Metabolism of a DDT metabolite via a chloroepoxide

The 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) metabolic intermediate 1-chloro-2,2-bis(p-chlorophenyl)ethene (DDMU) is partially metabolized in vivo by mice to 2-hydroxy-2,2-bis(p-chlorophenyl)acetic acid (αOH-DDA) and other metabolites which are excreted in urine. The subsequent DDT metabolic intermediates 1-chloro-2,2-bis(p-chlorophenyl)ethane (DDMS) and 1,1-bis(p-chlorophenyl)ethene (DDNU) are metabolized to αOH-DDA to a much lesser extent. These results imply that DDMU may be metabolized via an α-chloroepoxide. The authentic DDMU-epoxide, which after oral administration is excreted as αOH-DDA, is mutagenic in the Ames assay, and thermally rearranges rapidly to the corresponding α-chloroaldehyde, 2,2-bis(p-chlorophenyl)-2-chloroacetaldehyde (αCl-DDCHO). As expected αCl-DDCHO yielded the same urinary metabolites as DDMU-epoxide. This suggested metabolic pathway for DDMU via a chloroepoxide intermediate may account for the tumorigenicity of DDT in mice.     

Metabolism of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane in the mouse

The metabolites of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) found in the urine of female Swiss mice are reported. The metabolites of DDT are DDD, 1-chloro-2,2-bis(p-chlorophenyl)ethene (DDMU), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethene (DDE), 2,2-bis(p-chlorophenyl)acetic acid (DDA), 2-hydroxy-2,2-bis(p-chlorophenyl)acetic acid (αOH-DDA) and 2,2-bis(p-chlorophenyl)ethanol (DDOH), while DDD afforded DDMU, DDE, DDA, αOH-DDA and DDOH. The relative excreted levels of DDA and DDOH and the absence of 2,2-bis(p-chlorophenyl)acetaldehyde (DDCHO) are not consistent with the generally accepted path way for DDA formation, which involves sequential metabolism of DDT and DDD via DDOH to afford DDA. The quantitative results are interpreted to mean that DDA is formed by hydroxylation at the chlorinated sp³-side chain carbon of DDD to give 2,2-bis(p-chlorophenyl)acetyl chloride (DDA-Cl), which in turn is hydrolyzed to DDA. The excretion of αOH-DDA from both DDT- and DDD-treated mice has never been previously observed. It is suggested that this metabolite arises from the initial epoxidation of DDMU, a metabolite of DDT and DDD, to yield 1,2-epoxy-1-chloro-2,2-bis(p-chlorophenyl)ethane (DDMU-epoxide). This chloroepoxide is then hydrolyzed and oxidized to produce the αOH-DDA.     

Tissue Residue Guideline for ∑DDT for Protection of Aquatic Birds in China

DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) is a chlorinated hydrocarbon insecticide that has been used worldwide. While the use of DDT has been phased out in many countries, it is still produced in some parts of the world for use to control vectors of malaria. DDE (1,1,-dichloro-2,2-bis(p-chlorophenyl)ethylene) and DDD (1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) are primary metabolites of DDT and have similar chemical and physical properties. DDT and its metabolites (DDE and DDD) are collectively referred to as ∑DDT. The lipophilic nature and persistence of the ∑DDT result in biomagnification in wildlife that feed at higher trophic levels in the food chain. Wildlife in aquatic ecosystems depend on aquatic biota as their primary source of food, which provide the main route of exposure to ∑DDT. Studies about effects of ∑DDT on birds were reviewed. The tissue residue guidelines for DDT (TRGs) for protection of birds in China were derived using species sensitivity distribution (SSD) and toxicity percentile rank method (TPRM) based on the available toxicity data. Risks of ∑DDT to birds were assessed by comparing the TRGs and ∑DDT concentrations in fishes from China. The tissue residue guideline for protection of birds in China is recommended to be 12.0 ng ∑DDT/g food.     

Reactions of vitamin B12r with polyhalogenated hydrocarbon pesticides

The reaction of vitamin B_12r, generated by photolysis of methylcobalamin under a nitrogen atmosphere, with 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), results in extensive dechlorination and formation of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) as the major products. Minor quantities of 1,1-bis(p-chlorophenyl)-2-chloroethane (DDMS), 1,1-bis(p-chlorophenyl)-2-chloroethylene (DDMU), 1,1-bis(p-chlorophenyl)ethane (DDO), and 1,1-bis(p-chlorophenyl)ethylene (DDNU) were also formed. Reaction of vitamin B_12r with DDD results in the production of DDMU and DDMS, the latter of which can react to produce DDNU and DDO. DDE and DDMU do not react with vitamin B_12r. The results obtained are suggestive of a vitamin B_12r-mediated dechlorination pathway for polyhalogenated hydrocarbon pesticides.     

Metabolism of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene by Alcaligenes denitrificans

Metabolism of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), a persistent metabolite of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), by an Alcaligenes denitrificans was optimal under `non-shaking' conditions, was accelerated by adding 1 g glucose l^–1, and inhibited by 1 g sodium acetate l^–1 or 1 g sodium succinate l^–1. Addition of biphenyl, in the vapor form, to the reaction mixture did not enhance DDE metabolism. During the reaction, accumulation of conventional metabolites, 1-chloro-2,2-bis(4-chlorophenyl)ethylene (DDMU) and 4-chlorobenzoate, was not observed.     

Biodegradation of organochlorine pesticides by bacteria grown in microniches of the porous structure of green bean coffee

In this paper, the authors propose a model for DDT biodegradation by bacteria grown in microniches created in the porous structure of green bean coffee. Five bacteria isolated from coffee beans, identified as Pseudomonas aeruginosa, P. putida, Stenotrophomonas maltophilia, Flavimonas oryzihabitans, and Morganella morganii. P. aeruginosa and F. oryzihabitans, were selected for pesticide degradation. Bacteria were selected according to their ability to grow on mineral media amended with: (a) glucose (10 g l⁻¹), (b) peptone (2 g l⁻¹), and (c) ground coffee beans (2 g l⁻¹). These three media were supplemented with 50 mg l⁻¹ of 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) and endosulfan. GC/MS analysis demonstrated that the greatest DDT removal was obtained in the medium supplemented with coffee beans, where 1,1-dichloro-2,2′-bis (4-chlorophenyl)ethylene (DDE), 1-chloro-2,2-bis (4-chlorophenyl) ethane (DDMU) and 2,2′-bis (p-chlorophenyl)ethanol (DDOH) were detected. DDMU is a product of the reductive dechlorination of DDE, which in this system could be carried out under the anaerobic conditions in microniches present in the porous structure of the coffee bean. This was supported by scanning electron microscopy. Green bean coffee could be used as a nutrient source and as a support for bacterial growth in pesticide degradation.     

Evidence for Dual Binding Sites for 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) in Insect Sodium Channels

1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), the first organochlorine insecticide, and pyrethroid insecticides are sodium channel agonists. Although the use of DDT is banned in most of the world due to its detrimental impact on the ecosystem, indoor residual spraying of DDT is still recommended for malaria control in Africa. Development of resistance to DDT and pyrethroids is a serious global obstacle for managing disease vectors. Mapping DDT binding sites is necessary for understanding mechanisms of resistance and modulation of sodium channels by structurally different ligands. The pioneering model of the housefly sodium channel visualized the first receptor for pyrethroids, PyR1, in the II/III domain interface and suggested that DDT binds within PyR1. Previously, we proposed the second pyrethroid receptor, PyR2, at the I/II domain interface. However, whether DDT binds to both pyrethroid receptor sites remains unknown. Here, using computational docking of DDT into the K_v1.2-based mosquito sodium channel model, we predict that two DDT molecules can bind simultaneously within PyR1 and PyR2. The bulky trichloromethyl group of each DDT molecule fits snugly between four helices in the bent domain interface, whereas two p-chlorophenyl rings extend into two wings of the interface. Model-driven mutagenesis and electrophysiological analysis confirmed these propositions and revealed 10 previously unknown DDT-sensing residues within PyR1 and PyR2. Our study proposes a dual DDT-receptor model and provides a structural background for rational development of new insecticides.     

Metabolism of DDT [1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane] by <Emphasis Type="Italic">Alcaligenes denitrificans</Emphasis> ITRC-4 Under Aerobic and Anaerobic Conditions

An isolated bacterium, Alcaligenes denitrificans ITRC-4, metabolizes 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) under both aerobic and anaerobic conditions. The aerobic metabolism is inhibited by 38% and 47% in the presence of 1.0 g L⁻¹ of sodium acetate and sodium succinate, respectively, but remains uninhibited in the presence of 1.0 g L⁻¹ of glucose. Also, the metabolism is inhibited completely in the presence of biphenyl vapors, as well as 0.8 g L⁻¹ of 2,2′-bipyridyl. Under anaerobic conditions, DDT is metabolized into 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), which is further enhanced by 50% in the presence of 1.0 g L⁻¹ of glucose. Besides, the bacterium also metabolizes 4-chlorobenzoate, which is accompanied by the release of chloride ions. Received: 13 March 2002 / Accepted: 8 April 2002     

Enhancement of hepatic microsomal drug oxidation and glucuronidation in rat by 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT)

A single dose of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) (160 mg/kg i.p.) enhanced the monooxygenase step of drug biotransformation in rat liver. The O-demethylation of p-nitroanisole was especially increased, a peak in activity approximately 5-fold compared with controls being attained in 7 days. On the other hand, there was only a 2-fold increase in aryl hydrocarbon hydroxylase activity.DDT increased the cytochrome P-450 content of the liver, this increase coincided well with that in p-nitroanisole O-demethylation activity.The UDPglucuronosyltransferase activity of liver microsomes was not enhanced by DDT administration, unless the microsomes were pretreated to reveal latent activity prior to assay. After trypsin digestion of microsomes a maximum increase in activity of approximately 3-fold was observed as a result of DDT dosage. The canonic surfactant cetylpyridinium chloride was less active in revealing the latent UDP-glucuronosyltransferase activity, and two other membrane perturbants, the detergent digitonin and phospholipase A, were unable to show enhancement in UDPglucuronosyltransferase as a result of DDT dosage.     

A novel metabolic pathway for biodegradation of DDT by the white rot fungi, <Emphasis Type="Italic">Phlebia lindtneri</Emphasis> and <Emphasis Type="Italic">Phlebia brevispora</Emphasis>

1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) was used as the substrate for a degradation experiment with the white rot fungi Phlebia lindtneri GB-1027 and Phlebia brevispora TMIC34596, which are capable of degrading polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated biphenyls (PCBs). Pure culture of P. lindtneri and P. brevispora with DDT (25 μmol l⁻¹) showed that 70 and 30% of DDT, respectively, disappeared in a low-nitrogen medium after a 21-day incubation period. The metabolites were analyzed using gas chromatography/mass spectrometry (GC/MS). Both fungi metabolized DDT to 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 2,2-bis(4-chlorophenyl)acetic acid (DDA) and 4,4-dichlorobenzophenone (DBP). Additionally, DDD was converted to DDA and DBP. DDA was converted to DBP and 4,4-dichlorobenzhydrol (DBH). While DBP was treated as substrate, DBH and three hydroxylated metabolites, including one dihydroxylated DBP and two different isomers of monohydroxylated DBH, were produced from fungal cultures, and these hydroxylated metabolites were efficiently inhibited by the addition of a cytochrome P-450 inhibitor, piperonyl butoxide. These results indicate that the white rot fungi P. lindtneri and P. brevispora can degrade DBP/DBH through hydroxylation of the aromatic ring. Moreover, the single-ring aromatic metabolites, such as 4-chlorobenzaldehyde, 4-chlorobenzyl alcohol and 4-chlorobenzoic acid, were found as metabolic products of all substrate, demonstrating that the cleavage reaction of the aliphatic-aryl carbon bond occurs in the biodegradation process of DDT by white rot fungi.     

Products Formed from Analogues of 1,1,1-Trichloro-2,2-Bis(p-Chlorophenyl) Ethane (DDT) Metabolites by Pseudomonas putida

Cultures of Pseudomonas putida growing in solutions with diphenylmethane as sole carbon source formed 1,1,1′,1′-tetraphenyldimethyl ether. The product was identified by gas chromatography, mass spectrometry, and infrared and nuclear magnetic resonance spectrometry. The formation of benzophenone, benzhydrol, and phenylglycolic acid was established by gas chromatography and mass spectrometry. Similar techniques also revealed that phenylacetic acid was a major metabolite. Resting cell suspensions converted benzhydrol to phenyl-glycolic acid and products tentatively identified as hydroxybenzhydrols and a hydroxybenzophenone. Cell suspensions of the bacterium also converted the tetraphenyldimethyl ether to benzhydrol and benzophenone. Possible pathways for the degradation of these analogues of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) metabolites are discussed.     

Cytogenetic and mutagenic effects of DDT and DDE in a Chinese hamster cell line

The mutagenic and cytogenetic effects of the chlorinated hydrocarbon 1,11-trichloro-2,2-bis(p-chlorophenyl)ethane] (DDT), and its metabolite [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene] (DDE) were investigated in vitro using a Chinese hamster cell line. A forward mutation system utilizing the 8-azaguanine sensitive to 8-azaguanine resistant marker was used as the index of mutagenic action. Methyl methanesulfonate (MMS) was used as the positive control. In all experiments, DDE consistently produced a significant increase in the mutation frequency over the control level, while DDT proved inactive.Resultsof the cytogenetic studies indicated that DDE-treated cells had a significant increase in chromosome aberrations over those occuring in the control population; exchange figures and chromatid breaks wre evident. DDT produced no significant increase in chromosome abnormalities. The Chinese hamster cell populations exposed to DDE also manifested an increased number of polyploid cells over the control level.     

Metabolism of DDT and Kelthane in cell suspension cultures of parsley (Petroselinum hortense,Hoffm.) and soybean (Glycine max L.)

Cell suspension cultures of parsley and soybean were incubated for 44 to 48 h with¹⁴C-labeled DDT or Kelthane; autoclaved cultures were used as controls. Most of the radioactivity became associated with the cells, and metabolites were isolated by a sequential extraction procedure. The metabolites amounted to 0.6 to 2.2% of the applied pesticide. Relatively non-polar metabolites were identified as DDE in the case of DDT, and remained unidentified in the case of Kelthane. Polar metabolites were also isolated and are as yet unidentified. They were chromatographically different from the known and less polar metabolites of DDT and Kelthane reported from animal and insect studies. [DDT-1,1,1-Trichloro-2,2-bis-(4-chlorophenyl)-ethane; Kelthane=(1,1-bis-(4-chlorophenyl)-2,2,2-trichloro-ethanol; DDE=1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethylene.]Abbreviations DDT 1,1,1-Trichloro-2,2-bis-(4-chlorophenyl)-ethane - Kelthane (1,1-bis-(4-chlorophenyl)-2,2,2-trichloro-ethanol - DDE 1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethylene - DDA 2,2-bis-(4-chlorophenyl)-acetic acid - DDOH 2,2-bis-(4-chlorophenyl)-ethanol - DDD 1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethane - DBP 4,4-Dichloro-benzophenone - DDMU 1-Chloro-2,2-bis-(4-chlorophenyl)-ethylene - DDM Bis-(4-chlorophenyl)-methane - FW-152 1,1-Bis-(4-chlorophenyl)-2,2-dichloro-ethanol - SDS sodium dodecylsulphate     

Oxidation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5

Previous studies demonstrated that Alcaligenes eutrophus A5 transforms 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) to 4-chlorobenzoate via a meta-ring fission product. The initial reactions could be catalyzed by either monooxygenase or dioxygenase enzymes. In the present study, a transient intermediate that accumulated during the transformation of DDT by the biphenyl-grown cells was identified as 1,1,1-trichloro-2-(4-chlorophenyl-2,3-dihydro-4,6-cyclohexadiene)-2-(4′-chlorophenyl)ethane (DDT-2,3-dihydrodiol) on the basis of mass spectral analysis after n-butylboronic acid derivatization. The dihydrodiol undergoes a characteristic acid-catalyzed dehydration to produce phenols. ¹H-NMR indicated a cis-relative stereochemistry. The results indicate that the biphenyl dioxygenase from A. eutrophus A5 catalyzes the dihydroxylation of DDT at the unsubstituted carbons on the aromatic ring to produce DDT-2,3-dihydrodiol. Received: 22 July 1998 / Accepted: 6 October 1998     

Degradation of chlorinated pesticide DDT by litter-decomposing basidiomycetes

One hundred and two basidiomycete strains (93 species in 41 genera) that prefer a soil environment were examined for screening of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) biodegradation. Three strains within two litter-decomposing genera, Agrocybe and Marasmiellus, were selected for their DDT biotransformation capacity. Eight metabolites; 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), two monohydroxy-DDTs, monohydroxy-DDD, 2,2-dichloro-1,1-bis(4-chlorophenyl)ethanol, putative 2,2-bis(4-chlorophenyl)ethanol and two unidentified compounds were detected from the culture with Marasmiellus sp. TUFC10101. A P450 inhibitor, 1-ABT, inhibited the formation of monohydroxy-DDTs and monohydroxy-DDD from DDT and DDD, respectively. These results indicated that oxidative pathway which was catalyzed by P450 monooxygenase exist beside reductive dechlorination of DDT. Monohydroxylation of the aromatic rings of DDT (and DDD) by fungal P450 is reported here for the first time.     

Elucidating Turnover Pathways of Bioactive Small Molecules by Isotopomer Analysis: The Persistent Organic Pollutant DDT

The persistent organic pollutant DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) is still indispensable in the fight against malaria, although DDT and related compounds pose toxicological hazards. Technical DDT contains the dichloro congener DDD (1-chloro-4-[2,2-dichloro-1-(4-chlorophenyl)ethyl]benzene) as by-product, but DDD is also formed by reductive degradation of DDT in the environment. To differentiate between DDD formation pathways, we applied deuterium NMR spectroscopy to measure intramolecular deuterium distributions (²H isotopomer abundances) of DDT and DDD. DDD formed in the technical DDT synthesis was strongly deuterium-enriched at one intramolecular position, which we traced back to ²H/¹H fractionation of a chlorination step in the technical synthesis. In contrast, DDD formed by reductive degradation was strongly depleted at the same position, which was due to the incorporation of ²H-depleted hydride equivalents during reductive degradation. Thus, intramolecular isotope distributions give mechanistic information on reaction pathways, and explain a puzzling difference in the whole-molecule ²H/¹H ratio between DDT and DDD. In general, our results highlight that intramolecular isotope distributions are essential to interpret whole-molecule isotope ratios. Intramolecular isotope information allows distinguishing pathways of DDD formation, which is important to identify polluters or to assess DDT turnover in the environment. Because intramolecular isotope data directly reflect isotope fractionation of individual chemical reactions, they are broadly applicable to elucidate transformation pathways of small bioactive molecules in chemistry, physiology and environmental science.     

Cometabolism of 1,1-Dichloro-2,2-Bis(4-Chlorophenyl)Ethylene by Pseudomonas acidovorans M3GY Grown on Biphenyl

1,1-Dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), a toxic breakdown product of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), has traditionally been viewed as a dead-end metabolite: there are no published reports detailing enzymatic ring fission of DDE by bacteria in either soil or pure culture. In this study, we investigated the ability of Pseudomonas acidovorans M3GY to transform DDE and its unchlorinated analog, 1,1-diphenylethylene (DPE). While strain M3GY could grow on DPE, cells grown on DPE as a sole carbon source could not degrade DDE. Cells grown on biphenyl, however, did degrade DDE. Mass balance analysis of [¹⁴C]DDE showed transformation of more than 40% of the recoverable radioactivity. Nine chlorinated metabolites produced from DDE were identified by gas chromatography-mass spectrometry–Fourier-transform infrared spectrometry (GC-MS-FTIR) from cultures grown on biphenyl. Recovery of these metabolites demonstrates that biphenyl-grown cells degrade DDE through a meta-fission pathway. This study provides a possible model for biodegradation of DDE in soil by biphenyl-utilizing bacteria.     

Interaction of insecticides with human plasma lipoproteins

The binding of chlorinated hydrocarbon, carbamate and organophosphate insecticides to human low density plasma lipoproteins (LDL) and high density plasma lipoproteins (HDL) was studied at pH 7.0 and 16°C and 26°C by equilibrium dialysis, difference spectra and fluorescence. The results suggest interaction to be a partitioning rather than a stoichiometric binding process. Distribution is related to lipid content and composition of the lipoproteins. The K-values vary from 3 × 10⁵ M^?1 for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) to less than 10 M^?1 for nicotine and aldicarb, and ΔG_tr° is in the range of 7400 cal for DDT to less than 1000 cal for aldicarb and nicotine. The K and ΔG_tr° are inversely related to the water solubility of the insecticides. A significant role of plasma lipoproteins in the transport of slightly water soluble insecticides is suggested.     

The separation of multiple forms of housefly 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane (DDT) dehydrochlorinase from glutathione S-aryltransferase by electrofocusing and electrophoresis

Electrophoresis in a sucrose gradient at pH values between 5 and 8 separated housefly DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] dehydrochlorinase into two major fractions. GSH S-aryltransferase under similar conditions migrated as a single peak of activity. Separation of housefly homogenates or partially purified enzyme preparations by electrofocusing in a natural pH gradient also showed the presence of multiple forms of DDT dehydrochlorinase.     

Biodegradation of DDT by a <Emphasis Type="Italic">Pseudomonas</Emphasis> Species

A bacterial strain capable of degrading 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) was isolated from insecticide-contaminated soil by biphenyl enrichment culture and identified as a Pseudomonas species. The organism degraded DDT through the intermediate formation of 2,3-dihydroxy-DDT, which undergoes meta-ring cleavage, ultimately yielding 4-chlorobenzoic acid as a stable metabolite.