Cumulative exposure and dietary risk assessment of phthalates in bottled water and bovine milk samples: A preliminary case study in Tamil Nadu,India

Exposure to phthalates may cause adverse health effects in wildlife and humans. Study on phthalates exposure and risk is limited in the Indian context. Therefore, this preliminary investigation was performed to ascertain the phthalates exposure through bottled water and milk among the Indian sub-population. Phthalates were extracted from water and milk by solid-phase and ultrasonication methods, respectively, and analysis was performed using gas chromatography–mass spectrometry. Total phthalates in bottled water and milk were in the range of 39–7820 ng/L and 56–686 ng/g, respectively, with the highest contribution from diethylhexyl phthalate (DEHP). A substantial increase in phthalates concentration in bottled water was observed with increased shelf life. Total mean phthalates in packed milk (245 ng/g) and raw milk (134 ng/g) shows potential enrichment during “farm to table” process. Among phthalates, the lowest risk was expected from diethyl phthalate, whereas the highest risk was observed for DEHP with cumulative dietary exposure of 0.23 μg/kg bw/day (median). The human health risk based on tolerable daily intake and reference dose was found safe. This is the first study reporting phthalates migration in packed commodities from a developing country, India, which further warrants extensive phthalates exposure assessment to understand its effect on public health.     

Biodegradation of diethyl phthalate by an organic-solvent-tolerant Bacillus subtilis strain 3C3 and effect of phthalate ester coexistence

The contamination and distribution of phthalate esters - synthetic compounds widely used in plastic product production, including food and medical packaging - has raised safety concerns due to their endocrine-disrupting activity and mandated to be treated. Bacillus subtilis strain 3C3, isolated as an organic-solvent-tolerant bacterium, was capable of utilizing diethyl phthalate as a sole carbon source. Biodegradation of diethyl phthalate occurred constitutively without lag period, and its kinetics followed a first-order model. The biodegradability was significantly enhanced with the supplementation of yeast extract as a co-metabolic substrate. In the presence of Tween-80 as a solubilizing agent, cells rapidly degrade a range of short-chain phthalate esters at high concentrations (up to 1000 mg l⁻¹ for diethyl phthalate). The biodegradation of short-chain phthalates in the binary, ternary and quaternary substrate system revealed that the coexistence of other short-chain phthalates had no significant influence on the biodegradation of diethyl phthalate, and vice versa. These results substantiated that B. subtilis strain 3C3 has potential application as a bioaugmented bacterial culture for bioremediation of phthalates.     

Cloning of a dibutyl phthalate hydrolase gene from Acinetobacter sp. strain M673 and functional analysis of its expression product in Escherichia coli

A dibutyl phthalate (DBP) transforming bacterium, strain M673, was isolated and identified as Acinetobacter sp. This strain could not grow on dialkyl phthalates, including dimethyl, diethyl, dipropyl, dibutyl, dipentyl, dihexyl, di(2-ethylhexyl), di-n-octyl, and dinonyl phthalate, but suspensions of cells could transform these compounds to phthalate via corresponding monoalkyl phthalates. During growth in Luria–Bertani medium, M673 produced the high amounts of non-DBP-induced intracellular hydrolase in the stationary phase. One DBP hydrolase gene containing an open reading frame of 1,095 bp was screened from a genomic library, and its expression product hydrolyzed various dialkyl phthalates to the corresponding monoalkyl phthalates.     

Biodegradation of endocrine-disrupting phthalates by Pleurotus ostreatus

Biodegradation of endocrine-disrupting phthalates [diethyl phthalate (DEP), dimethyl phthalate (DMP), butylbenzyl phthalate (BBP)] was investigated with 10 white rot fungi isolated in Korea. When the fungal mycelia were added together with 100 mg/l of phthalate into yeast extract-malt extract-glucose (YMG) medium, Pleurotus ostreatus, Irpex lacteus, Polyporus brumalis, Merulius tremellosus, Trametes versicolor, and T. versicolor MrP1 and MrP13 (transformant of the Mn-repressed peroxidase gene of T. versicolor) could remove almost all of the 3 kinds of phthalates within 12 days of incubation. When the phthalates were added to 5-day pregrown fungal cultures, most fungi except I. lacteus showed the increased removal of the phthalates compared with those of the nonpregrown cultures. In both culture conditions, P. ostreatus showed the highest degradation rates for the 3 phthalates tested. BBP was degraded with the highest rates among the 3 phthalates by all fungal strains. Only 14.9% of 100 mg/l BBP was degraded by the supernatant of P. ostreatus culture in YMG medium in 4 days of incubation, but the washed or homogenized mycelium of P. ostreatus could remove 100% of BBP within 2 days even in distilled water, indicating that the initial BBP biodegradation by P. ostreatus may be attributed to mycelium-associated enzymes rather than extracellular enzymes. The biodegradation rate of BBP by the immobilized cells of P. ostreatus was almost the same as that in the suspended culture. The estrogenic activity of 100 mg/l DMP decreased during biodegradation by P. ostreatus.     

电子垃圾拆解地区土壤和植物中邻苯二甲酸酯分布特征

近年来,电子垃圾不当拆解带来的环境问题引起国际社会的极大关注.本研究对浙江省台州市不同电子垃圾拆解地区土壤和植物样品中5种邻苯二甲酸酯类（PAEs）污染物进行了测定分析.结果表明：土壤（以干质量计）中PAEs类污染物的浓度为12.566~46.669 mg·kg^-1,其中邻苯二甲酸二异辛酯（DEHP）、邻苯二甲酸二丁酯（DBP）和邻苯二甲酸二乙酯（DEP）相对含量较高,约占PAEs总量的94%以上.拆解地区蚕豆（Vicia faba L.）中PAEs总量明显高于同地区其他植物,且土壤和所有植物体内PAEs浓度相关性均不显著(P>0.05).与美国国家环保局制定的土壤PAEs治理标准比较,台州电子垃圾拆解地区土壤PAEs污染较为严重.     

Biodegradation of diethyl phthalate in soil by a novel pathway

Biodegradation of diethyl phthalate (DEP) has been shown to occur as a series of sequential steps common to the degradation of all phthalates. Primary degradation of DEP to phthalic acid (PA) has been reported to involve the hydrolysis of each of the two diethyl chains of the phthalate to produce the monoester monoethyl phthalate (MEP) and then PA. However, in soil co-contaminated with DEP and MeOH, biodegradation of the phthalate to PA resulted in the formation of three compounds, in addition to MEP. These were characterised by gas chromatography-electron ionisation mass spectrometry and nuclear magnetic resonance as ethyl methyl phthalate, dimethyl phthalate and monomethyl phthalate, and indicated the existence of an alternative pathway for the degradation of DEP in soil co-contaminated with MeOH. Transesterification or demethylation were proposed as the mechanisms for the formation of the three compounds, although the 7:1 ratio of H(2)O to MeOH means that transesterification is unlikely.     

Children’s Phthalate Intakes and Resultant Cumulative Exposures Estimated from Urine Compared with Estimates from Dust Ingestion,Inhalation and Dermal Absorption in Their Homes and Daycare Centers

Total daily intakes of diethyl phthalate (DEP), di(n-butyl) phthalate (DnBP), di(isobutyl) phthalate (DiBP), butyl benzyl phthalate (BBzP) and di(2-ethylhexyl) phthalate (DEHP) were calculated from phthalate metabolite levels measured in the urine of 431 Danish children between 3 and 6 years of age. For each child the intake attributable to exposures in the indoor environment via dust ingestion, inhalation and dermal absorption were estimated from the phthalate levels in the dust collected from the child’s home and daycare center. Based on the urine samples, DEHP had the highest total daily intake (median: 4.42 µg/d/kg-bw) and BBzP the lowest (median: 0.49 µg/d/kg-bw). For DEP, DnBP and DiBP, exposures to air and dust in the indoor environment accounted for approximately 100%, 15% and 50% of the total intake, respectively, with dermal absorption from the gas-phase being the major exposure pathway. More than 90% of the total intake of BBzP and DEHP came from sources other than indoor air and dust. Daily intake of DnBP and DiBP from all exposure pathways, based on levels of metabolites in urine samples, exceeded the Tolerable Daily Intake (TDI) for 22 and 23 children, respectively. Indoor exposures resulted in an average daily DiBP intake that exceeded the TDI for 14 children. Using the concept of relative cumulative Tolerable Daily Intake (TDI_cum), which is applicable for phthalates that have established TDIs based on the same health endpoint, we examined the cumulative total exposure to DnBP, DiBP and DEHP from all pathways; it exceeded the tolerable levels for 30% of the children. From the three indoor pathways alone, several children had a cumulative intake that exceeded TDI_cum. Exposures to phthalates present in the air and dust indoors meaningfully contribute to a child’s total intake of certain phthalates. Such exposures, by themselves, may lead to intakes exceeding current limit values.     

Endocrine Disruption: Computational Perspectives on Human Sex Hormone-Binding Globulin and Phthalate Plasticizers

Phthalates are a class of high volume production chemicals used as plasticizers for household and industrial use. Several members of this chemical family have endocrine disrupting activity. Owing to ubiquitous environmental distribution and exposure of human population at all stages of life, phthalate contamination is a continuous global public health problem. Clinical and experimental studies have indicated that several phthalates are associated with adverse effects on development and function of human and animal systems especially the reproductive system and exposures during pregnancy and early childhood are by far of utmost concern. Sex hormone-binding globulin (SHBG) is a plasma carrier protein that binds androgens and estrogens and represents a potential target for phthalate endocrine disruptor function in the body. In the present study, the binding mechanism of the nine phthalates i.e. DMP, DBP, DIBP, BBP, DNHP, DEHP, DNOP, DINP, DIDP with human SHBG was delineated by molecular docking simulation. Docking complexes of the nine phthalates displayed interactions with 15–31 amino acid residues of SHBG and a commonality of 55–95% interacting residues between natural ligand of SHBG, dihydrotestosterone, and the nine phthalate compounds was observed. The binding affinity values were more negative for long chain phthalates DEHP, DNOP, DINP, and DIDP compared to short chain phthalates such as DMP and DBP. The Dock score and Glide score values were also higher for long chain phthalates compared to short chain phthalates. Hence, overlapping of interacting amino acid residues between phthalate compounds and natural ligand, dihydrotestosterone, suggested potential disrupting activity of phthalates in the endocrine homeostasis function of SHBG, with long chain phthalates expected to be more potent than the short chain phthalates.     

Direct LC-ES-MS/MS determination of phthalates in physiological saline solutions

A method for determining a group of phthalic esters (PAEs) in physiological saline solutions has been developed. The PAEs studied were dimethyl phthalate, diethyl phthalate, butyl benzyl phthalate and dibutyl phthalate. These groups of phthalates were determined by liquid chromatography–electrospray ionization-tandem mass spectrometry, working in positive ion mode. The compounds were separated by liquid chromatography working in gradient mode with acetonitrile–ultrapure water as a mobile phase. The separation was performed starting with 5% of acetonitrile and increasing to 75% in 5 min, followed by isocratic elution for 8 min. The method was precise (with relative standard deviation (RSD) from 1.0 to 6.8%) and sensitive, with LODs of 0.05, 0.38, 0.05 and 0.82 μg L^?1 for DMP, DEP, BBP and DBP, respectively. The proposed analytical method has been applied to determine these compounds in different physiological saline solutions commercialized in plastic bottles.     

The effects of selected phenol and phthalate derivatives on steroid hormone production by cultured porcine granulosa cells

We have investigated the effects of several phenols (octylphenol [OP], nonylphenol [NP], tert-octylphenol [tOP]) and phthalates (dioctylphthalate [DOP], diisodecylphthalate [DiDP], diisononylphthalate [DiNP]) on steroid hormone production by porcine ovarian granulosa cells after a 72-hour incubation. These chemicals are widely used as plasticisers and are suspected to possess endocrine disrupting properties. No changes were exhibited in basal progesterone production after treatment with NP or tOP, or with the tested phthalates. However, OP tended to decrease progesterone levels, while DOP and DiDP, at the lowest concentration used (10(-8)M), increased progesterone levels in the culture media. Neither of the tested phenols affected follicle stimulating hormone (FSH)-stimulated progesterone production, except for OP and NP at 10(-4)M, which decreased progesterone levels. The phthalates, tested at higher concentrations, were able to amplify FSH-stimulated progesterone release into the culture medium. An inhibitory action on oestradiol production by porcine granulosa cells was observed after the treatment with both groups of test chemicals. The results obtained in the experiments on primary granulosa cell cultures indicate that ovarian steroidogenesis might be one of the possible sites affected by the endocrine disrupting actions of phenols and phthalates.     

In silico evidences for the binding of phthalates onto human estrogen receptor α, β subtypes and human estrogen-related receptor γ

Being lipophilic xenobiotic chemicals, phthalates from the surrounding environments can easily be absorbed into the biological system, thereby causing various health problems including cancer and endocrine disruption in test animals and also in humans. In the present in silico study employing Glide, Schrödinger Suite 2012, we analysed in detail the binding affinities of 12 commonly used diphthalates and their metabolites (corresponding mono ester and phthalic acid) onto the ligand-binding domain (LBD) of the human estrogen receptor α (hERα), human estrogen receptor β (hERβ) and human estrogen related receptor γ (hERRγ). Natural ligand 17β estradiol (E2), known xenoestrogen bisphenol A, the phytoestrogen genistein, the agonists/antagonists 4-hydroxy tamoxifen and raloxifene were also docked onto these receptors as positive controls for comparing the binding efficiencies with that of phthalates and their metabolites. Results revealed that E2 had less binding affinity to the receptors in comparison to certain phthalates, i.e. maximum binding scores (G score, kcal/mol) were diisononyl phthalate ( ? 9.44) to hERα, monophenyl phthalate ( ? 8.66) to hERβ and di(2-ethylhexyl)phthalate ( ? 9.38) to hERRγ. The most concerned monophthalates established additional H bonds with certain surrounding crucial amino acid residues in the LBD, and thus showed more affinity to all the receptors than even the natural ligand and other well-characterised xenoestrogens as demonstrated in this study. Briefly, this study gives an insight into the virtual binding behaviours of commonly used phthalates and their metabolites onto hERs and hERRγ, which would accelerate further in vitro mechanistic, preclinical and clinical studies on real in vitro or in vivo platforms.     

Sex Steroid Hormone Levels and Reproductive Development of Eight-Year-Old Children following In Utero and Environmental Exposure to Phthalates

In utero exposure to phthalates may adversely affect reproductive development in children due to the anti-androgenic properties of the pthalates. Accordingly, we aimed to determine the effects of in utero and environmental phthalate exposure on the reproductive development of eight-year-old children. We recruited 180 children in central Taiwan during November 2001 and followed them until August 2009 when all children became eight years old. Birth outcomes were collected. Bone age, hormone concentrations, and reproductive developmental stages were determined. Phthalate metabolite levels, including mono-2-ethylhexyl phthalate [MEHP], mono-n-butyl phthalate [MnBP], and mono-benzyl phthalate [MBzP], were assessed. No significant gender differences were found in in utero phthalate exposure. Maternal urinary levels of phthalate metabolites did not correlate significantly with birth outcomes, physical characteristics, and reproductive hormones of the eight-year-old children. Regarding the urinary phthalate metabolite levels of the eight-year-old children, MEHP correlated significantly with serum progesterone levels. MEHP levels in girls correlated significantly with serum progesterone levels. MnBP correlated significantly with serum FSH in all children. In girls, MnBP correlated with serum FSH, and MBzP correlated with serum progesterone and FSH levels. Urinary phthalate metabolite levels did not correlate with female developmental stages or the development of female reproductive organs. Phthalate metabolites did not correlate with the physical characteristics and reproductive hormones in boys. Therefore, environmental exposure to phthalates, as determined by urinary phthalate metabolite levels of eight-year-old children, may affect reproductive hormone levels in children, indicating that further studies on the environmental health effects of phthalates are warranted.     

Phthalates biodegradation in the environment

Phthalates are synthesized in massive amounts to produce various plastics and have become widespread in environments following their release as a result of extensive usage and production. This has been of an environmental concern because phthalates are hepatotoxic, teratogenic, and carcinogenic by nature. Numerous studies indicated that phthalates can be degraded by bacteria and fungi under aerobic, anoxic, and anaerobic conditions. This paper gives a review on the biodegradation of phthalates and includes the following aspects: (1) the relationship between the chemical structure of phthalates and their biodegradability, (2) the biodegradation of phthalates by pure/mixed cultures, (3) the biodegradation of phthalates under various environments, and (4) the biodegradation pathways of phthalates.     

Urinary concentration of endocrine-disrupting phthalates and breast cancer risk in Indian women: A case-control study with a focus on mutations in phthalate-responsive genes

BackgroundPhthalates are known endocrine-disrupting chemicals used indiscriminately as constituents in consumer products including food processing, and packaging, cosmetics, personal care and household items. Although, few studies have assessed the risk of breast cancer on exposure to phthalates, their association with breast cancer risk in Indian women have not yet been evaluated.MethodsWe conducted a case-control study involving 171 participants. Urinary concentrations of six phthalate dieters; DMP (Dimethyl phthalate), DEP (Diethyl phthalate), DBP (Dibutyl phthalate), BBP (benzyl butyl phthalate), DEHP (Di-2-ethyl-hexyl phthalate), DINOP (Di-n-octyl phthalate) were estimated by GC-MS and geometric means were calculated. Univariate and multivariable logistic regression was performed to assess breast cancer risk on exposure to phthalates. Genes responsive to phthalates were identified through literature search and matched with NGS data, and gene-enrichment analysis was performed.ResultsSignificant associations were observed between urinary phthalate concentrations and increased risk of breast cancer for di-butyl phthalate (OR=1.5, 95% CI; 1.06, 2.11, p = 0.002) and di-2-ethyl-hexyl phthalate (>median vs ≤ median; OR=2.97, 95% CI; 1.18, 7.47, p = 0.005) in multivariable analyses. We also found several phthalate-responsive gene mutations in paired breast tumor tissues, which include PTPRD (76.19%), AR (42.86%), CYP1A1 (42.86%), CYP19A1 (23.81%), AHRR (19.05%), PIK3CA (19.05%), CYP1B1 (9.52%), RB1 (9.52%) and MMP9 (9.52%). Gene-enrichment analysis revealed that these genes form a major part of ER/PR, PPAR and HIF-1α-TGF-β signaling cascades involved in breast cancerConclusionAlthough the sample size is small, in this first case-control study from India, DBP and DEHP were found to be associated with increased risk of invasive breast cancer and tumor tissues revealed mutations in several phthalate-responsive genes. It is, therefore suggested that human biomonitoring in India and larger studies evaluating the early life genetic and epigenetic alterations on phthalates exposure are required to establish their role in breast carcinogenesis.     

Phthalates: toxicogenomics and inferred human diseases

Phthalates are widely used as plasticizers to soften and increase the flexibility in polyvinyl chloride plastics, but they can leach into the surrounding environment. There is sufficient evidence in rodents that phthalate exposure causes developmental and reproductive toxicity. The curated interactions between 16 phthalates and genes/proteins were obtained from Comparative Toxicogenomics Database (CTD), and a total of 445 interactions between the five most frequently curated phthalates (DEHP/MEHP and DBP/BBP/MBP) and 249 unique genes/proteins were found. The GeneOntology, pathways and networks of these 249 unique genes/proteins were fully analyzed. The pathways and networks of top 34 genes/proteins were found to be very similar to those of the 249 unique genes/proteins. Thus, the top 34 genes/proteins may serve as molecular biomarkers of phthalate toxicity. The top three phthalate toxicity categories were found to be cardiotoxicity, hepatotoxicity and nephrotoxicity, and the top 20 diseases included cardiovascular, liver, urologic, endocrine and genital diseases.     

What is an endocrine disruptor?

Endocrine disrupting chemicals (EDCs) and potential EDCs are mostly man-made found in various materials. By interfering with the body's endocrine system, endocrine disruptors produce adverse developmental, reproductive, neurological, and immune effects in humans, abnormal growth patterns and neurodevelopmental delays in children. Thus, diethylstilbestrol (DES) a non-steroidal estrogen, which is regarded as a proof of concept, induces clear cell carcinoma among young women. EDCS may be found in plastic bottles and metal food cans (BPA), medical devices (phthalates), detergents, flame retardants (polybrominated diphenyl ethers), food (BPA), toys (phthalates), cosmetics and drugs (parabens), and pesticides (alkyl phenols such as nonylphenol). The deleterious effects of endocrine disruptors constitute a real public health issue. However concerning the mechanisms of action of EDCs, many questions remain unanswered and need further investigations.     

Chemical Analysis and Risk Assessment of Diethyl Phthalate in Alcoholic Beverages with Special Regard to Unrecorded Alcohol

Background

Phthalates are synthetic compounds with a widespread field of applications. For example, they are used as plasticizers in PVC plastics and food packaging, or are added to personal care products. Diethyl phthalate (DEP) may be used to denature alcohol, e.g., for cosmetic purposes. Public health concerns of phthalates include carcinogenic, teratogenic, hepatotoxic and endocrine effects. The aim of this study was to develop and validate a method for determining phthalates in alcohol samples and to provide a risk assessment for consumers of such products.

Methodology/Principal Findings

A liquid-liquid extraction procedure was optimized by varying the following parameters: type of extraction solvent (cyclohexane, n-hexane, 1,1,2-trichlorotrifluoroethane), the ratio extraction solvent/sample volume (1∶1 to 50∶1) and the number of extraction repetitions (1–10). The best extraction yield (99.9%) was achieved with the solvent 1,1,2-trichlorotrifluoroethane, an extraction solvent volume/sample volume ratio of 10∶1 and a double extraction. For quantification, gas chromatography/mass spectrometry with deuterated internal standards was used. The investigated samples were alcoholic beverages and unrecorded alcohol products from different countries (n = 257). Two unrecorded alcohol samples from Lithuania contained diethyl phthalate in concentrations of 608 mg/L and 210 mg/L.

Conclusions/Significance

The consumption of the phthalate-positive unrecorded alcohols would exceed tolerable daily intakes as derived from animal experiments. Both positive samples were labelled as cosmetic alcohol, but had clearly been offered for human consumption. DEP seems to be unsuitable as a denaturing agent as it has no effect on the organoleptic properties of ethanol. In light of our results that DEP might be consumed by humans in unrecorded alcohols, the prohibition of its use as a denaturing agent should be considered.     

Influence of Phthalates on in vitro Innate and Adaptive Immune Responses

Phthalates are a group of endocrine disrupting chemicals, suspected to influence the immune system. The aim of this study was to investigate the influence of phthalates on cytokine secretion from human peripheral blood mononuclear cells. Escherichia coli lipopolysaccharide and phytohemagglutinin-P were used for stimulation of monocytes/macrophages and T cells, respectively. Cells were exposed for 20 to 22 hours to either di-ethyl, di-n-butyl or mono-n-butyl phthalate at two different concentrations. Both diesters were metabolised to their respective monoester and influenced cytokine secretion from both monocytes/macrophages and T cells in a similar pattern: the secretion of interleukin (IL)-6, IL-10 and the chemokine CXCL8 by monocytes/macrophages was enhanced, while tumour necrosis factor (TNF)-α secretion by monocytes/macrophages was impaired, as was the secretion of IL-2 and IL-4, TNF-α and interferon-γ by T cells. The investigated phthalate monoester also influenced cytokine secretion from monocytes/macrophages similar to that of the diesters. In T cells, however, the effect of the monoester was different compared to the diesters. The influence of the phthalates on the cytokine secretion did not seem to be a result of cell death. Thus, results indicate that both human innate and adaptive immunity is influenced in vitro by phthalates, and that phthalates therefore may affect cell differentiation and regenerative and inflammatory processes in vivo.     

An erythromycin process improvement using the diethyl methylmalonate-responsive (Dmr) phenotype of the <Emphasis Type="BoldItalic">Saccharopolyspora erythraea mutB</Emphasis> strain

The Saccharopolyspora erythraea mutB knockout strain, FL2281, having a block in the methylmalonyl-CoA mutase reaction, was found to carry a diethyl methylmalonate-responsive (Dmr) phenotype in an oil-based fermentation medium. The Dmr phenotype confers the ability to increase erythromycin A (erythromycin) production from 250–300% when the oil-based medium is supplemented with 15 mM levels of this solvent. Lower concentrations of the solvent stimulated proportionately less erythromycin production, while higher concentrations had no additional benefit. Although the mutB strain is phenotypically a low-level erythromycin producer, diethyl methylmalonate supplementation allowed it to produce up to 30% more erythromycin than the wild-type (control) strain—a strain that does not show the Dmr phenotype. The Dmr phenotype represents a new class of strain improvement phenotype. A theory to explain the biochemical mechanism for the Dmr phenotype is proposed. Other phenotypes found to be associated with the mutB knockout were a growth defect and hyper-pigmentation, both of which were restored to normal by exposure to diethyl methylmalonate. Furthermore, mutB fermentations did not significantly metabolize soybean oil in the presence of diethyl methylmalonate. Finally, a novel method is proposed for the isolation of additional mutants with the Dmr phenotype.