoreum to assess the effect of the surfactant structure on those properties. Aerobic biodegradation was evaluated by means of the CO₂ headspace test and anaerobic biodegradation was assessed by a method based on the ECETOC test. All the surfactants tested were readily biodegraded under aerobic conditions. No clear effect of the surfactant structures on the toxicity to the aquatic organisms tested was found. The most significant differences in the surfactants studied were observed in their behaviour under anaerobic conditions. Alkane sulphonates, alpha-olefin sulphonates and methyl ester sulphonates were not mineralized in lab anaerobic digesters despite the fact that the last one showed a certain degree of primary degradation. Nevertheless, these surfactants did not significantly inhibit methanogenic activity at concentrations up to 15 g surfactant/kg dry sludge, a concentration that is much higher than the expected concentrations of these surfactants in real anaerobic digesters. Sulphosuccinates showed a high level of primary biodegradation in anaerobic conditions. However, linear alkyl sulphosuccinates were completely mineralized whereas branched alkyl sulphosuccinates achieved percentages of ultimate biodegradation ≤50%.     

Biodegradation of a cationic surfactant in activated sludge

The biodegradation of DTDMAC (ditallowdimethylammonium chloride, a fabric softening agent) was established in semi-batch activated sludge reactors. Three ¹⁴C-forms of DTDMAC were studied separately under the simulated organic loading rates of conventional and extended aeration activated sludge treatment. Primary biodegradation was shown by means of a specific analytical technique in combination with radiochemical procedures. Ultimate biodegradation for each carbon position in the DTDMAC molecule was established by the detection of ¹⁴CO₂. Intermediate metabolites were followed throughout the study by both radio thin-layer chromatography and radiochemical procedures. Each carbon position of the DTDMAC molecule was equally accessible to metabolism and ultimate degradation by acclimated microorganisms. Degradation occurred more rapidly under extended aeration conditions and was influenced by the strong tendency of DTDMAC to associate with the microbial population. The low levels of metabolites observed were not of a single classification or characteristic and they did not persist in the activated sludge. This study suggests that DTDMAC removal in an activated sludge plant is a result of both sorption/precipitation and biodegradation mechanisms.     

Anaerobic degradation of nonionic and anionic surfactants in enrichment cultures and fixed-bed reactors

Anaerobic biodegradation and inhibitory effects of nonionic and anionic surfactants on methanogenic fermentation were tested in incubation experiments with anoxic sediment samples and sewage sludge. Alkylsulfonates and alkylbenzenesulfonates were not degraded but inhibited methanogenesis from sludge constituents at concentrations ≥10 mgl⁻¹. Sodium dodecylsulfate was at least partly degraded after adaptation at concentrations 100 mgl⁻¹ and the sulfate group was reduced to sulfide. The polyethyleneglycol moiety of alkylphenolethoxylates was fermented to methane at concentrations 500 mgl⁻¹ whereas the alkylphenol residue probably remained unchanged. Alkylethoxylates were completely degraded to methane and CO₂ at concentrations up to 1.0 gl⁻¹. Complete anaerobic degradation of this surfactant type to methane, CO₂, and traces of acetate and propionate was demonstrated in a lab scale anaerobic fixed-bed reactor, either with prereduced mineral salts medium or with air-saturated artificial wastewater. This process lends itself as a suited, inexpensive means for treatment of wastewaters containing enhanced loads of nonionic surfactants, e.g. from the surfactant manufacturing or processing industry.     

Biodegradation of 3,4,4′-trichlorocarbanilide, TCC, in sewage and activated sludge

As part of the studies to elucidate the environmental consequences from bacteriostat usage the extent of biodegradation of 3,4,4′-trichlorocarbanilide, TCC^®, in sewage systems was examined. TCC samples uniformly labeled in either the p-chloroaniline ring (¹⁴C PCA TCC) or the dichloroaniline ring (¹⁴C DCA-TCC) were monitored in activated sludge systems by measurements of ¹⁴CO₂ evolution. As was expected, the p-chloroaniline (PCA) ring of TCC was more rapidly degraded than the dichloroaniline (DCA) ring. In a continuous flow activated sludge system (10 h retention time, 200 μg1⁻¹ TCC) acclimation to primary biodegradation was readily gained. ¹⁴CO₂ evolution from ¹⁴C PCA TCC was consistent with complete metabolism of the PCA ring while that from ¹⁴C-PCA-TCC indicated about 50% biodegradation of the DCA ring. Analysis of effluents from continuous flow activated sludge units established that TCC undergoes primary biodegradation to its chloroaniline components which are in turn biodegraded.     

Fossil organic carbon in wastewater and its fate in treatment plants

This study reports the presence of fossil organic carbon in wastewater and its fate in wastewater treatment plants. The findings pinpoint the inaccuracy of current greenhouse gas accounting guidelines which defines all organic carbon in wastewater to be of biogenic origin. Stable and radiocarbon isotopes (¹³C and ¹⁴C) were measured throughout the process train in four municipal wastewater treatment plants equipped with secondary activated sludge treatment. Isotopic mass balance analyses indicate that 4–14% of influent total organic carbon (TOC) is of fossil origin with concentrations between 6 and 35 mg/L; 88–98% of this is removed from the wastewater. The TOC mass balance analysis suggests that 39–65% of the fossil organic carbon from the influent is incorporated into the activated sludge through adsorption or from cell assimilation while 29–50% is likely transformed to carbon dioxide (CO₂) during secondary treatment. The fossil organic carbon fraction in the sludge undergoes further biodegradation during anaerobic digestion with a 12% decrease in mass. 1.4–6.3% of the influent TOC consists of both biogenic and fossil carbon is estimated to be emitted as fossil CO₂ from activated sludge treatment alone. The results suggest that current greenhouse gas accounting guidelines, which assume that all CO₂ emission from wastewater is biogenic may lead to underestimation of emissions.     

Fate of C-triclocarban in biosolids-amended soils

Triclocarban (TCC) is an antibacterial compound commonly detected in biosolids at parts-per-million concentrations. Approximately half of the biosolids produced in the United States are land-applied, resulting in a systematic release of TCC into the soil environment. The extent of biosolids-borne TCC environmental transport and potential human/ecological exposures will be greatly affected by its bioavailability and the rate of degradation in amended soils. To investigate these factors, radiolabeled TCC (¹⁴C-TCC) was incorporated into anaerobically digested biosolids, amended to two soils, and incubated under aerobic conditions. The evolution of ¹⁴CO2 (biodegradation) and changes in chemical extractability (bioavailability) was measured over time. Water extractable TCC over the study period was low and significantly decreased over the first 3 weeks of the study (from 14% to 4% in a fine sand soil and from 3 to < 1% in a silty clay loam soil). Mineralization (i.e. ultimate degradation), as measured by evolution of ¹⁴CO₂, was < 4% over 7.5 months. Methanol extracts of the amended soils were analyzed by radiolabel thin-layer chromatography (RAD-TLC), but no intermediate degradation products were detected. Approximately 20% and 50% of the radioactivity in the amended fine sand and silty clay loam soils, respectively, was converted to bound residue as measured by solids combustion. These results indicate that biosolids-borne TCC becomes less bioavailable over time and biodegrades at a very slow rate.     

The effect of water hardness on nitrilotriacetate removal and microbial acclimation in activated sludge

The effect of water hardness on microbial acclimation to the removal of nitrilotriacetate (NTA) in the wastewater treatment process was examined in 4 laboratory-scale activated sludge reactors. Consistent high removal (i.e. >90%) was observed in all 4 reactors after acclimation occurred. Removals of greater than 60% of added NTA after 8 days and greater than 90% after 15 days were observed in a continuously fed, hard water (250 mg l⁻¹ as CaCO₃) reactor. In soft water (50 mg l⁻¹ as CaCO₃) comparable removals were observed after 20 and 28 days respectively. In 24 h, semi-continuous activated sludge reactors (24 h, fill-and-draw type) removal was greater in hard water than in soft water initially, but was greater than 95% in both hard and soft water after 4 weeks.The proportion of bacteria in the activated sludge with the ability to utilize NTA increased approx. 100-fold during the acclimation period. This proportion increased faster in hard water than in soft water and faster in semi-continuous activated sludge reactors than continuous activated sludge reactors.Ultimate biodegradation of ¹⁴C[U]NTA was greater than 85% in both hard and soft water acclimated units. Accumulation of ¹⁴C in the sludge solids was negligible (<1%).     

Degradation of 14c‐maneb in sediment from a Nicaraguan estuary

Sediment samples were collected at 5 sites in the Nicaraguan estuary “El Naranjo” in July 94 and September 94. The samples were incubated with ¹⁴C‐maneb (0.08 μg.g^‐1 dw sediment), and evolved ¹⁴CO₂ and residual ¹⁴C‐ETU in soil were measured. Mineralization kinetics of ^I4C‐maneb was best described with kinetic models which include growth of microorganisms. The amounts of ¹⁴C‐maneb mineralized were highest at the sites closest to the mouth of the river. No significant differences in degradation between July and September were seen. After 67 days between 9.73 and 16.18% of added ¹⁴C had evolved as ¹⁴CO₂ in the July samples and after 150 days between 11.18 and 27.37% of added ¹⁴C had evolved as ¹⁴CO₂ from the September samples. When 4.61–8.20% of added ^I4C was found in the soil extract, 0.00–2.72% was ¹⁴C‐ETU.     

A novel separation of tritiated water formed by biodegradation of surfactant samples and its application to a sample preservation study

Primary biodegradation of a NEODOL^® ethoxylate labeled with tritium primarily in the α and γ positions of the alkyl chain and uniformly with ¹⁴C in the polyoxyethylene chain resulted in a very rapid release of tritiated water (HTO) by cleavage of the surfactant at the hydrophobe-hydrophile junction. This reaction was used to investigate the conditions necessary to preserve samples of biotreater clarifier effluent and mixed liquor suspended solids (MLSS). Treatment of the sample with granular activated carbon, followed by filtration, readily isolated the tritiated water. The amount of HTO formed indicated the extent of continued degradation of the surfactant. It was concluded that refrigeration alone was not adequate to prevent primary degradation of the surfactant. Refrigeration in combination with either formalin or 200 ppm HgCl₂ prevented short-term degradation of the surfactant. Formalin, but not HgCl₂, was found adequate for preventing degradation in samples of mixed liquor suspended solids preserved in excess of 14 days.     

Biodegradation of the endogenous residue of activated sludge

This study evaluated the potential biodegradability of the endogenous residue in activated sludge subjected to batch digestion under either non-aerated or alternating aerated and non-aerated conditions. Mixed liquor for the tests was generated in a 200 L pilot-scale aerobic membrane bioreactor (MBR) operated at a 5.2 days SRT. The MBR system was fed a soluble and completely biodegradable synthetic influent composed of sodium acetate as the sole carbon source. This influent, which contained no influent unbiodegradable organic or inorganic materials, allowed to generate sludge composed of essentially two fractions: a heterotrophic biomass X_H and an endogenous residue X_E, the nitrifying biomass being negligible (less than 2%). The endogenous decay rate and the active biomass fraction of the MBR sludge were determined in 21-day aerobic digestion batch tests by monitoring the VSS and OUR responses. Fractions of X_H and X_E: 68% and 32% were obtained, respectively, at a 5.2 days SRT. To assess the biodegradability of X_E, two batch digestion units operated at 35 °C were run for 90 days using thickened sludge from the MBR system. In the first unit, anaerobic conditions were maintained while in the second unit, alternating aerated and non-aerated conditions were applied. Data for both units showed apparent partial biodegradation of the endogenous residue. Modeling the batch tests indicated endogenous residue decay rates of 0.005 d⁻¹ and 0.012 d⁻¹ for the anaerobic unit and the alternating aerated and non-aerated conditions, respectively.     

The application of an ether cleavage technique to a study of the biodegradation of a linear alcohol ethoxylate nonionic surfactant

The biodegradation of a linear alcohol ethoxylate nonionic surfactant (C₁₂-C₁₅, eo₉) was studied by gas chromatography of the alkyl bromides produced by acid cleavage of the surfactant ether linkages with hydrogen bromide. In laboratory cultures and in a lake environment, a conventional method showed rapid loss of surface activity. The gas chromatographic method revealed a rapid degradation of the surfactant alkyl moiety but a slow and incomplete removal of the polyethoxylate moiety.     

Evaluation and modeling of benzalkonium chloride inhibition and biodegradation in activated sludge

The inhibitory effect and biodegradation of benzalkonium chloride (BAC), a mixture of alkyl benzyl dimethyl ammonium chlorides with different alkyl chain lengths, was investigated at a concentration range from 5 to 20 mg/L and different biomass concentrations in an activated sludge system. A solution containing glucose and mineral salts was used as the wastewater in all the assays performed. The inhibition of respiratory enzymes was identified as the mode of action of BAC as a result of oxygen uptake rate analysis performed at BAC concentrations ranging between 5 and 70 mg/L. The glucose degradation in the activated sludge at different BAC and biomass concentrations was well-described with Monod kinetics with competitive inhibition. The half-saturation inhibition constant (K_I) which is equivalent to EC₅₀ of BAC for the activated sludge tested ranged between 0.12 and 3.60 mg/L. The high K_I values were recorded at low BAC-to-biomass ratios, i.e. less than 10 mg BAC/g VSS, at which BAC was almost totally adsorbed to biomass and not bioavailable. BAC degradation started as soon as glucose was totally consumed. Although BAC was almost totally adsorbed on the biomass, it was degraded completely. Therefore, BAC degradation was modeled using two-phase biodegradation kinetics developed in this study. This model involves rapid partitioning of BAC to biomass and consecutive degradation in both aqueous and solid phases. The aqueous phase BAC degradation rate was twenty times, on average, higher than the solid phase degradation rate. The specific aqueous (k_I1) and solid (k_I2) phase BAC utilization rate constants were 1.25 and 0.31 mg BAC/g VSS h, respectively. The findings of this study would help to understand the reason of extensive distribution of quaternary ammonium compounds in wastewater treatment plant effluents and in natural water systems although QACs are biodegradable, and develop strategies to avoid their release and accumulation in the environment.     

Analyse des anaeroben Benzolabbaus: Vergleich von In-situ-Mikrokosmen, Elektronenakzeptorbilanzen und Isotopenfraktionierungsprozessen

Geochemical and isotope chemical methods were applied to assess the in situ biodegradation of benzene in a shallow aquifer (Zeitz, Germany). The vertical structure of the plume was investigated in a multi level sampling approach. Benzene degradation was investigated using in situ microcosms incubated with [¹³C₆]-labelled benzene. The transformation of ¹³C into fatty acids proved biodegradation was occurring, and indicated that a complex microbial community is colonising the in situ microcosms. The depletion of sulphate in the plume indicated sulphate-reducing conditions may account for the oxidation of 1.7?mmol/l benzene. The concentration and isotopic composition of dissolved inorganic carbon indicated a degradation of 2.2?mmol/l benzene within the plume. The isotopic composition of benzene suggested an average degradation of 3.0?mmol/l benzene. The various approaches used to assess benzene degradation were within the same order, and illustrated significant benzene degradation in the anaerobic aquifer.     

Synergetic pretreatment of sewage sludge by microwave irradiation in presence of H2O2 for enhanced anaerobic digestion

A microwave-enhanced advanced hydrogen peroxide oxidation process (MW/H₂O₂-AOP) was studied in order to investigate the synergetic effects of MW irradiation on H₂O₂ treated waste activated sludges (WAS) in terms of mineralization (permanent stabilization), sludge disintegration/solubilization, and subsequent anaerobic biodegradation as well as dewaterability after digestion. Thickened WAS sample pretreated with 1 g H₂O₂/g total solids (TS) lost 11-34% of its TS, total chemical oxygen demand (COD) and total biopolymers (humic acids, proteins and sugars) via advanced oxidation. In a temperature range of 60-120 °C, elevated MW temperatures (>80 °C) further increased the decomposition of H₂O₂ into OH^• radicals and enhanced both oxidation of COD and solubilization of particulate COD (>0.45 micron) of WAS indicating that a synergetic effect was observed when both H₂O₂ and MW treatments were combined. However, at all temperatures tested, MW/H₂O₂ treated samples had lower first-order mesophilic (33 ± 2 °C) biodegradation rate constants and ultimate (after 32 days of digestion) methane yields (mL per gram sample) compared to control and MW irradiated WAS samples, indicating that synergistically (MW/H₂O₂-AOP) generated soluble organics were slower to biodegrade or more refractory than those generated during MW irradiation.     

Biodegradation of oxo-alcohol ethoxylates in the continuous flow activated sludge simulation test

Biodegradation of two alpha-methyl branched oxo-alcohol ethoxylates (OAE) of different polydispersity: LIAL 125/14 BRD (LIALB) (broad M.W. distribution) and LIAL 125/14 NRD (LIALN) (narrow M.W. distribution), both having an average of 14 oxyethylene subunits (EO) and a C(12-15) alkyl moiety were tested under the continuous flow activated sludge conditions of the classical Husmann plant. Primary biodegradation and concentration of metabolites: free oxo-alcohol fraction (FOA) and poly(ethylene glycols) (PEG), were measured. PEG were divided into two fractions: short-chained PEG (PEGshch) (1-4 EO) and long-chained PEG (PEGlch) (>4 EO). The indirect tensammetric technique combined with an adequate separation was used for analysis. Central fission was found to be a highly dominating pathway, as is the case with fatty alcohol ethoxylates. OAE are highly primarily biodegraded (above 95%). High concentrations of FOA and PEG are formed. Once formed the PEGlch are further fragmented into the PEGshch. Free alcohol fraction compounds are biodegraded sooner when alkyl moiety is shorter. OAE polydispersity has an influence on the kinetics of biodegradation; PEG formed from LIALN are biodegraded slower and to a lower degree than those from LIALB.     

Effect of linear alkylbenzene sulphonates (LAS) on the anaerobic digestion of sewage sludge

Batch anaerobic biodegradation tests with different alkylbenzene sulphonates (LAS) at increasing concentrations were performed in order to investigate the effect of LAS homologues on the anaerobic digestion process of sewage sludge. Addition of LAS homologues to the anaerobic digesters increased the biogas production at surfactant concentrations 5-10 g/kg dry sludge and gave rise to a partial or total inhibition of the methanogenic activity at higher surfactant loads. Therefore, at the usual LAS concentration range in sewage sludge, no adverse effects on the anaerobic digesters functioning of a wastewater treatment plant (WWTP) can be expected. The increase of biogas production at low surfactant concentrations was attributed to an increase of the bioavailability and subsequent biodegradation of organic pollutants associated with the sludge, promoted by the surfactant adsorption at the solid/liquid interface. When the available surfactant fraction in the aqueous phase instead of the nominal surfactant concentration was used to evaluate the toxicity of LAS homologues, a highly significant relationship between toxicity and alkyl chain length was obtained. Taking into account the homologue distribution of commercial LAS in the liquid phase of the anaerobic digesters of a WWTP, an EC(50) value of 14 mg/L can be considered for LAS toxicity on the anaerobic microorganisms.     

Intrinsic biodegradation potential of aromatic hydrocarbons in an alluvial aquifer--potentials and limits of signature metabolite analysis and two stable isotope-based techniques

Three independent techniques were used to assess the biodegradation of monoaromatic hydrocarbons and low-molecular weight polyaromatic hydrocarbons in the alluvial aquifer at the site of a former cokery (Flémalle, Belgium).Firstly, a stable carbon isotope-based field method allowed quantifying biodegradation of monoaromatic compounds in situ and confirmed the degradation of naphthalene. No evidence could be deduced from stable isotope shifts for the intrinsic biodegradation of larger molecules such as methylnaphthalenes or acenaphthene. Secondly, using signature metabolite analysis, various intermediates of the anaerobic degradation of (poly-) aromatic and heterocyclic compounds were identified. The discovery of a novel metabolite of acenaphthene in groundwater samples permitted deeper insights into the anaerobic biodegradation of almost persistent environmental contaminants. A third method, microcosm incubations with ¹³C-labeled compounds under in situ-like conditions, complemented techniques one and two by providing quantitative information on contaminant biodegradation independent of molecule size and sorption properties. Thanks to stable isotope labels, the sensitivity of this method was much higher compared to classical microcosm studies. The ¹³C-microcosm approach allowed the determination of first-order rate constants for ¹³C-labeled benzene, naphthalene, or acenaphthene even in cases when degradation activities were only small. The plausibility of the third method was checked by comparing ¹³C-microcosm-derived rates to field-derived rates of the first approach. Further advantage of the use of ¹³C-labels in microcosms is that novel metabolites can be linked more easily to specific mother compounds even in complex systems. This was achieved using alluvial sediments where ¹³C-acenaphthyl methylsuccinate was identified as transformation product of the anaerobic degradation of acenaphthene.     

Biodegradation potential of bulking agents used in sludge bio-drying and their contribution to bio-generated heat

Straw and sawdust are commonly used bulking agents in sludge composting or bio-drying. It is important to determine if they contribute to the biodegradable volatile solids pool. A sludge bio-drying process was performed in this study using straw, sawdust and their combination as the bulking agents. The results revealed that straw has substantial biodegradation potential in the aerobic process and sawdust has poor capacity to be degraded. The temperature profile and bio-drying efficiency were highest in the trial that straw was added, as indicated by a moisture removal ratio and VS loss ratio of 62.3 and 31.0%, respectively. In separate aerobic incubation tests, straw obtained the highest oxygen uptake rate (OUR) of 2.14 and 4.75 mg O₂ g⁻¹VS h⁻¹ at 35 °C and 50 °C, respectively, while the highest OUR values of sludge were 12.1 and 5.68 mg O₂ g⁻¹VS h⁻¹ at 35 °C and 50 °C and those of sawdust were 0.286 and 0.332 mg O₂ g⁻¹VS h⁻¹, respectively. The distribution of biochemical fractions revealed that soluble fractions in hot water and hot neutral detergent were the main substrates directly attacked by microorganisms, which accounted for the initial OUR peak. The cellulose-like fraction in straw was transformed to soluble fractions, resulting in an increased duration of aerobic respiration. Based on the potential VS degradation rate, no bio-generated heat was contributed by sawdust, while that contribution by straw was about 41.7% and the ratio of sludge/straw was 5:1 (w/w, wet basis).     

Transformation of C-pyrimidynyloxybenzoic herbicide ZJ0273 in aerobic soils

A soil metabolism study of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate (ZJ0273), a novel broad-spectrum herbicide, was carried out using ¹⁴C labeled on two different rings, i.e., [pyrimidine-4,6-¹⁴C] ZJ0273 and [benzyl-U-¹⁴C] ZJ0273. Ultralow liquid scintillation counting and LC-MS/MS were used to identify the degradation intermediates and quantify their dynamics in aerobic soils. Four aromatic intermediates, 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoic acid (M1), 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzamido)benzoic acid (M2), 2-(4,6-dimethoxypyrimidin-2-yloxy)benzoic acid (M3), and 4,6-dimethoxypyrimidin-2-ol (M4), were identified and their identity was further confirmed against authentic standards. Analysis of metabolites suggested two degradation pathways: (1) Upon loss of the propyl group, M1 was produced via hydrolysis of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate after which the C-N bond between rings A and B was cleaved by oxidation and biochemical degradation to yield M3, which was further converted into M4 and finally mineralized to CO₂; and (2) the first step was the same as in pathway 1, but M1 first underwent a carbonylation to form M2. The C-N bond between rings A and B of M2 was cleaved by hydrolysis to yield M3. Dynamic changes in the four metabolites in aerobic soils were also investigated by HPLC coupled analysis of radioactivity of isolated peaks. After a 100-day incubation, 1.7-9.7% of applied ¹⁴C was found as M1, 0.3-1.1% as M2, 14.5-20.9% as M3, and 3.7-6.7% as M4 in the soils, and pH appeared to be the most influential soil property affecting the formation and dissipation of these metabolites.